Note: Descriptions are shown in the official language in which they were submitted.
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A NOVEL PHARMACEUTICAL COMPOUND CONTAINING
ABACAVIR SULFATE AND METHODS OF MAIKING AND USING SAME
FIELD OF THE INVENTION
The present invention relates to a novel pharmaceutical compound that
comprises
a polypeptide that is preferably covalently attached to abacavir sulfate, as
well as
methods for protecting and administering abacavir sulfate. This novel
compound,
referred to as a CARRIERWAVETM Molecular Analogue (C1VIA), has the benefit of
taking a known effective pharmaceutical agent that is both well studied and
occupies a
known segment of the pharmaceutical market, and combining it with a carrier
compound
that enhances the usefulness of the pharmaceutical agent without compromising
its
pharmaceutical effectiveness.
BACKGROUND OF THE INVENTION
Abacavir sulfate is a known pharmaceutical agent - a carbocyclic 2'-
deoxyguanosine nucleoside analogue that is a reverse transcriptase inhibitor
used in the
treatment of HIV. Its chemical name is (1S,4R)-4-[2-amino-6-(cyclopropylamino)-
9H-
purin-9-yl]-2-cyclopentene-1-methanol. Its structure is as follows:
~N
N \N
N ~N
O
The novel pharmaceutical compound of the present invention is useful in
accomplishing one or more of the following goals: enhancement of the chemical
stability
of the original compound; alteration of the release profile of an orally
administered
product; enhanced digestion or absorbtion; targeted delivery to particular
tissuelcell type;
and provision for an oral dosage form when none exists. The novel
pharmaceutical
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compound may contain one or more of the following: another active
pharmaceutical
agent, an adjuvant, or an inhibitor.
Active agent delivery systems are often critical for the effective delivery of
a
biologically active agent (active agent) to the appropriate target. The
importance of these
systems becomes magnified when patient compliance and active agent stability
are taken
under consideration. For instance, one would expect patient compliance to
increase
markedly if an active agent is administered orally in lieu of an injection or
another
invasive technique. Increasing the stability of the active agent, such as
prolonging shelf
life or survival in the stomach, will assure dosage reproducibility and
perhaps even
reduce the number of dosages required which could improve patient compliance.
Absorption of an orally administered active agent is often blocked by the
harshly
acidic stomach milieu, powerful digestive enzymes in the GI tract,
permeability of
cellular membranes and transport across lipid bilayers. Incorporating
adjuvants such as
resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance
permeability of
cellular membranes. Microencapsulating active agents using protenoid
microspheres,
liposomes or polysaccharides have been effective in abating enzyme degradation
of the
active agent. Enzyme inhibiting adjuvants have also been used to prevent
enzyme
degradation. Enteric coatings have been used as a protector of pharmaceuticals
in the
stomach.
2o Active agent delivery systems also provide the ability to control the
release of the
active agent. For example, formulating diazepam with a copolymer of glutamic
acid and
aspartic acid enables a sustained release of the active agent. As another
example,
copolymers of lactic acid and glutaric acid are used to provide timed release
of human
growth hormone. A wide range of pharmaceuticals purportedly provide sustained
release
through microencapsulation of the active agent in amides of dicarboxylic
acids, modified
amino acids or thermally condensed amino acids. Slow release rendering
additives can
also be intermixed with a large array of active agents in tablet formulations.
Each of these technologies imparts enhanced stability and time-release
properties
to active agent substances. Unfortunately, these technologies suffer from
several
2
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shortcomings. Incorporation of the active agent is often dependent on
diffusion into the
microencapsulating matrix, which may not be quantitative and may complicate
dosage
reproducibility. In addition, encapsulated drugs rely on diffusion out of the
matrix, which
is highly dependant on the water solubility of the active agent. Conversely,
water-soluble
microspheres swell by an infinite degree and, unfortunately, may release the
active agent
in bursts with little active agent available for sustained release.
Furthermore, in some
technologies, control of the degradation process required for active agent
release is
unreliable. For example, an enterically coated active agent depends on pH to
release the
active agent and, as such, is difficult to control the rate of release.
In the past, use has been made of amino acid side chains of polypeptides as
pendant groups to which active agents can be attached. These technologies
typically
require the use of spacer groups between the amino acid pendant group and the
active
agent. The peptide-drug conjugates of this class of drug delivery system rely
on enzymes
in the bloodstream for the release of the drug and, as such, are not used for
oral
administration. Examples of timed and targeted release of injectable or
subcutaneous
pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer,
to the
gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a
peptide
spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been
covalently
attached directly to the beta carboxylate of polyaspartic acid without a
spacer group.
This prodrug formulation was designed as a colon-specific drug delivery system
where
the drug is released by bacterial hydrolytic enzymes residing in the large
intestines. The
released dexamethasone active agent, in turn, was targeted to treat large
bowel disorders
and was not intended to be absorbed into the bloodstream. Yet another
technology
combines the advantages of covalent drug attachment with liposome formation
where the
active ingredient is attached to highly ordered lipid films (known as HARs)
via a peptide
linker. Thus, there has been no drug delivery system, heretofore reported,
that
incorporates the concept of attaching an active ingredient to a polypeptide
pendant group
with its targeted delivery into the bloodstream via oral administration.
It is also important to control the molecular weight, molecular size and
particle
size of the active agent delivery system. Variable molecular weights have
unpredictable
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diffusion rates and pharmacokinetics. High molecular weight carriers are
digested slowly
or late, as in the case of naproxen-linked dextran, which is digested almost
exclusively in
the colon by bacterial enzymes. High molecular weight microspheres usually
have high
moisture content which may present a problem with water labile active
ingredients.
Particle size not only becomes a problem with injectable drugs, as in the HAR
application, but absorption through the brush-border membrane of the
intestines is limited
to less than 5 microns.
SUMMARY OF THE INVENTION
The present invention provides covalent attachment of the active agent
(abacavir
sulfate) to a polymer of peptides or amino acids. The invention is
distinguished from the
above mentioned technologies by virtue of covalently attaching abacavir
sulfate to the N-
terminus, the C-terminus or directly to the amino acid side chain of an
oligopeptide or
polypeptide, also referred to herein as a carrier peptide: In certain
applications, the
polypeptide will stabilize the active agent, primarily in the stomach, through
conformational protection. In these applications, delivery of the active agent
is
controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon
entry into the
upper intestinal tract, indigenous enzymes release the active ingredient for
absorption by
the body by selectively hydrolyzing the peptide bonds of the carrier peptide.
This
enzymatic action introduces a second order sustained release mechanism.
Alternatively, the present invention provides a pharmaceutical composition
comprising abacavir microencapsulated by a polypeptide.
The invention provides a composition comprising a polypeptide and abacavir
sulfate covalently attached to the polypeptide. Preferably, the polypeptide is
(i) an
oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring
amino acids,
(iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a
homopolymer
of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino
acids or
(vi) a heteropolymer of one or more naturally occurring amino acids and one or
more
synthetic amino acids.
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abacavir sulfate preferably is covalently attached to a side chain, the N-
terminus
or the C-terminus of the polypeptide. In a preferred embodiment, the active
agent is a
carboxylic acid and is covalently attached to the N-terminus of the
polypeptide. In
another preferred embodiment, the active agent is an amine and is covalently
attached to
the C-ternunus of the polypeptide. In another preferred embodiment, the active
agent is
an alcohol and is covalently attached to the C-terminus of the polypeptide. In
yet another
preferred embodiment, the active agent is an alcohol and is covalently
attached to the N-
terminus of the polypeptide.
The composition of the invention can also include one or more of a
microencapsulating agent, an adjuvant and a pharmaceutically acceptable
excipient. The
xnicroencapsulating agent can be selected from polyethylene glycol {PEG), an
amino
acid, a sugar and a salt. When an adjuvant is included in the composition, the
adjuvant
preferably activates an intestinal transporter.
Preferably, the composition of the invention is in the form of an ingestable
tablet,
an intravenous preparation or an oral suspension. The active agent can be
conformationally protected by folding of the polypeptide about the active
agent. In
another embodiment, the polypeptide is capable of releasing the active agent
from the
composition in a pH-dependent manner.
The invention also provides a method for protecting abacavir sulfate from
degradation comprising covalently attaching it to a polypeptide.
The invention also provides a method for delivering abacavir sulfate to a
patient,
the patient being a human or a non-human animal, comprising administering to
the
patient a composition comprising a polypeptide and an active agent covalently
attached to
the polypeptide. In a preferred embodiment, abacavir sulfate is released from
the
composition by an enzyme-catalyzed release. In another preferred embodiment,
abacavir
sulfate is released in a time-dependent manner based on the pharmacokinetics
of the
enzyme-catalyzed release. In another preferred embodiment, the composition
further
comprises a microencapsulating agent and abacavir sulfate is released from the
composition by dissolution of the microencapsulating agent. In another
preferred
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embodiment, abacavir sulfate is released from the composition by a pH-
dependent
unfolding of the polypeptide. In another preferred embodiment, abacavir
sulfate is
released from the composition in a sustained release. In yet another preferred
embodiment, the composition further comprises an adjuvant covalently attached
to the
polypeptide and release of the adjuvant from the composition is controlled by
the
polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drug
conjugate for biphasic release of active ingredients.
The invention also provides a method for preparing a composition comprising a
polypeptide and an active agent covalently attached to the polypeptide. The
method
to comprises the steps of:
(a) attaching abacavir sulfate to a side chain of an amino acid to form an
active
agent/amino acid complex;
(b) forming an active agentlamino acid complex N-carboxyanhydride (NCA)
from the active agent/amino acid complex; and
15 (c) polymerizing the active agent/amino acid complex N-carboxyanhydride
(NCA).
In a preferred embodiment, steps (a) and (b) are repeated prior to step (c)
with a
second active agent. When steps (a) and (b) are repeated prior to step (c)
with a second
agent, abacavir sulfate and a second active agent can be copolymerized in step
(c). In
2o another preferred embodiment, the amino acid is glutamic acid and the
active agent is
released from the glutamic acid as a dimer upon a hydrolysis of the
polypeptide and
wherein the active agent is released from the glutamic acid by coincident
intramolecular
transamination. In another preferred embodiment, the glutamic acid is replaced
by an
amino acid selected from the group consisting of aspartic acid, arginine,
asparagine,
25 cysteine, lysine, threonine, and serine, and wherein the active agent is
attached to the side
chain of the amino acid to form an amide, a thioester, an ester, an ether, a
urethane, a
carbonate, an anhydride or a carbamate. In yet another preferred embodiment,
the
glutamic acid is replaced by a synthetic amino acid with a pendant group
comprising an
amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.
6
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It is to be understood that both the foregoing general description and the
following detailed description are exemplary, but are not restrictive, of the
invention.
The general applications of this invention to other active pharmaceutical
agents is
described in U.S. Patent Application Serial Number 091642,820, filed August
22, 2000,
incorporated herein by reference.
DETAILED DESCRIPTION OF INVENTION
The present invention provides several benefits for active agent delivery.
First,
the invention can stabilize abacavir sulfate and prevent its digestion in the
stomach. In
addition, the pharmacologic effect can be prolonged by delayed release of
abacavir
sulfate. Furthermore, active agents can be combined to produce synergistic
effects.
Also, absorption of the active agent in the intestinal tract can be enhanced.
The invention
also allows targeted delivery of active agents to specifics sites of action.
Abacavir sulfate is the subject of U.S. Patent Numbers 5,034,394 and
5,089,500,
herein incorporated by reference, which describes how to make that drug.
The composition of the invention comprises abacavir sulfate covalently
attached
to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a
homopolymer of
one of the twenty naturally occurring amino acids, (iii) a heteropolymer of
two or more
naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid,
(v) a
heteropolymer of two or more synthetic amuno acids~or (vi) a heteropolymer of
one or
more naturally occurring amino acids and one or more synthetic amino acids.
Proteins, oligopeptides and polypeptides are polymers of amino acids that have
primary, secondary and tertiary structures. The secondary structure of the
protein is the
local conformation of the polypeptide chain and consists of helices, pleated
sheets and
turns. 'The protein's amino acid sequence and the structural constraints on
the
. conformations of the chain determine the spatial arrangement of the
molecule. The
folding of the secondary structure and the spatial arrangement of the side
chains
constitute the tertiary structure.
7
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Proteins fold because of the dynamics associated between neighboring atoms on
the protein and solvent molecules. The thermodynamics of protein folding and
unfolding
are defined by the free energy of a particular condition of the protein that
relies on a
particular model. The process of protein folding involves, amongst other
things, amino
acid residues packing into a hydrophobic core. The amino acid side chains
inside the
protein core occupy the same volume as they do in amino acid crystals. The
folded
protein interior is therefore more like a crystalline solid than an oil drop
and so the best
model for determining forces contributing to protein stability is the solid
reference state.
The major forces contributing to the thermodynamics of protein folding are Van
to der Waals interactions, hydrogen bonds, electrostatic interactions,
configurational
entropy and the hydrophobic effect. Considering protein stability, the
hydrophobic effect
refers to the energetic consequences of removing apolar groups from the
protein interior
and exposing them to water. Comparing the energy of amino acid hydrolysis with
protein unfolding in the solid reference state, the hydrophobic effect is the
dominant
15 force. Hydrogen bonds are established during the protein fold process and
intramolecular
bonds are formed at the expense of hydrogen bonds with water. Water molecules
are
"pushed out" of the packed, hydrophobic protein core. All of these forces
combine and
contribute to the overall stability of the folded protein where the degree to
which ideal
packing occurs determines the degree of relative stability of the protein. The
result of
2o maximum packing is to produce a center of residues or hydrophobic core that
has
maximum shielding from solvent.
Since it is likely that lipophilic drugs would reside in the hydrophobic core
of a
peptide, it would require energy to unfold the peptide before the drug can be
released.
The unfolding process requires overcoming the hydrophobic effect by hydrating
the
25 amino acids or achieving the melting temperature of the protein. The heat
of hydration is
a destabilization of a protein. Typically, the folded state of a protein is
favored by only
5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at
neutral pH and
at room temperature requires chemical reagents. In fact, partial unfolding of
a protein is
often observed prior to the onset of irreversible chemical or conformation
processes.
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Moreover, protein conformation generally controls the rate and extent of
deleterious
chemical reactions.
Conformational protection of active agents by proteins depends on the
stability of
the protein's folded state and the thermodynamics associated with the agent's
decomposition. Conditions necessary for the agent's decomposition should be
different
than for protein unfolding.
Selection of the amino acids will depend on the physical properties desired.
For
instance, if increase in bulk or lipophilicity is desired, then the carrier
polypeptide will be
enriched in the amino acids in the table provided below. Polar amino acids, on
the other
to hand, can be selected to increase the hydrophilicity of the polypeptide.
Ionizing amino acids can be selected for pH controlled peptide unfolding.
Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the
stomach, but will
ionize upon entry into the intestine. Conversely, basic amino acids, such as
histidine,
lysine and arginine, ionize in the stomach and are neutral in an alkaline
environment.
15 Other factors such as ~-~ interactions between aromatic residues, kinking
of the
peptide chain by addition of proline, disulfide crosslinking and hydrogen
bonding can all
be used to select the optimum amino acid sequence for a given application.
Ordering of
the linear sequence can influence how these interactions can be maximized and
is
important in directing the secondary and tertiary structures of the
polypeptide.
20 Furthermore, amino acids with reactive side chains (e.g., glutamic acid,
lysine,
aspartic acid, serine, threonine and cysteine~ can be incorporated for
attaching multiple
active agents or adjuvants to the same carrier peptide. This is particularly
useful if a
synergistic effect between two or more active agents is desired.
As stated above, variable molecular weights of the carrier compound can have
25 profound effects on the active agent release kinetics. As a result, low
molecular weight
active agent delivery systems are preferred. An advantage of this invention is
that chain
length and molecular weight of the polypeptide can be optimized depending on
the level
of conformational protection desired. This property can be optimized in
concert with the
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kinetics of the first order release mechanism. Thus, another advantage of this
invention is
that prolonged release time can be imparted by increasing the molecular weight
of the
carrier polypeptide. Another, significant advantage of the invention is that
the kinetics of
active agent release is primarily controlled by the enzymatic hydrolysis of
the key bond
5 between the carrier peptide and the active agent.
Dextran is the only polysaccharide known that has been explored as a
macromolecular carrier for the covalent binding of drug for colon specific
drug delivery.
Generally, it was only possible to load up to 1/10 of the total drug-dextran
conjugate
weight with drug. As stated earlier, polysaccharides are digested mainly in
the colon and
to drug absorption is mainly limited to the colon. As compared to dextran,
this invention
has two major advantages. First, peptides are hydrolyzed by any one of several
aminopeptidases found in the intestinal lumen or associated with the brush-
border
membrane and so active agent release and subsequent absorption can occur in
the
jejunum or the ileum. Second, the molecular weight of the carrier molecule can
be
controlled and, thus, active agent loading can also be controlled.
As a practical example, the following table lists the molecular weights of
lipophilic amino acids (less one water molecule) and selected analgesics and
vitamins.
TABLE
Amino acid MW Active agent ~r
Glycine 57 Acetaminophen 151
Alanine 71 Vitamin B6 (Pyroxidine) 169
Valine 99 Vitamin C (Ascorbic acid) 176
Leucine 113 Aspirin 180
Isoleucine 113 Ibuprofen 206
Phenylalanine147 Retinoic acid 300
Tyrosine 163 Vitamin BZ (Riboflavin) 376
Vitamin D2 397
Vitamin E (Tocopherol) 431
Lipophilic amino acids are preferred because conformational protection through
the
2o stomach is important for the selected active agents, which were selected
based on ease of
covalent attachment to an oligopeptide. Eighteen was subtracted from the amino
acid's
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molecular weight so that their condensation into a polypeptide is considered.
For
example, a decamer of glycine (MW=588) linked to aspirin would have a total
molecular
weight of 750 and aspirin would represent 24% of the total weight of the
active agent
delivery composition or over two times the maximum drug loading for dextran.
This is
only for an N- or C- terminus application, for those active agents attached to
pendant
groups of decaglutamic acid, for instance, a drug with a molecular weight of
180 could
conceivably have a loading of 58%, although this may not be entirely
practical.
The alcohol, amine or carboxylic acid group of an active agent may be
covalently
attached to the N-terminus, the C-terminus or the side chain of the
oligopeptide or
l0 polypeptide. The location of attachment depends somewhat on the functional
group
selection. For instance, if the active drug is a carboxylic acid (e.g.,
aspirin) then the N-
terminus of the oligopeptide is the preferred point of attachment. If the
active agent is an
amine (e.g., ampicillin), then the C-terminus is the preferred point of
attachment in order
to achieve a stable peptide linked active agent. In both, the C- and N-
terminus examples,
15 the peptide is, in essence, extended by one monomeric unit forming a new
peptide bond.
If the active agent is an alcohol, then either the C-terminus or the N-
terminus is the
preferred point of attachment in order to achieve a stable composition. As in
the example
above where the alcohol, norethindrone, was covalently attached to
poly(hydroxypropylglutamine), an alcohol can be converted into an
alkylchloroformate
20 with phosgene. This invention, then, pertains to the reaction of this key
intermediate
with the N-terminus of the peptide carrier. The active ingredient can be
released from
the peptide carrier by intestinal peptidases.
The alcohol can be selectively bound to the gamma carboxylate of glutamic acid
and then this conjugate covalently attached to the C-terminus of the peptide
carrier.
25 Because the glutamic acid-drug conjugate can be considered a dimer, this
product adds
two monomeric units to the C-terminus of the peptide carrier where the
glutamic acid
moiety serves as a spacer between the peptide and the drug as shown in Fig. 4.
Intestinal
enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drug
moiety
from the peptide carrier. The newly formed free amine of the glutamic acid
residue will
30 then undergo an intramolecular transamination reaction, thereby, releasing
the active
11
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agent with coincident formation of pyroglutamic acid as shown in Fig. 5.
Alternatively,
the glutamic acid-drug dimer can be converted into the gamma ester of glutamic
acid N-
carboxyanhydride. This intermediate can then be polymerized, as described
above, using
any suitable initiator as shown in Fig. 4. The product of this polymerization
is
polyglutamic acid with active ingredients attached to multiple pendant groups.
Hence,
maximum drug loading of the carrier peptide can be achieved. In addition,
other amino
acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to
impart
specific properties to the drug delivery system.
The invention also provides a method of imparting the same mechanism of action
l0 for other polypeptides containing functional side chains. Examples include,
but are not
limited to, polylysine, polyasparagine, polyarginine, polyserine,
polycysteine,
polytyrosine, polythreonine and polyglutamine. The mechanism can translate to
these
polypeptides through a spacer or linker on the pendant group, which is
terminated,
preferably, by the glutamic acid-drug dimer. This carrier peptide-drug
conjugate is
15 distinguished from the prior art by virtue of the fact that the primary
release of the drug
moiety relies on peptidases and not on esterases. Alternatively, the active
agent can be
attached directly to the pendant group where some other indigenous enzymes in
the
alimentary tract can affect release.
The active agent can be covalently attached to the N-terminus, the C-terminus
or
20 the side chain of the polypeptide using known techniques. Examples of
linking organic
compounds to the N-terminus type of a peptide include, but are not limited to,
the
attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides
and 1,3-
dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example,
there are
known techniques for forming peptide linked biotin and peptide linked
acridine.
25 In the present invention, abacavir sulfate is covalently attached to the
polypeptide
via its alcohol group or, alternatively, its amino group.
The polypeptide carrier can be prepared using conventional techniques. A
preferred technique is copolymerization of mixtures of amino acid N-
carboxyanhydrides.
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Alternatively, if a specific sequence is desired, a solid state automated
peptide synthesizer
can be used.
The addition of stabilizers to the composition has the potential of
stabilizing the
polypeptide further. Stabilizers such as sugar, amino acids, polyethylene
glycol (PEG)
and salts have been shown to prevent protein unfolding. In another embodiment
of the
invention, a pre-first order release of the active agent is imparted by
microencapsulating
the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid
complex,
PEG or salts.
There is evidence that hydrophilic compounds are absorbed through the
intestinal
epithelia efficiently via specialized transporters. The entire membrane
transport system is
intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one
can expect
that excitation of the membrane transport system will involve some sort of
specialized
adjuvant resulting in localized delivery of active agents. There are seven
known
intestinal transport systems classified according to the physical properties
of the
transported substrate. They include the amino acid, oligopeptide, glucose,
monocarboxic
acid, phosphate, bile acid and the P-glycoprotein transport systems and each
has its own
associated mechanism of transport. The mechanisms can depend on hydrogen ions,
sodium ions, binding sites or other cofactors. The invention also allows
targeting the
mechanisms for intestinal epithelial transport systems to facilitate
absorption of active
2o agents.
In another embodiment of the invention, the composition includes one or more
adjuvants to enhance the bioavailability of the active agent. Addition of an
adjuvant is
particularly preferred when using an otherwise poorly absorbed active agent.
Suitable
adjuvants, for example, include: papain, which is a potent enzyme for
releasing the
catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which
activate
enzymes in the BBM; and bile acids, which have been attached to peptides to
enhance
absorption of the peptides.
Preferably, the resultant peptide-abacavir sulfate conjugate is formulated
into a
tablet using suitable excipients and can either be wet granulated or dry
compressed.
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Compositions of the invention are, in essence, the formation of amides from
acids
and amines and can be prepared by the following examples.
AcidlN-terminus conjugation
An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to
0°C. The solution can then be treated with diisopropylcarbodiimide and
hydroxybenzotriazole followed by the amine peptide carrier. The reaction can
then be
stirred for several hours at room temperature, the urea by-product filtered
off, the product
precipitated out in ether and purified using gel permeation chromatography
(GPC) or
dialysis.
l0 AminelC-terminus conjugation
The peptide carrier can be dissolved in D1VIF under nitrogen and cooled to
0°C.
The solution can then be treated with diisopropylcarbodiimide and
hydroxybenzotriazole
followed by the amine bioactive agent. The reaction can then be stirred for
several hours
at room temperature, the urea by-product filtered off, the product
precipitated out in ether
and purified using GPC or dialysis.
Alcohol/N-Terminus Conjugation
In the following example the combination of the alcohol with triphosgene
produces a chloroformate, which when reacted with the N-terminus of the
peptide
produces a carbamate. Pursuant to this, an alcohol bioactive agent can be
treated with
triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier
is then
added slowly and the solution stirred at room temperature for several hours.
The product
is then precipitated out in ether. The crude product is suitably deprotected
and purified
using GPC.
Other solvents, activating agents, cocatalysts and bases can be used. Examples
of
other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or
chlorinated
solvents such as chloroform. Examples of other activating agents include
dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst
is N-
14
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hydroxysuccinimide. Examples of bases include pyrrolidinopyridine,
dimethylaminopyridine, triethylamine or tributylamine.
Preparation of Y Alkyl Glutamate
There have been over 30 different y alkyl glutamates prepared any one of which
may be suitable for the drug alcohol of choice. For example, a suspension of
glutamic
acid, the alcohol and concentrated hydrochloric acid can be prepared and
heated for
several hours. The y alkyl glutamate product can be precipitated out in
acetone, filtered,
dried and recrystallized from hot water.
y Alkyl Glutamate/C-Terminus Conjugation
10 The peptide Garner can be dissolved in DMF under nitrogen and cooled to
0°C.
The solution can then be treated with diisopropylcarbodiimide and
hydroxybenzotriazole
followed by the y alkyl glutamate bioactive agent. The reaction can then be
stirred for
several hours at room temperature, the urea by-product filtered off, the
product
precipitated out in ether and purified using GPC or dialysis.
15 Preparation of y Alkyl Glutamate-NCA
'y Alkyl glutamate can be suspended in dry THF where triphosgene is added and
the mixture refluxed under a nitrogen atmosphere until the mixture becomes
homogenous. The solution can be poured into heptane to precipitate the NCA
product,
which is filtered, dried and recrystallized from a suitable solvent.
2o Preparation of Poly['y Alkyl Glutamate]
y Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of
a primary amine can be added to the solution until it becomes viscous
(typically
overnight). The product can be isolated from the solution by pouring it into
water and
filtering. The product can be purified using GPC or dialysis.
Although illustrated and described above with reference to specific
embodiments,
the invention is nevertheless not intended to be limited to the details shown.
Rather,
is
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various modifications may be made in the details within the scope and range of
equivalents of the claims and without departing from the spirit of the
invention.
16
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CLAIMS
What is claimed is:
1. A pharmaceutical composition comprising:
a polypeptide; and
abacavir sulfate covalently attached to said polypeptide.
2. The composition of claim 1 wherein said polypeptide is an oligopeptide.
3. The composition of claim 1 wherein said polypeptide is a homopolymer of a
naturally occurring amino acid.
4. The composition of claim 1 wherein said polypeptide is a heteropolymer of
two or more naturally occurring amino acids.
5. The composition of claim 1 wherein said polypeptide is a homopolymer of a
synthetic amino acid.
6. The composition of claim 1 wherein said polypeptide is a heteropolymer of
two or more synthetic amino acids.
7. The composition of claim 1 wherein said polypeptide is a heteropolymer of
one or more naturally occurring amino acids and one or more synthetic amino
acids.
8. The composition of claim 1 wherein abacavir sulfate is covalently attached
to a
side chain, the N-terminus or the C-terminus of said polypeptide.
9. The composition of claim 1 further comprising a microencapsulating agent.
10. The composition of claim 9 wherein said microencapsulating agent is
selected from the group consisting of polyethylene glycol (PEG), an amino
acid, a sugar
and a salt.
11. The composition of claim 1 further comprising an adjuvant.
17
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12. The composition of claim 11 wherein said adjuvant activates an intestinal
transporter.
13. The composition of claim 1 further comprising a pharmaceutically
acceptable
excipient.
14. The composition of claim 1 wherein said composition is in the form of an
ingestable tablet.
15. The composition of claim 1 wherein said composition is in the form of an
intravenous preparation.
16. The composition of claim 1 wherein said composition is in the form of an
l0 oral suspension.
17. The composition of claim 1 wherein abacavir sulfate is conformationally
protected by folding of said polypeptide about said active agent.
18. The composition of claim 1 wherein said polypeptide is capable of
releasing
abacavir sulfate from said composition in a pH-dependent manner.
19. A method for protecting abacavir sulfate from degradation comprising
covalently attaching said active agent to a polypeptide.
20. A method for controlling release of abacavir sulfate from a composition
wherein said composition comprises a polypeptide, said method comprising
covalently
attaching abacavir sulfate to said polypeptide.
21. A method for delivering abacavir sulfate to a patient comprising
administering to said patient a composition comprising:
a polypeptide; and
abacavir sulfate covalently attached to said polypeptide.
22. The method of claim 21 wherein abacavir sulfate is released from said
composition by an enzyme-catalyzed release.
18
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23. The method of claim 21 wherein abacavir sulfate is released from said
composition by a pH-dependent unfolding of said polypeptide.
24. The method of claim 21 wherein said active agent is released from said
composition in a sustained release.
25. The method of claim 21 wherein said composition further comprises an
adjuvant covalently attached to said polypeptide and wherein release of said
adjuvant
from said composition is controlled by said polypeptide.
Abstract
A composition comprising a polypeptide and abacavir sulfate covalently
attached
to the polypeptide. Also provided is a method for delivery of abacavir sulfate
to a patient
comprising administering to the patient a composition comprising a polypeptide
and
abacavir sulfate covalently attached to the polypeptide. Also provided is a
method for
protecting abacavir sulfate from degradation comprising covalently attaching
it to a
polypeptide. Also provided is a method for controlling release of abacavir
sulfate from a
composition comprising covalently attaching it to a polypeptide.
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A NOVTEL PHARMACEUTICAL COMPOUND CONTAINING ABARELIX AND
METHODS OF MAKING AND USING SAME
FP'F,LD OF THE INVENTION
5 The present invention relates to a novel pharmaceutical compound that
comprises
a polypeptide that is preferably covalently attached to abarelix, as well as
methods for
protecting and administering abarelix. This novel compound, referred to as a
CARRIERWAVETM Molecular Analogue (CMA), has the benefit of taking a known
effective pharmaceutical agent that is both well studied and occupies a known
segment of
to the pharmaceutical market, and combining it with a carrier compound that
enhances the
usefulness of the pharmaceutical agent without compromising its pharmaceutical
effectiveness.
BACKGROUND OF THE INVENTION
Abarelix is a known pharmaceutical agent that is used in the treatment of
prostate
15 cancer, acting as a gonadotropin-releasing hormone antagonist. Its chemical
name is N-
acetyl-3-(2-naphthalenyl)-D-alanyl-4-chloro-D-phenylalanyl-3-(3-pyridinyl)-D-
alanyl-L-
seryl-N-methyl-L-tyrosyl-D-asparagynyl-L-N6-( 1-methylethyl)-L-lysyl-L-prolyl-
D-
alaninamide. Abarelix is both commercially available and readily manufactured
using
published synthetic schemes by those of ordinary skill in the art. Its
structure is as
2o follows:
C1
0
0
0 \ 0 I O N O
N Id N N
~N ~N ~N N
N / 0 0 0 0 0 N II
/ I ~N
O
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The novel pharmaceutical compound of the present invention is useful in
accomplishing one or more of the following goals: enhancement of the chemical
stability
of the original compound; alteration of the release profile of an orally
administered
product; enhanced digestion or absorbtion; targeted delivery to particular
tissue/cell type;
and provision for an oral dosage form when none exists. The novel
pharmaceutical
compound may contain one or more of the following: another active
pharmaceutical
agent, an adjuvant, or an inhibitor.
Active agent delivery systems are often critical for the effective delivery of
a
biologically active agent (active agent) to the appropriate target. The
importance of these
to systems becomes magnified when patient compliance and active agent
stability are taken
under consideration. For instance, one would expect patient compliance to
increase
markedly if an active agent is administered orally in lieu of an injection or
another
invasive technique. Increasing the stability of the active agent, such as
prolonging shelf
life or survival in the stomach, will assure dosage reproducibility and
perhaps even
15 reduce the number of dosages required which could improve patient
compliance.
Absorption of an orally administered active agent is often blocked by the
harshly
acidic stomach milieu, powerful digestive enzymes in the GI tract,
permeability of
cellular membranes and transport across lipid bilayers. Incorporating
adjuvants such as
resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance
permeability of
20 cellular membranes. Microencapsulating active agents using protenoid
microspheres,
liposomes or polysaccharides have been effective in abating enzyme degradation
of the
active agent. Enzyme inhibiting adjuvants have also been used to prevent
enzyme
degradation. Enteric coatings have been used as a protector of pharmaceuticals
in the
stomach.
25 Active agent delivery systems also provide the ability to control the
release of the
active agent. For example, formulating diazepam with a copolymer of glutamic
acid and
aspartic acid enables a sustained release of the active agent. As another
example,
copolymers of lactic acid and glutaric acid are used to provide timed release
of human
growth hormone. A wide range of pharmaceuticals purportedly provide sustained
release
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through microencapsulation of the active agent in amides of dicarboxylic
acids, modified
amino acids or thermally condensed amino acids. Slow release rendering
additives can
also be intermixed with a large array of active agents in tablet formulations.
Each of these technologies imparts enhanced stability and time-release
properties
to active agent substances. Unfortunately, these technologies suffer from
several
shortcomings. Incorporation of the active agent is often dependent on
diffusion into the
microencapsulating matrix, which may not be quantitative and may complicate
dosage
reproducibility. In addition, encapsulated drugs rely on diffusion out of the
matrix, which
is highly dependant on the water solubility of the active agent. Conversely,
water-soluble
microspheres swell by an infinite degree and, unfortunately, may release the
active agent
in bursts with little active agent available for sustained release.
Furthermore, in some
technologies, control of the degradation process required for active agent
release is
unreliable. For example, an enterically coated active agent depends on pH to
release the
active agent and, as such, is difficult to control the rate of release.
In the past, use has been made of amino acid side chains of polypeptides as
pendant groups to which active agents can be attached. These technologies
typically
require the use of spacer groups between the amino acid pendant group and the
active
agent. The peptide-drug conjugates of this class of drug delivery system rely
on enzymes
in the bloodstream for the release of the drug and, as such, are not used for
oral
administration. Examples of timed and targeted release of injectable or
subcutaneous
pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer,
to the
gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a
peptide
spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been
covalently
attached directly to the beta carboxylate of polyaspartic acid without a
spacer group.
This prodrug formulation was designed as a colon-specific drug delivery system
where
the drug is released by bacterial hydrolytic enzymes residing in the large
intestines. The
released dexamethasone active agent, in turn, was targeted to treat large
bowel disorders
and was not intended to be absorbed into the bloodstream. Yet another
technology
combines the advantages of covalent drug attachment with liposome formation
where the
active ingredient is attached to highly ordered lipid films (known as HARs)
via a peptide
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linker. Thus, there has been no drug delivery system, heretofore reported,
that
incorporates the concept of attaching an active ingredient to a polypeptide
pendant group
with its targeted delivery into the bloodstream via oral administration.
It is also important to control the molecular weight, molecular size and
particle
size of the active agent delivery system. Variable molecular weights have
unpredictable
diffusion rates and pharmacokinetics. High molecular weight carriers are
digested slowly
or late, as in the case of naproxen-linked dextran, which is digested almost
exclusively in
the colon by bacterial enzymes. High molecular weight microspheres usually
have high
moisture content which may present a problem with water labile active
ingredients.
to Particle size not only becomes a problem with injectable drugs, as in the
HAR
application, but absorption through the brush-border membrane of the
intestines is limited
to less than 5 microns.
SUMMARY OF THE INVENTION
The present invention provides covalent attachment of the active agent
(abarelix)
' to a polymer of peptides or amino acids. The invention is distinguished from
the above
mentioned technologies by virtue of covalently attaching abarelix to the N-
terminus, the
C-terminus or directly to the amino acid side chain of an oligopeptide or
polypeptide,
also referred to herein as a carrier peptide. In certain applications, the
polypeptide will
stabilize the active agent, primarily in the stomach, through conformational
protection.
In these applications, delivery of the active agent is controlled, in part, by
the kinetics of
unfolding of the carrier peptide. Upon entry into the upper intestinal tract,
indigenous
enzymes release the active ingredient for absorption by the body by
selectively
hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action
introduces a
second order sustained release mechanism.
Alternatively, the present invention provides a pharmaceutical composition
comprising abarelix microencapsulated by a polypeptide.
The invention provides a composition comprising a polypeptide and abarelix
covalently attached to the polypeptide. Preferably, the polypeptide is (i) an
oligopeptide,
4
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(ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii)
a
heteropolymer of two or more naturally occurring amino acids, (iv) a
homopolymer of a
synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids
or (vi) a
heteropolymer of one or more naturally occurring amino acids and one or more
synthetic
amino acids.
Abarelix preferably is covalently attached to a side chain, the N-terminus or
the
C-terminus of the polypeptide. In a preferred embodiment, the active agent is
a
carboxylic acid and is covalently attached to the N-terminus of the
polypeptide. In
another preferred embodiment, the active agent is an amine and is covalently
attached to
to the C-terminus of the polypeptide. In another preferred embodiment, the
active agent is
an alcohol and is covalently attached to the C-terminus of the polypeptide. In
yet another
preferred embodiment, the active agent is an alcohol and is covalently
attached to the N-
terminus of the polypeptide.
The composition of the invention can also include one or more of a
15 microencapsulating agent, an adjuvant and a pharmaceutically acceptable
excipient. The
microencapsulating agent can be selected from polyethylene glycol (PEG), an
amino
acid, a sugar and a salt. When an adjuvant is included in the composition, the
adjuvant
preferably activates an intestinal transporter.
Preferably, the composition of the invention is in the form of an ingestable
tablet,
20 an intravenous preparation or an oral suspension. The active agent can be
conformationally protected by folding of the polypeptide about the active
agent. In
another embodiment, the polypeptide is capable of releasing the active agent
from the
composition in a pH-dependent manner.
The invention also provides a method for protecting abarelix from degradation
25 comprising covalently attaching it to a polypeptide.
The invention also provides a method for delivering abarelix to a patient, the
patient being a human or a non-human animal, comprising administering to the
patient a
composition comprising a polypeptide and an active agent covalently attached
to the
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polypeptide. In a preferred embodiment, abarelix is released from the
composition by an
enzyme-catalyzed release. In another preferred embodiment, abarelix is
released in a
time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed
release.
In another preferred embodiment, the composition further comprises a
5 microencapsulating agent and abarelix is released from the composition by
dissolution of
the microencapsulating agent. In another preferred embodiment, abarelix is
released
from the composition by a pH-dependent unfolding of the polypeptide. In
another
preferred embodiment, abarelix is released from the composition in a sustained
release.
In yet another preferred embodiment, the composition further comprises an
adjuvant
10 covalently attached to the polypeptide and release of the adjuvant from the
composition is
controlled by the polypeptide. The adjuvant can be microencapsulated into a
carrier
peptide-drug conjugate for biphasic release of active ingredients.
The invention also provides a method for preparing a composition comprising a
polypeptide and an active agent covalently attached to the polypeptide. The
method
15 comprises the steps of:
(a) attaching abarelix to a side chain of an amino acid to form an active
agent/amino acid complex;
(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA)
from the active agent/amino acid complex; and
20 (c) polymerizing the active agentlamino acid complex N-carboxyanhydride
(NCA).
In a preferred embodiment, steps (a) and (b) are repeated prior to step (c)
with a
second active agent. When steps (a) and (b) are repeated prior to step (c)
with a second
agent, abarelix and a second active agent can be copolymerized in step (c). In
another
25 preferred embodiment, the amino acid is glutamic acid and the active agent
is released
from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and
wherein the
active agent is released from the glutamic acid by coincident intramolecular
transamination. In another preferred embodiment, the glutamic acid is replaced
by an
amino acid selected from the group consisting of aspartic acid, arginine,
asparagine,
cysteine, lysine, threonine, and serine, and wherein the active agent is
attached to the side
chain of the amino acid to form an amide, a thioester, an ester, an ether, a
urethane, a
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carbonate, an anhydride or a carbamate. In yet another preferred embodiment,
the
glutamic acid is replaced by a synthetic amino acid with a pendant group
comprising an
amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.
It is to be understood that both the foregoing general description and the
following detailed description are exemplary, but are not restrictive, of the
invention.
The general applications of this invention to other active pharmaceutical
agents is
described in U.S. Patent Application Serial Number 09/642,820, filed August
22, 2000,
incorporated herein by reference.
DETAILED DESCRIPTION OF INVENTION
The present invention provides several benefits for active agent delivery.
First,
the invention can stabilize abarelix and prevent its digestion in the stomach.
In addition,
the pharmacologic effect can be prolonged by delayed release of abarelix.
Furthermore,
active agents can be combined to produce synergistic effects. Also, absorption
of the
active agent in the intestinal tract can be enhanced. The invention also
allows targeted
delivery of active agents to specifics sites of action.
The composition of the invention comprises abarelix covalently attached to a
polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a
homopolymer of one
of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or
more
naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid,
(v) a
heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of
one or
more naturally occurring amino acids and one or more synthetic amino acids.
Proteins, oligopeptides and polypeptides are polymers of amino acids that have
primary, secondary and tertiary structures. The secondary structure of the
protein is the
local conformation of the polypeptide chain and consists of helices, pleated
sheets and
turns. The protein's amino acid sequence and the structural constraints on the
conformations of the chain determine the spatial arrangement of the molecule.
The
folding of the secondary structure and the spatial arrangement of the side
chains
constitute the tertiary structure.
7
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Proteins fold because of the dynamics associated between neighboring atoms on
the protein and solvent molecules. The thermodynamics of protein folding and
unfolding
are defined by the free energy of a particular condition of the protein that
relies on a
particular model. The process of protein folding involves, amongst other
things, amino
acid residues packing into a hydrophobic core. The amino acid side chains
inside the
protein core occupy the same volume as they do in amino acid crystals. The
folded
protein interior is therefore more like a crystalline solid than an oil drop
and so the best
model for determining forces contributing to protein stability is the solid
reference state.
The major forces contributing to the thermodynamics of protein folding are Van
der Waals interactions, hydrogen bonds, electrostatic interactions,
configurational
entropy and the hydrophobic effect. Considering protein stability, the
hydrophobic effect
refers to the energetic consequences of removing apolar groups from the
protein interior
and exposing them to water. Comparing the energy of amino acid hydrolysis with
protein unfolding in the solid reference state, the hydrophobic effect is the
dominant
force. Hydrogen bonds are established during the protein fold process and
intramolecular
bonds are formed at the expense of hydrogen bonds with water. Water molecules
are
"pushed out" of the packed, hydrophobic protein core. All of these forces
combine and
contribute to the overall stability of the folded protein where the degree to
which. ideal
packing occurs determines the degree of relative stability of the protein. The
result of
maximum packing is to produce a center of residues or hydrophobic core that
has
maximum shielding from solvent.
Since it is likely that lipophilic drugs would reside in the hydrophobic core
of a
peptide, it would require energy to unfold the peptide before the drug can be
released.
The unfolding process requires overcoming the hydrophobic effect by hydrating
the
amino acids or achieving the melting temperature of the protein. The heat of
hydration is
a destabilization of a protein. Typically, the folded state of a protein is
favored by only
5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at
neutral pH and
at room temperature requires chemical reagents. In fact, partial unfolding of
a protein is
often observed prior to the onset of irreversible chemical or conformation
processes.
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Moreover, protein conformation generally controls the rate and extent of
deleterious
chemical reactions.
Conformational protection of active agents by proteins depends on the
stability of
the protein's folded state and the thermodynamics associated with the agent's
decomposition. Conditions necessary for the agent's decomposition should be
different
than for protein unfolding.
Selection of the amino acids will depend on the physical properties desired.
For
instance, if increase in bulk or lipophilicity is desired, then the carrier
polypeptide will be
enriched in the amino acids in the table provided below. Polar amino acids, on
the other
hand, can be selected to increase the hydrophilicity of the polypeptide.
Ionizing amino acids can be selected for pH controlled peptide unfolding.
Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the
stomach, but will
ionize upon entry into the intestine. Conversely, basic amino acids, such as
histidine,
lysine and arginine, ionize in the stomach and are neutral in an alkaline
environment.
Other factors such as ~-rt interactions between aromatic residues, kinking of
the
peptide chain by addition of proline, disulfide crosslinking and hydrogen
bonding can all
be used to select the optimum amino acid sequence for a given application.
Ordering of
the linear sequence can influence how these interactions can be maximized and
is
important in directing the secondary and tertiary structures of the
polypeptide.
Furthermore, amino acids with reactive side chains (e.g., glutamic acid,
lysine,
aspartic acid, serine, threonine and cysteine) can be incorporated for
attaching multiple
active agents or adjuvants to the same carrier peptide. This is particularly
useful if a
synergistic effect between two or more active agents is desired.
As stated above, variable molecular weights of the carrier compound can have
profound effects on the active agent release kinetics. As a result, low
molecular weight
active agent delivery systems are preferred. An advantage of this invention is
that chain
length and molecular weight of the polypeptide can be optimized depending on
the level
of conformational protection desired. This property can be optimized in
concert with the
9
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kinetics of the first order release mechanism. Thus, another advantage of this
invention is
that prolonged release time can be imparted by increasing the molecular weight
of the
carrier polypeptide. Another, significant advantage of the invention is that
the kinetics of
active agent release is primarily controlled by the enzymatic hydrolysis of
the key bond
between the carrier peptide and the active agent.
Dextran is the only polysaccharide known that has been explored as a
macromolecular carrier for the covalent binding of drug for colon specific
drug delivery.
Generally, it was only possible to load up to 1/10 of the total drug-dextran
conjugate
weight with drug. As stated earlier, polysaccharides are digested mainly in
the colon and
l0 drug absorption is mainly limited to the colon. As compared to dextran,
this invention
has two major advantages. First, peptides are hydrolyzed by any one of several
aminopeptidases found in the intestinal lumen or associated with the brush-
border
membrane and so active agent release and subsequent absorption can occur in
the
jejunum or the ileum. Second, the molecular weight of the carrier molecule can
be
controlled and, thus, active agent loading can also be controlled.
As a practical example, the following table lists the molecular weights of
lipophilic amino acids (less one water molecule) and selected analgesics and
vitamins.
TABLE
Amino acid MW Active agent MW
Glycine 57 Acetaminophen 151
Alanine 71 Vitamin B6 (Pyroxidine) 169
Valine 99 Vitamin C (Ascorbic acid) 176
Leucine 113 Aspirin 180
Isoleucine 113 Ibuprofen 206
Phenylalanine147 Retinoic acid 300
Tyrosine 163 Vitamin BZ (Riboflavin) 376
Vitamin D2 397
Vitamin E (Tocopherol) 431
Lipophilic amino acids are preferred because conformational protection through
the
stomach is important for the selected active agents, which were selected based
on ease of
covalent attachment to an oligopeptide. Eighteen was subtracted from the amino
acid's
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molecular weight so that their condensation into a polypeptide is considered.
For
example, a decamer of glycine (MW=588) linked to aspirin would have a total
molecular
weight of 750 and aspirin would represent 24% of the total weight of the
active agent
delivery composition or over two times the maximum drug loading for dextran.
This is
only for an N- or C- terminus application, for those active agents attached to
pendant
groups of decaglutamic acid, for instance, a drug with a molecular weight of
180 could
conceivably have a loading of 58%, although this may not be entirely
practical.
The alcohol, amine or carboxylic acid group of an active agent may be
covalently
attached to the N-ternunus, the C-terminus or the side chain of the
oligopeptide or
10 polypeptide. The location of attachment depends somewhat on the functional
group
selection. For instance, if the active drug is a carboxylic acid (e.g.,
aspirin) then the N-
terminus of the oligopeptide is the preferred point of attachment. If the
active agent is an
amine (e.g., ampicillin), then the C-terminus is the preferred point of
attachment in order
to achieve a stable peptide linked active agent. In both, the C- and N-
terminus examples,
15 the peptide is, in essence, extended by one monomeric unit forming a new
peptide bond.
If the active agent is an alcohol, then either the C-terminus or the N-
terminus is the
preferred point of attachment in order to achieve a stable composition. As in
the example
above where the alcohol, norethindrone, was covalently attached to
poly(hydroxypropylglutamine), an alcohol can be converted into an
alkylchloroformate
20 with phosgene. This invention, then, pertains to the reaction of this key
intermediate
with the N-terminus of the peptide carrier. The active ingredient can be
released from
the peptide carrier by intestinal peptidases.
The alcohol can be selectively bound to the gamma carboxylate of glutamic acid
and then this conjugate covalently attached to the C-terminus of the peptide
carrier.
25 Because the glutamic acid-drug conjugate can be considered a dimer, this
product adds
two monomeric units to the C-terminus of the peptide carrier where the
glutamic acid
moiety serves as a spacer between the peptide and the drug as shown in Fig. 4.
Intestinal
enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drug
moiety
from the peptide carrier. The newly formed free amine of the glutamic acid
residue will
30 then undergo an intramolecular transamination reaction, thereby, releasing
the active
11
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agent with coincident formation of pyroglutamic acid as shown in Fig. 5.
Alternatively,
the glutamic acid-drug dimer can be converted into the gamma ester of glutamic
acid N-
carboxyanhydride. This intermediate can then be polymerized, as described
above, using
any suitable initiator as shown in Fig. 4. The product of this polymerization
is
polyglutamic acid with active ingredients attached to multiple pendant groups.
Hence,
maximum drug loading of the carrier peptide can be achieved. In addition,
other amino
acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to
impart
specific properties to the drug delivery system.
The invention also provides a method of imparting the same mechanism of action
for other polypeptides containing functional side chains. Examples include,
but are not
limited to, polylysine, polyasparagine, polyarginine, polyserine,
polycysteine,
polytyrosine, polythreonine and polyglutamine. The mechanism can translate to
these
polypeptides through a spacer or linker on the pendant group, which is
terminated,
preferably, by the glutamic acid-drug dimer. This carrier peptide-drug
conjugate is
distinguished from the prior art by virtue of the fact that the primary
release of the drug
moiety relies on peptidases and not on esterases. Alternatively, the active
agent can be
attached directly to the pendant group where some other indigenous enzymes in
the
alimentary tract can affect release.
The active agent can be covalently attached to the N-terminus, the C-terminus
or
the side chain of the polypeptide using known techniques. Examples of linking
organic
compounds to the N-terminus type of a peptide include, but are not limited to,
the
attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides
and 1,3-
dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example,
there are
known techniques for forming peptide linked biotin and peptide linked
acridine.
In the present invention, abarelix is covalently attached to the polypeptide
via the
free alcohol group or, alternatively, through one of its amino groups.
The polypeptide carrier can be prepared using conventional techniques. A
preferred technique is copolymerization of mixtures of amino acid N-
carboxyanhydrides.
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Alternatively, if a specific sequence is desired, a solid state automated
peptide synthesizer
can be used.
The addition of stabilizers to the composition has the potential of
stabilizing the
polypeptide further. Stabilizers such as sugar, amino acids, polyethylene
glycol (PEG)
and salts have been shown to prevent protein unfolding. In another embodiment
of the
invention, a pre-first order release of the active agent is imparted by
microencapsulating
the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid
complex,
PEG or salts.
There is evidence that hydrophilic compounds are absorbed through the
intestinal
epithelia efficiently via specialized transporters. The entire membrane
transport system is
intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one
can expect
that excitation of the membrane transport system will involve some sort of
specialized
adjuvant resulting in localized delivery of active agents. There are seven
known
intestinal transport systems classified according to the physical properties
of the
' transported substrate. They include the amino acid, oligopeptide, glucose,
monocarboxic
acid, phosphate, bile acid and the P-glycoprotein transport systems and each
has its own
associated mechanism of transport. The mechanisms can depend on hydrogen ions,
sodium ions, binding sites or other cofactors. The invention also allows
targeting the
mechanisms for intestinal epithelial transport systems to facilitate
absorption of active
2o agents.
In another embodiment of the invention, the composition includes one or more
adjuvants to enhance the bioavailability of the active agent. Addition of an
adjuvant is
particularly preferred when using an otherwise poorly absorbed active agent.
Suitable
adjuvants, for example, include: papain, which is a potent enzyme for
releasing the
catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which
activate
enzymes in the BBM; and bile acids, which have been attached to peptides to
enhance
absorption of the peptides.
Preferably, the resultant peptide-abarelix conjugate is formulated into a
tablet
using suitable excipients and can either be wet granulated or dry compressed.
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Compositions of the invention are, in essence, the formation of amides from
acids
and amines and can be prepared by the following examples.
Acid/N-terminus conjugation
An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to
0°C. The solution can then be treated with diisopropylcarbodiimide and
hydroxybenzotriazole followed by the amine peptide carrier. The reaction can
then be
stirred for several hours at room temperature, the urea by-product filtered
off, the product
precipitated out in ether and purified using gel permeation chromatography
(GPC) or
dialysis.
to Amine/C-terminus conjugation
The peptide carrier can be dissolved in DMF under nitrogen and cooled to
0°C.
The solution can then be treated with diisopropylcarbodiimide and
hydroxybenzotriazole
followed by the amine bioactive agent. The reaction can then be stirred for
several hours
at room temperature, the urea by-product filtered off, the product
precipitated out in ether
15 and purified using GPC or dialysis.
Alcohol/N-Terminus Conjugation
In the following example the combination of the alcohol with triphosgene
produces a chloroformate, which when reacted with the N-terminus of the
peptide
produces a carbamate. Pursuant to this, an alcohol bioactive agent can be
treated with
20 triphosgene in dry DMF under nitrogen. The suitably protected peptide
carrier is then
added slowly and the solution stirred at room temperature for several hours.
The product
is then precipitated out in ether. The crude product is suitably deprotected
and purified
using GPC.
Other solvents, activating agents, cocatalysts and bases can be used. Examples
of
25 other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or
chlorinated
solvents such as chloroform. Examples of other activating agents include
dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst
is N-
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hydroxysuccinimide. Examples of bases include pyrrolidinopyridine,
dimethylaminopyridine, triethylamine or tributylamine.
Preparation of ~y Alkyl Glutamate
There have been over 30 different y alkyl glutamates prepared any one of which
may be suitable for the drug alcohol of choice. For example, a suspension of
glutamic
acid, the alcohol and concentrated hydrochloric acid can be prepared and
heated for
several hours. The Y alkyl glutamate product can be precipitated out in
acetone, filtered,
dried and recrystallized from hot water.
y Alkyl Glutamate/C-Terminus Conjugation
The peptide carrier can be dissolved in DMF under nitrogen and cooled to
0°C.
The solution can then be treated with diisopropylcarbodiimide and
hydroxybenzotriazole
followed by the y alkyl glutamate bioactive agent. The reaction can then be
stirred for
several hours at room temperature, the urea by-product filtered off, the
product
precipitated out in ether and purified using GPC or dialysis.
Preparation of y Alkyl Glutamate-NCA
y Alkyl glutamate can be suspended in dry THF where triphosgene is added and
the mixture refluxed under a nitrogen atmosphere until the mixture becomes
homogenous. The solution can be poured into heptane to precipitate the NCA
product,
which is filtered, dried and recrystallized from a suitable solvent.
2o Preparation of Poly['y Alkyl Glutamate]
y Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of
a primary amine can be added to the solution until it becomes viscous
(typically
overnight). The product can be isolated from the solution by pouring it into
water and
filtering. The product can be purified using GPC or dialysis.
Although illustrated and described above with reference to specific
embodiments,
the invention is nevertheless not intended to be limited to the details shown.
Rather,
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various modifications may be made in the details within the scope and range of
equivalents of the claims and without departing from the spirit of the
invention.
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CLAIMS
What is claimed is:
1. A pharmaceutical composition comprising:
a polypeptide; and
abarelix covalently attached to said polypeptide.
2. The composition of claim 1 wherein said polypeptide is an oligopeptide.
3. The composition of claim 1 wherein said polypeptide is a homopolymer of a
naturally occurring amino acid.
4. The composition of claim 1 wherein said polypeptide is a heteropolymer of
l0 two or more naturally occurring amino acids.
5. The composition of claim 1 wherein said polypeptide is a homopolymer of a
synthetic amino acid.
6. The composition of claim 1 wherein said polypeptide is a heteropolymer of
two or more synthetic amino acids.
7. The composition of claim 1 wherein said polypeptide is a heteropolymer of
one or more naturally occurring amino acids and one or more synthetic amino
acids.
8. The composition of claim 1 wherein abarelix is covalently attached to a
side
chain, the N-terminus or the C-terminus of said polypeptide.
9. The composition of claim 1 further comprising a microencapsulating agent.
10. The composition of claim 9 wherein said microencapsulating agent is
selected from the group consisting of polyethylene glycol (PEG), an amino
acid, a sugar
and a salt.
11. The composition of claim 1 further comprising an adjuvant.
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12. The composition of claim 11 wherein said adjuvant activates an intestinal
transporter.
13. The composition of claim 1 further comprising a pharmaceutically
acceptable
excipient.
14. The composition of claim 1 wherein said composition is in the form of an
ingestable tablet.
15. The composition of claim 1 wherein said composition is in the form of an
intravenous preparation.
16. The composition of claim 1 wherein said composition is in the form of an
oral suspension.
17. The composition of claim 1 wherein abarelix is conformationally protected
by folding of said polypeptide about said active agent.
l~. The composition of claim 1 wherein said polypeptide is capable of
releasing
abarelix from said composition in a pH-dependent manner.
19. A method for protecting abarelix from degradation comprising covalently
attaching said active agent to a polypeptide.
20. A method for controlling release of abarelix from a composition wherein
said
composition comprises a polypeptide, said method comprising covalently
attaching
abarelix to said polypeptide.
21. A method for delivering abarelix to a patient comprising administering to
said
patient a composition comprising:
a polypeptide; and
abarelix covalently attached to said polypeptide.
22. The method of claim 21 wherein abarelix is released from said composition
by an enzyme-catalyzed release.
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23. The method of claim 21 wherein abarelix is released from said composition
by a pH-dependent unfolding of said polypeptide.
24. The method of claim 21 wherein said active agent is released from said
composition in a sustained release.
25. The method of claim 21 wherein said composition further comprises an
adjuvant covalently attached to said polypeptide and wherein release of said
adjuvant
from said composition is controlled by said polypeptide.
to
Abstract
A composition comprising a polypeptide and abarelix covalently attached to the
polypeptide. Also provided is a method for delivery of abarelix to a patient
comprising
administering to the patient a composition comprising a polypeptide and
abarelix .
covalently attached to the polypeptide. Also provided is a method for
protecting abarelix
from degradation comprising covalently attaching it to a polypeptide. Also
provided is a
method for controlling.release of abarelix from a composition comprising
covalently
attaching it to the polypeptide.
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A NOVEL PHARMACEUTICAL COMPOUND CONTAINING ACARBOSE AND
METHODS OF MAKING AND USING SAME
FIELD OF THE INVENTION
The present invention relates to a novel pharmaceutical compound that
comprises
a polypeptide that is preferably covalently attached to acarbose, as well as
methods for
protecting and administering acarbose. This novel compound, referred to as a
CARRIERWAVETM Molecular Analogue (CMA), has the benefit of taking a known
effective pharmaceutical agent that is both well studied and occupies a known
segment of
to the pharmaceutical market, and combining it with a carrier compound that
enhances the
usefulness of the pharmaceutical agent without compromising its pharmaceutical
effectiveness.
BACKGROUND OF THE INVENTION
Acarbose is a known pharmaceutical agent that is used in the treatment of type
II
diabetes. Its chemical name is O-4,6-dideoxy-4-[[[1S-
(lalpha,4alpha,5beta,6alpha)]-
4,5,6-trihydroxy-3-(hydroxymethyl)-2-cyclohexen-1-yl]amino]-alpha-D-
glucopyranosyl-
(1-4)-O-alpha-D-glucopyranosyl-(1-4)-D-glucose. Its structure is as follows:
0
O. N O\
O O
\ O
O O O O
O
O O O ~O
O 'O
The novel pharmaceutical compound of the present invention is useful in
accomplishing one or more of the following goals: enhancement of the chemical
stability
of the original compound; alteration of the release profile of an orally
administered
product; enhanced digestion or absorbtion; targeted delivery to particular
tissue/cell type;
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and provision for an oral dosage form when none exists. The novel
pharmaceutical
compound may contain one or more of the following: another active
pharmaceutical
agent, an adjuvant, or an inhibitor.
Active agent delivery systems are often critical for the effective delivery of
a
5 biologically active agent (active agent) to the appropriate target. The
importance of these
systems becomes magnified when patient compliance and active agent stability
are taken
under consideration. For instance, one would expect patient compliance to
increase
markedly if an active agent is administered orally in lieu of an injection or
another
invasive technique. Increasing the stability of the active agent, such as
prolonging shelf
10 life or survival in the stomach, will assure dosage reproducibility and
perhaps even
reduce the number of dosages required which could improve patient compliance.
Absorption of an orally administered active agent is often blocked by the
harshly
acidic stomach milieu, powerful digestive enzymes in the GI tract,
permeability of
cellular membranes and transport across lipid bilayers. Incorporating
adjuvants such as
15 resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance
permeability of
cellular membranes. Microencapsulating active agents using protenoid
microspheres,
liposomes or polysaccharides have been effective in abating enzyme degradation
of the
active agent. Enzyme inhibiting adjuvants have also been used to prevent
enzyme
degradation. Enteric coatings have been used as a protector of pharmaceuticals
in the
2o stomach.
Active agent delivery systems also provide the ability to control the release
of the
active agent. For example, formulating diazepam with a copolymer of glutamic
acid and
aspartic acid enables a sustained release of the active agent. As another
example,
copolymers of lactic acid and glutaric acid are used to provide timed release
of human
25 growth hormone. A wide range of pharmaceuticals purportedly provide
sustained release
through microencapsulation of the active agent in amides of dicarboxylic
acids, modified
amino acids or thermally condensed amino acids. Slow release rendering
additives can
also be intermixed with a large array of active agents in tablet formulations.
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Each of these technologies imparts enhanced stability and time-release
properties
to active agent substances. Unfortunately, these technologies suffer from
several
shortcomings. Incorporation of the active agent is often dependent on
diffusion into the
microencapsulating matrix, which may not be quantitative and may complicate
dosage
reproducibility. In addition, encapsulated drugs rely on diffusion out of the
matrix, which
is highly dependant on the water solubility of the active agent. Conversely,
water-soluble
microspheres swell by an infinite degree and, unfortunately, may release the
active agent
in bursts with little active agent available for sustained release.
Furthermore, in some
technologies, control of the degradation process required for active agent
release is
unreliable. For example, an enterically coated active agent depends on pH to
release the
active agent and, as such, is difficult to control the rate of release.
In the past, use has been made of amino acid side chains of polypeptides as
pendant groups to which active agents can be attached. These technologies
typically
require the use of spacer groups between the amino acid pendant group and the
active
agent. The peptide-drug conjugates of this class of drug delivery system rely
on enzymes
in the bloodstream for the release of the drug and, as such, are not used for
oral
administration. Examples of timed and targeted release of injectable or
subcutaneous
pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer,
to the
gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a
peptide
2o spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been
covalently
attached directly to the beta carboxylate of polyaspartic acid without a
spacer group.
This prodrug formulation was designed as a colon-specific drug delivery system
where
the drug is released by bacterial hydrolytic enzymes residing in the large
intestines. The
released dexamethasone active agent, in turn, was targeted to treat large
bowel disorders
and was not intended to be absorbed into the bloodstream. Yet another
technology
combines the advantages of covalent drug attachment with liposome formation
where the
active ingredient is attached to highly ordered lipid films (known as HARs)
via a peptide
linker. Thus, there has been no drug delivery system, heretofore reported,
that
incorporates the concept of attaching an active ingredient to a polypeptide
pendant group
3o with its targeted delivery into the bloodstream via oral administration.
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It is also important to control the molecular weight, molecular size and
particle
size of the active agent delivery system. Variable molecular weights have
unpredictable
diffusion rates and pharmacokinetics. High molecular weight carriers are
digested slowly
or late, as in the case of naproxen-linked dextran, which is digested almost
exclusively in
the colon by bacterial enzymes. High molecular weight microspheres usually
have high
moisture content which may present a problem with water labile active
ingredients.
Particle size not only becomes a problem with injectable drugs, as in the HAR
application, but absorption through the brush-border membrane of the
intestines is limited
to less than 5 microns.
1o SUMMARY OF THE INVENTION
The present invention provides covalent attachment of the active agent
(acarbose)
to a polymer of peptides or amino acids. The invention is distinguished from
the above
mentioned technologies by virtue of covalently attaching acarbose to the N-
terminus, the
C-terminus or directly to the amino acid side chain of an oligopeptide or
polypeptide,
15 also referred to herein as a carrier peptide. In certain applications, the
polypeptide will
stabilize the active agent, primarily in the stomach, through conformational
protection.
In these applications, delivery of the active agent is controlled, in part, by
the kinetics of
unfolding of the carrier peptide. Upon entry into the upper intestinal tract,
indigenous
enzymes release the active ingredient for absorption by the body by
selectively
2o hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action
introduces a
second order sustained release mechanism.
Alternatively, the present invention provides a pharmaceutical composition
comprising acarbose microencapsulated by a polypeptide.
The invention provides a composition comprising a polypeptide and acarbose
25 covalently attached to the polypeptide. Preferably, the polypeptide is (i)
an oligopeptide,
(ii) a homopolymer of one of the twenty naturally occurnng amino acids, (iii)
a
heteropolymer of two or more naturally occurring amino acids, (iv) a
homopolymer of a
synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids
or (vi) a
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heteropolymer of one or more naturally occurnng amino acids and one or more
synthetic
amino acids.
acarbose preferably is covalently attached to a side chain, the N-terminus or
the
C-terminus of the polypeptide. In a preferred embodiment, the active agent is
a
carboxylic acid and is covalently attached to the N-terminus of the
polypeptide. In
another preferred embodiment, the active agent is an amine and is covalently
attached to
the C-terminus of the polypeptide. In another preferred embodiment, the active
agent is
an alcohol and is covalently attached to the C-terminus of the polypeptide. In
yet another
preferred embodiment, the active agent is an alcohol and is covalently
attached to the N-
terminus of the polypeptide.
The composition of the invention can also include one or more of a
microencapsulating agent, an adjuvant and a pharmaceutically acceptable
excipient. The
microencapsulating agent can be selected from polyethylene glycol (PEG), an
amino
acid, a sugar and a salt. When an adjuvant is included in the composition, the
adjuvant
preferably activates an intestinal transporter.
Preferably, the composition of the invention is in the form of an ingestable
tablet,
an intravenous preparation or an oral suspension. The active agent can be
conformationally protected by folding of the polypeptide about the active
agent. In
another embodiment, the polypeptide is capable of releasing the active agent
from the
2o composition in a pII-dependent manner.
The invention also provides a method for protecting acarbose from degradation
comprising covalently attaching it to a polypeptide.
The invention also provides a method for delivering acarbose to a patient, the
patient being a human or a non-human animal, comprising administering to the
patient a
composition comprising a polypeptide and an active agent covalently attached
to the
polypeptide. In a preferred embodiment, acarbose is released from the
composition by an
enzyme-catalyzed release. In another preferred embodiment, acarbose is
released in a
time-dependent manner based on the pharmacolcinetics of the enzyme-catalyzed
release.
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In another preferred embodiment, the composition further comprises a
microencapsulating agent and acarbose is released from the composition by
dissolution of
the microencapsulating agent. In another preferred embodiment, acarbose is
released
from the composition by a pH-dependent unfolding of the polypeptide. In
another
preferred embodiment, acarbose is released from the composition in a sustained
release.
In yet another preferred embodiment, the composition further comprises an
adjuvant
covalently attached to the polypeptide and release of the adjuvant from the
composition is
controlled by the polypeptide. The adjuvant can be microencapsulated into a
carrier
peptide-drug conjugate for biphasic release of active ingredients.
to The invention also provides a method for preparing a composition comprising
a
polypeptide and an active agent covalently attached to the polypeptide. The
method
comprises the steps of:
(a) attaching acarbose to a side chain of an amino acid to form an active
agent/amino acid complex;
15 (b) forming an active agent/amiiio acid complex N-carboxyanhydride (NCA)
from the active agendamino acid complex; and
(c) polymerizing the active agent/amino acid complex N-carboxyanhydride
(NCA).
In a preferred embodiment, steps (a) and (b) are repeated prior to step (c)
with a
2o second active agent. When steps (a) and (b) are repeated prior to step (c)
with a second
agent, acarbose and a second active agent can be copolymerized in step (c). In
another
preferred embodiment, the amino acid is glutamic acid and the active agent is
released
from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and
wherein the
active agent is released from the glutamic acid by coincident intramolecular
25 transamination. In another preferred embodiment, the glutamic acid is
replaced by an
amino acid selected from the group consisting of aspartic acid, arginine,
asparagine,
cysteine, lysine, threonine, and serine, and wherein the active agent is
attached to the side
chain of the amino acid to form an amide, a thioester, an ester, an ether, a
urethane, a
carbonate, an anhydride or a carbamate. In yet another preferred embodiment,
the
3o glutamic acid is replaced by a synthetic amino acid with a pendant group
comprising an
amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.
6
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It is to be understood that both the foregoing general description and the
following detailed description are exemplary, but are not restrictive, of the
invention.
The general applications of this invention to other active pharmaceutical
agents is
described in U.S. Patent Application Serial Number 09/642,820, filed August
22, 2000,
incorporated herein by reference.
DETAILED DESCRIPTION OF INVENTION
The present invention provides several benefits for active agent delivery.
First,
the invention can stabilize acarbose and prevent its digestion in the stomach.
In addition,
the pharmacologic effect can be prolonged by delayed release of acarbose.
Furthermore,
to active agents can be combined to produce synergistic effects. Also,
absorption of the
active agent in the intestinal tract can be enhanced. The invention also
allows targeted
delivery of active agents to specifics sites of action.
Acarbose is the subject of U.S. Patent Number 4,904,769, herein incorporated
by
reference, which describes how to make that drug.
15 The composition of the invention comprises acarbose covalently attached to
a
polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a
homopolymer of one
of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or
more
naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid,
(v) a
heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of
one or
2o more naturally occurring amino acids and one or more synthetic amino acids.
Proteins, oligopeptides and polypeptides are polymers of amino acids that have
primary, secondary and tertiary structures. The secondary structure of the
protein is the
local conformation of the'polypeptide chain and consists of helices, pleated
sheets and
turns. The protein's amino acid sequence and the structural constraints on the
25 conformations of the chain determine the spatial arrangement of the
molecule. The
folding of the secondary structure and the spatial arrangement of the side
chains
constitute the tertiary structure.
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Proteins fold because of the dynamics associated between neighboring atoms on
the protein and solvent molecules. The thermodynamics of protein folding and
unfolding
are defined by the free energy of a particular condition of the protein that
relies on a
particular model. The process of protein folding involves, amongst other
things, amino
acid residues packing into a hydrophobic core. The amino acid side chains
inside the
protein core occupy the same volume as they do in amino acid crystals. The
folded
protein interior is therefore more like a crystalline solid than an oils drop
and so the best
model for determining forces contributing to protein stability is the solid
reference state.
The major forces contributing to the thermodynamics of protein folding are Van
to der Waals interactions, hydrogen bonds, electrostatic interactions,
configurational
entropy and the hydrophobic effect. Considering protein stability, the
hydrophobic effect
refers to the energetic consequences of removing apolar groups from the
protein interior
and exposing them to water. Comparing the energy of amino acid hydrolysis with
protein unfolding in the solid reference state, the hydrophobic effect is the
dominant
15 force. Hydrogen bonds are established during the protein fold process and
intramolecular
bonds are formed at the expense of hydrogen bonds with water. Water molecules
are
"pushed out" of the packed, hydrophobic protein core. All of these forces
combine and
contribute to the overall stability of the folded protein where the degree to
which ideal
packing occurs determines the degree of relative stability of the protein. The
result of
2o maximum packing is to produce a center of residues or hydrophobic core that
has
maximum shielding from solvent.
Since it is likely that lipophilic drugs would reside in the hydrophobic core
of a
peptide, it would require energy to unfold the peptide before the drug can be
released.
The unfolding process requires overcoming the hydrophobic effect by hydrating
the
25 amino acids or achieving the melting temperature of the protein. The heat
of hydration is
a destabilization of a protein. Typically, the folded state of a protein is
favored by only
5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at
neutral pH and
at room temperature requires chemical reagents. In fact, partial unfolding of
a protein is
often observed prior to the onset of irreversible chemical or conformation
processes.
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Moreover, protein conformation generally controls the rate and extent of
deleterious
chemical reactions.
Conformational protection of active agents by proteins depends on the
stability of
the protein's folded state and the thermodynamics associated with the agent's
decomposition. Conditions necessary for the agent's decomposition should be
different
than for protein unfolding.
Selection of the amino acids will depend on the physical properties desired.
For
instance, if increase in bulk or lipophilicity is desired, then the carrier
polypeptide will be
enriched in the amino acids in the table provided below. Polar amino acids, on
the other
hand, can be selected to increase the hydrophilicity of the polypeptide.
Ionizing amino acids can be selected for pH controlled peptide unfolding.
Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the
stomach, but will
ionize upon entry into the intestine. Conversely, basic amino acids, such as
histidine,
lysine and arginine, ionize in the stomach and are neutral in an alkaline
environment.
Other factors such as ~t-~ interactions between aromatic residues, kinking of
the
peptide chain by addition of proline, disulfide crosslinking and hydrogen
bonding can all
be used to select the optimum amino acid sequence for a given application.
Ordering of
the linear sequence can influence how these interactions can be maximized and
is
important in directing the secondary and tertiary structures of the
polypeptide.
Furthermore, amino acids with reactive side chains (e.g., glutamic acid,
lysine,
aspartic acid, serine, threonine and cysteine) can be incorporated for
attaching multiple
active agents or adjuvants to the same carrier peptide. This is particularly
useful if a
synergistic effect between two or more active agents is desired.
As stated above, variable molecular weights of the carrier compound can have
profound effects on the active agent release kinetics. As a result, low
molecular weight
active agent delivery systems are preferred. An advantage of this invention is
that chain
length and molecular weight of the polypeptide can be optimized depending on
the level
of conformational protection desired. This property can be optimized in
concert with the
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kinetics of the first order release mechanism. Thus, another advantage of this
invention is
that prolonged release time can be imparted by increasing the molecular weight
of the
carrier polypeptide. Another, significant advantage of the invention is that
the kinetics of
active agent release is primarily controlled by the enzymatic hydrolysis of
the key bond
between the carrier peptide and the active agent.
Dextran is the only polysaccharide known that has been explored as a
macromolecular carrier for the covalent binding of drug for colon specific
drug delivery.
Generally, it was only possible to load up to 1/10 of the total drug-dextran
conjugate
weight with drug. As stated earlier, polysaccharides are digested mainly in
the colon and
drug absorption is mainly limited to the colon. As compared to dextran, this
invention
has two major advantages. First, peptides are hydrolyzed by any one of several
aminopeptidases found in the intestinal lumen or associated with the brush-
border
membrane and so active agent release and subsequent absorption can occur in
the
jejunum or the ileum. Second, the molecular weight of the carrier molecule can
be
controlled and, thus, active agent loading can also be controlled.
As a practical example, the following table lists the molecular weights of
lipophilic amino acids (less one water molecule) and selected analgesics and
vitamins.
TABLE
Amino acid MW Active a.
Glycine 57 Acetaminophen 151
Alanine 71 Vitamin B6 (Pyroxidine) 169
Valine 99 Vitamin C (Ascorbic acid) 176
Leucine 113 Aspirin 180
Isoleucine 113 Ibuprofen 206
Phenylalanine147 Retinoic acid 300
Tyrosine 163 Vitamin B2 (Riboflavin) 376
Vitamin D2 397
Vitamin E (Tocopherol) 431
Lipophilic amino acids are preferred because conformational protection through
the
2o stomach is important for the selected active agents, which were selected
based on ease of
covalent attachment to an oligopeptide. Eighteen was subtracted from the amino
acid's
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molecular weight so that their condensation into a polypeptide is considered.
For
example, a decamer of glycine (MW=588) linked to aspirin would have a total
molecular
weight of 750 and aspirin would represent 24% of the total weight of the
active agent
delivery composition or over two times the maximum drug loading for dextran.
This is
only for an N- or C- terminus application, for those active agents attached to
pendant
groups of decaglutamic acid, for instance, a drug with a molecular weight of
18,0 could
conceivably have a loading of 58%, although this may not be entirely
practical.
The alcohol, amine or carboxylic acid group of an active agent may be
covalently
attached to the N-terminus, the C-terminus or the side chain of the
oligopeptide or
to polypeptide. The location of attachment depends somewhat on the functional
group
selection. For instance, if the active drug is a carboxylic acid (e.g.,
aspirin) then the N-
terminus of the oligopeptide is the preferred point of attachment. If the
active agent is an
amine (e.g., ampicillin), then the C-terminus is the preferred point of
attachment in order
to achieve a stable peptide linked active agent. In both, the C- and N-
terminus examples,
15 the peptide is, in essence, extended by one monomeric unit forming a new
peptide bond.
If the active agent is an alcohol, then either the C-terminus or the N-
terminus is the
preferred point of attachment in order to achieve a stable composition. As in
the example
above where the alcohol, norethindrone, was covalently attached to
poly(hydroxypropylglutamine), an alcohol can be converted into an
alkylchloroformate
2o with phosgene. This invention, then, pertains to the reaction of this key
intermediate
with the N-terminus of the peptide carrier. The active ingredient can be
released from
the peptide carrier by intestinal peptidases.
The alcohol can be selectively bound to the gamma carboxylate of glutamic acid
and then this conjugate covalently attached to the C-terminus of the peptide
carrier.
25 Because the glutamic acid-drug conjugate can be considered a dimer, this
product adds
two monomeric units to the C-terminus of the peptide carrier where the
glutamic acid
moiety serves as a spacer between the peptide and the drug as shown in Fig. 4.
Intestinal
enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drug
moiety
from the peptide carrier. The newly formed free amine of the glutamic acid
residue will
30 then undergo an intramolecular transamination reaction, thereby, releasing
the active
11
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agent with coincident formation of pyroglutamic acid as shown in Fig. 5.
Alternatively,
the glutamic acid-drug dimer can be converted into the gamma ester of glutamic
acid N-
carboxyanhydride. This intermediate can then be polymerized, as described
above, using
any suitable initiator as shown in Fig. 4. The product of this polymerization
is
polyglutamic acid with active ingredients attached to multiple pendant groups.
Hence,
maximum drug loading of the Garner peptide can be achieved. In addition, other
amino
acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to
impart
specific properties to the drug delivery system.
The invention also provides a method of imparting the same mechanism of action
10 for other polypeptides containing functional side chains. Examples include,
but are not
limited to, polylysine, polyasparagine, polyarginine, polyserine,
polycysteine,
polytyrosine, polythreonine and polyglutamine. The mechanism can translate to
these
polypeptides through a spacer or linker on the pendant group, which is
terminated,
preferably, by the glutamic acid-drug dimer. This carrier peptide-drug
conjugate is
15 distinguished from the prior art by virtue of the fact that the primary
release of the drug
moiety relies on peptidases and not on esterases. Alternatively, the active
agent can be
attached directly to the pendant group where some other indigenous enzymes in
the
alimentary tract can affect release.
The active agent can be covalently attached to the N-terminus, the C-terminus
or
20 the side chain of the polypeptide using known techniques. Examples of
linking organic
compounds to the N-terminus type of a peptide include, but are not limited to,
the
attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides
and 1,3-
dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example,
there are
known techniques for forming peptide linked biotin and peptide linked
acridine.
25 In the present invention, acarbose is covalently attached to the
polypeptide via
any of the free hydroxyl groups.
The polypeptide carrier can be prepared using conventional techniques. A
preferred technique is copolymerization of mixtures of amino acid N-
carboxyanhydrides.
12
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Alternatively, if a specific sequence is desired, a solid state automated
peptide synthesizer
can be used.
The addition of stabilizers to the composition has the potential of
stabilizing the
polypeptide further. Stabilizers such as sugar, amino acids, polyethylene
glycol (PEG)
and salts have been shown to prevent protein unfolding. In another embodiment
of the
invention, a pre-first order release of the active agent is imparted by
microencapsulating
the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid
complex,
PEG or salts.
There is evidence that hydrophilic compounds are absorbed through the
intestinal
to epithelia efficiently via specialized transporters. The entire membrane
transport system is
intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one
can expect
that excitation of the membrane transport system will involve some sort of
specialized
adjuvant resulting in localized delivery of active agents. There are seven
known
intestinal transport systems classified according to the physical properties
of the
transported substrate. They include the amino acid, oligopeptide, glucose,
monocarboxic
acid, phosphate, bile acid and the P-glycoprotein transport systems and each
has its own
associated mechanism of transport. The mechanisms can depend on hydrogen ions,
sodium ions, binding sites or other cofactors. The invention also allows
targeting the
mechanisms for intestinal epithelial transport systems to facilitate
absorption of active
agents.
In another embodiment of the invention, the composition includes one or more
adjuvants to enhance the bioavailability of the active agent. Addition of an
adjuvant is
particularly preferred when using an otherwise poorly absorbed active agent.
Suitable
adjuvants, for example, include: papain, which is a potent enzyme for
releasing the
catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which
activate
enzymes in the BBM; and bile acids, which have been attached to peptides to
enhance
absorption of the peptides.
Preferably, the resultant peptide-acarbose conjugate is formulated into a
tablet
using suitable excipients and can either be wet granulated or dry compressed.
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Compositions of the invention are, in essence, the formation of amides from
acids
and amines and can be prepared by the following examples.
Acid/N-terminus conjugation
An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to
OoC. The solution can then be treated with diisopropylcarbodiimide and
hydroxybenzotriazole followed by the amine peptide carrier. The reaction can
then be
stirred for several hours at room temperature, the urea by-product filtered
off, the product
precipitated out in ether and purified using gel permeation chromatography
(GPC) or
dialysis.
Amine/C-terminus conjugation
The peptide carrier can be dissolved in DMF under nitrogen and cooled to
0°C.
The solution can then be treated with diisopropylcarbodiimide and
hydroxybenzotriazole
followed by the amine bioactive agent. The reaction can then be stirred for
several hours
at room temperature, the urea by-product filtered off, the product
precipitated out in ether
and purified using GPC or dialysis.
AlcohollN-Terminus Conjugation
In the following example the combination of the alcohol with triphosgene
produces a chloroformate, which when reacted with the N-terminus of the
peptide
produces a carbamate. Pursuant to this, an alcohol bioactive agent can be
treated with
triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier
is then
added slowly and the solution stirred at room temperature for several hours.
The product
is then precipitated out in ether. The crude product is suitably deprotected
and purified
using GPC.
Other solvents, activating agents, cocatalysts and bases can be used. Examples
of
other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or
chlorinated
solvents such as chloroform. Examples of other activating agents include
dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst
is N-
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hydroxysuccinimide. Examples of bases include pyrrolidinopyridine,
dimethylaminopyridine, triethylamine or tributylamine.
Preparation of 'y Alkyl Glutamate
There have been over 30 different y alkyl glutamates prepared any one of which
may be suitable for the drug alcohol of choice. For example, a suspension of
glutamic
acid, the alcohol and concentrated hydrochloric acid can be prepared and
heated for
several hours. The ~y alkyl glutamate product can be precipitated out in
acetone, filtered,
dried and recrystallized from hot water.
y Alkyl Glutamate/C-Terminus Conjugation
to The peptide carrier can be dissolved in DMF under nitrogen and cooled to
0°C.
The solution can then be treated with diisopropylcarbodiimide and
hydroxybenzotriazole
followed by the y alkyl glutamate bioactive agent. The reaction can then be
stirred for
several hours at room temperature, the urea by-product filtered off, the
product
precipitated out in ether and purified using GPC or dialysis.
Preparation of ~y Alkyl Glutamate-NCA
y Alkyl glutamate can be suspended in dry THF where triphosgene is added and
the mixture refluxed under a nitrogen atmosphere until the mixture becomes
homogenous. The solution can be poured into heptane to precipitate the NCA
product,
which is filtered, dried and recrystallized from a suitable solvent.
Preparation of Poly[~y Alkyl Glutamate]
y Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of
a primary amine can be added to the solution until it becomes viscous
(typically
overnight). The product can be isolated from the solution by pouring it into
water and
filtering. The product can be purified using GPC or dialysis.
Although illustrated and described above with reference to specific
embodiments,
the invention is nevertheless not intended to be limited to the details shown.
Rather,
is
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various modifications may be made in the details within the scope and range of
equivalents of the claims and without departing from the spirit of the
invention.
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CLAIMS
What is claimed is:
1. A pharmaceutical composition comprising:
a polypeptide; and
5 acarbose covalently attached to said polypeptide.
2. The composition of claim 1 wherein said polypeptide is an oligopeptide.
3. The composition of claim 1 wherein said polypeptide is a homopolymer of a
naturally occurring amino acid.
4. The composition of claim 1 wherein said polypeptide is a heteropolymer of
1o two or more naturally occurring amino acids.
5. The composition of claim 1 wherein said polypeptide is a homopolymer of a
synthetic amino acid.
6. The composition of claim 1 wherein said polypeptide is a heteropolymer of
two or more synthetic amino acids.
15 7. The composition of claim 1 wherein said polypeptide is a heteropolymer
of
one or more naturally occurring amino acids and one or more synthetic amino
acids.
8. The composition of claim 1 wherein acarbose is covalently attached to a
side
chain, the N-terminus or the C-terminus of said polypeptide.
9. The composition of claim 1 further comprising a microencapsulating agent.
20 10. The composition of claim 9 wherein said microencapsulating agent is
selected from the group consisting of polyethylene glycol (PEG), an amino
acid, a sugar
and a salt.
11. The composition of claim 1 further comprising an adjuvant.
17
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12. The composition of claim 11 wherein said adjuvant activates an intestinal
transporter.
13. The composition of claim 1 further comprising a pharmaceutically
acceptable
excipient.
14. The composition of claim 1 wherein said composition is in the form of an
ingestable tablet.
15. The composition of claim 1 wherein said composition is in the form of an
intravenous preparation.
16. The composition of claim 1 wherein said composition is in the form of an
i0 oral suspension.
17. The composition of claim 1 wherein acarbose is conformationally protected
by folding of said polypeptide about said active agent.
18. The composition of claim 1 wherein said polypeptide is capable of
releasing
acarbose from said composition in a pH-dependent manner.
19. A method for protecting acarbose from degradation comprising covalently
attaching said active agent to a polypeptide.
20. A method for controlling release of acarbose from a composition wherein
said composition comprises a polypeptide, said method comprising covalently
attaching
acarbose to said polypeptide.
21. A method for delivering acarbose to a patient comprising administering to
said patient a composition comprising:
a polypeptide; and
acarbose covalently attached to said polypeptide.
22. The method of claim 21 wherein acarbose is released from said composition
by an enzyme-catalyzed release.
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23. The method of claim 21 wherein acarbose is released from said composition
by a pH-dependent unfolding of said polypeptide.
24. The method of claim 21 wherein said active agent is released from said
composition in a sustained release.
25. The method of claim 21 wherein said composition further comprises an
adjuvant covalently attached to said polypeptide and wherein release of said
adjuvant
from said composition is controlled by said polypeptide.
to
Abstract
A composition comprising a polypeptide and acarbose covalently attached to the
polypeptide. Also provided is a method for delivery of acarbose to a patient
comprising
administering to the patient a composition comprising a polypeptide and
acarbose
covalently attached to the polypeptide. Also provided is a method for
protecting acarbose
from degradation comprising covalently attaching it to a polypeptide. Also
provided is a
method for controlling release of acarbose from a composition comprising
covalently
2o attaching it to the polypeptide.
19
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A NOVEL PHARMACEUTICAL COMPOUND CONTAINING
ACETAMINOPHEN AND METHODS OF MAKING AND USING SAME
FIELD OF THE INVENTION
The present invention relates to a novel pharmaceutical compound that
comprises
a polypeptide that is preferably covalently attached to acetaminophen, as well
as methods
for protecting and administering acetaminophen. This novel compound, referred
to as a
CARRIERWAVE'''~' Molecular Analogue (CMA), has the benefit of taking a known
effective pharmaceutical agent that is both well studied and occupies a known
segment of
1o the pharmaceutical market, and combining it with a carrier compound that
enhances the
usefulness of the pharmaceutical agent without compromising its pharmaceutical
effectiveness.
BACKGROUND OF THE INVENTION
Acetaminophen is a known pharmaceutical agent that is used in the treatment of
~5 minor aches and pains. Its chemical name is N-acetyl-p-aminophenol.
Acetaminophen
is both commercially available and readily manufactured using published
synthetic
schemes by those of ordinary skill in the art.
The novel pharmaceutical compound of the present invention is useful in
accomplishing one or more of the following goals: enhancement of the chemical
stability
20 of the original compound; alteration of the release profile of an orally
administered
product; enhanced digestion or absorbtion; targeted delivery to particular
tissue/cell type;
and provision for an oral dosage form when none exists. The novel
pharmaceutical
compound may contain one or more of the following: another active
pharmaceutical
agent, an adjuvant, or an inhibitor.
25 Active agent delivery systems are often critical for the effective delivery
of a
biologically active agent (active agent) to the appropriate target. The
importance of these
systems becomes magnified when patient compliance and active agent stability
are taken
under consideration. For instance, one would expect patient compliance to
increase
markedly if an active agent is administered orally in lieu of an injection or
another
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invasive technique. Increasing the stability of the active agent, such as
prolonging shelf
life or survival in the stomach, will assure dosage reproducibility and
perhaps even
reduce the number of dosages required which could improve patient compliance.
Absorption of an orally administered active agent is often blocked by the
harshly
acidic stomach milieu, powerful digestive enzymes in the GI tract,
permeability of
cellular membranes and transport across lipid bilayers. Incorporating
adjuvants such as
resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance
permeability of
cellular membranes. Microencapsulating active agents using protenoid
microspheres,
liposomes or polysaccharides have been effective in abating enzyme degradation
of the
to active agent. Enzyme inhibiting adjuvants have also been used to prevent
enzyme
degradation. Enteric coatings have been used as a protector of pharmaceuticals
in the
stomach.
Active agent delivery systems also provide the ability to control the release
of the
active agent. For example, formulating diazepam with a copolymer of glutamic
acid and
t5 aspartic acid enables a sustained release of the active agent. As another
example,
copolymers of lactic acid and glutaric acid are used to provide timed release
of human
growth hormone. A wide range of pharmaceuticals purportedly provide sustained
release
through microencapsulation of the active agent in amides of dicarboxylic
acids, modified
amino acids or thermally condensed amino acids. Slow release rendering
additives can
2o also be intermixed with a large array of active agents in tablet
formulations.
Each of these technologies imparts enhanced stability and time-release
properties
to active agent substances. Unfortunately, these technologies suffer from
several
shortcomings. Incorporation of the active agent is often dependent on
diffusion into the
microencapsulating matrix, which may not be quantitative and may complicate
dosage
25 reproducibility. In addition, encapsulated drugs rely on diffusion out of
the matrix, which
is highly dependant on the water solubility of the active agent. Conversely,
water-soluble
microspheres swell by an infinite degree and, unfortunately, may release the
active agent
in bursts with little active agent available for sustained release.
Furthermore, in some
technologies, control of the degradation process required for active agent
release is
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unreliable. For example, an enterically coated active agent depends on pH to
release the
active agent and, as such, is difficult to control the rate of release.
In the past, use has been made of amino acid side chains of polypeptides as
pendant groups to which active agents can be attached. These technologies
typically
5 require the use of spacer groups between the amino acid pendant group and
the active
agent. The peptide-drug conjugates of this class of drug delivery system rely
on enzymes
in the bloodstream for the release of the drug and, as such, are not used for
oral
administration. Examples of timed and targeted release of injectable or
subcutaneous
pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer,
to the
10 gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard,
via a peptide
spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been
covalently
attached directly to the beta carboxylate of polyaspartic acid without a
spacer group.
This prodrug formulation was designed as a colon-specific drug delivery system
where
the drug is released by bacterial hydrolytic enzymes residing in the large
intestines. The
15 released dexamethasone active agent, in turn, was targeted to treat large
bowel disorders
and was not intended to be absorbed into the bloodstream. Yet another
technology
combines the advantages of covalent drug attachment with liposome formation
where the
active ingredient is attached to highly ordered lipid films (known as HARs)
via a peptide
linker. Thus, there has been no drug delivery system, heretofore reported,
that
2o incorporates the concept of attaching an active ingredient to a polypeptide
pendant group
with its targeted delivery into the bloodstream via oral administration.
It is also important to control the molecular weight, molecular size and
particle
size of the active agent delivery system. Variable molecular weights have
unpredictable
diffusion rates and pharmacokinetics. High molecular weight Garners are
digested slowly
25 or late, as in the case of naproxen-linked dextran, which is digested
almost exclusively in
the colon by bacterial enzymes. High molecular weight microspheres usually
have high
moisture content which may present a problem with water labile active
ingredients.
Particle size not only becomes a problem with injectable drugs, as in the HAR
application, but absorption through the brush-border membrane of the
intestines is limited
30 to less than 5 microns.
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SUMMARY OF THE INVENTION
The present invention provides covalent attachment of the active agent
(acetaminophen) to a polymer of peptides or amino acids. The invention is
distinguished
from the above mentioned technologies by virtue of covalently attaching
acetaminophen
to the N-terminus, the C-terminus or directly to the amino acid side chain of
an
oligopeptide or polypeptide, also referred to herein as a carrier peptide. In
certain
applications, the polypeptide will stabilize the active agent, primarily in
the stomach,
through conformational protection. In these applications, delivery of the
active agent is
controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon
entry into the
1o upper intestinal tract, indigenous enzymes release the active ingredient
for absorption by
the body by selectively hydrolyzing the peptide bonds of the Garner peptide.
This
enzymatic action introduces a second order sustained release mechanism.
Alternatively, the present invention provides a pharmaceutical composition
comprising acetaminophen microencapsulated by a polypeptide.
The invention provides a composition comprising a polypeptide and
acetaminophen covalently attached to the polypeptide. Preferably, the
polypeptide is (i)
an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring
amino acids,
(iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a
homopolymer
of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino
acids or
(vi) a heteropolymer of one or more naturally occurring amino acids and one or
more
synthetic amino acids.
acetaminophen preferably is covalently attached to a side chain, the N-
terminus or
the C-terminus of the polypeptide. In a preferred embodiment, the active agent
is a
carboxylic acid and is covalently attached to the N-terminus of the
polypeptide. In
another preferred embodiment, the active agent is an amine and is covalently
attached to
the C-terminus of the polypeptide. In another preferred embodiment, the active
agent is
an alcohol and is covalently attached to the C-terminus of the polypeptide. In
yet another
preferred embodiment, the active agent is an alcohol and is covalently
attached to the N-
terminus of the polypeptide.
4
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The composition of the invention can also include one or more of a
microencapsulating agent, an adjuvant and a pharmaceutically acceptable
excipient. The
microencapsulating agent can be selected from polyethylene glycol (PEG), an
amino
acid, a sugar and a salt. When an adjuvant is included in the composition, the
adjuvant
preferably activates an intestinal transporter.
Preferably, the composition of the invention is in the form of an ingestable
tablet,
an intravenous preparation or an oral suspension. The active agent can be
conformationally protected by folding of the polypeptide about the active
agent. In
another embodiment, the polypeptide is capable of releasing the active agent
from the
composition in a pH-dependent manner.
The invention also provides a method for protecting acetaminophen from
degradation comprising covalently attaching it to a polypeptide.
The invention also provides a method for delivering acetaminophen to a
patient,
the patient being a human or a non-human animal, comprising administering to
the
patient a composition comprising a polypeptide and an active agent covalently
attached to
the polypeptide. In a preferred embodiment, acetaminophen is released from the
composition by an enzyme-catalyzed release. In another preferred embodiment,
acetaminophen is released in a time-dependent manner based on the
pharmacokinetics of
the enzyme-catalyzed release. In another preferred embodiment, the composition
further
2o comprises a microencapsulating agent and acetaminophen is released from the
composition by dissolution of the microencapsulating agent. In another
preferred
embodiment, acetaminophen is released from the composition by a pH-dependent
unfolding of the polypeptide. In another preferred embodiment, acetaminophen
is
released from the composition in a sustained release. In yet another preferred
embodiment, the composition further comprises an adjuvant covalently attached
to the
polypeptide and release of the adjuvant from the composition is controlled by
the
polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drug
conjugate for biphasic release of active ingredients.
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The invention also provides a method for preparing a composition comprising a
polypeptide and an active agent covalently attached to the polypeptide. The
method
comprises the steps of:
(a) attaching acetaminophen to a side chain of an amino acid to form an active
agentJamino acid complex;
(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA)
from the active agent/amino acid complex; and
(c) polymerizing the active agent/amino acid complex N-carboxyanhydride
(NCA).
In a preferred embodiment, steps (a) and (b) are repeated prior to step (c)
with a
second active agent. When steps (a) and (b) are repeated prior to step (c)
with a second
agent, acetaminophen and a second active agent can be copolymerized in step
(c). In
another preferred embodiment, the amino acid is glutamic acid and the active
agent is
released from the glutamic acid as a dimer upon a hydrolysis of the
polypeptide and
wherein the active agent is released from the glutamic acid by coincident
intramolecular
transamination. In another preferred embodiment, the glutamic acid is replaced
by an
amino acid selected from the group consisting of aspartic acid, arginine,
asparagine,
cysteine, lysine, threonine, and serine, and wherein the active agent is
attached to the side
chain of the amino acid to form an amide, a thioester, an ester, an ether, a
urethane, a
carbonate, an anhydride or a carbamate. In yet another preferred embodiment,
the
glutamic acid is replaced by a synthetic amino acid with a pendant group
comprising an
amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.
It is to be understood that both the foregoing general description and the
following detailed description are exemplary, but are not restrictive, of the
invention.
The general applications of this invention to other active pharmaceutical
agents is
described in U.S. Patent Application Serial Number 09/642,820, filed August
22, 2000,
incorporated herein by reference.
DETAILED DESCRIPTION OF INVENTION
The present invention provides several benefits for active agent delivery.
First,
the invention can stabilize acetaminophen and prevent its digestion in the
stomach. In
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addition, the pharmacologic effect can be prolonged by delayed release of
acetaminophen. Furthermore, active agents can be combined to produce
synergistic
effects. Also, absorption of the active agent in the intestinal tract can be
enhanced. The
invention also allows targeted delivery of active agents to specifics sites of
action.
The composition of the invention comprises acetaminophen covalently attached
to
a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a
homopolymer of
one of the twenty naturally occurring amino acids, (iii) a heteropolymer of
two or more
naturally occurnng amino acids, (iv) a homopolymer of a synthetic amino acid,
(v) a
heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of
one or
more naturally occurring amino acids and one or more synthetic amino acids.
Proteins, oligopeptides and polypeptides are polymers of amino acids that have
primary, secondary and tertiary structures. The secondary structure of the
protein is the
local conformation of the polypeptide chain and consists of helices, pleated
sheets and
turns. The protein's amino acid sequence and the structural constraints on the
t 5 conformations of the chain determine the spatial arrangement of the
molecule. The
folding of the secondary structure and the spatial arrangement of the side
chains
constitute the tertiary structure.
Proteins fold because of the dynamics associated between neighboring atoms on
the protein and solvent molecules. The thermodynamics of protein folding and
unfolding
are defined by the free energy of a particular condition of the protein that
relies on a
particular model. The process of protein folding involves, amongst other
things, amino
acid residues packing into a hydrophobic core. The amino acid side chains
inside the
protein core occupy the same volume as they do in amino acid crystals. The
folded
protein interior is therefore more like a crystalline solid than an oil drop
and so the best
model for determining forces contributing to protein stability is the solid
reference state.
The major forces contributing to the thermodynamics of protein folding are Van
der Waals interactions, hydrogen bonds, electrostatic interactions,
configurational
entropy and the hydrophobic effect. Considering protein stability, the
hydrophobic effect
refers to the energetic consequences of removing apolar groups from the
protein interior
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and exposing them to water. Comparing the energy of amino acid hydrolysis with
protein unfolding in the solid reference state, the hydrophobic effect is the
dominant
force. Hydrogen bonds are established during the protein fold process and
intramolecular
bonds are formed at the expense of hydrogen bonds with water. Water molecules
are
"pushed out" of the packed, hydrophobic protein core. All of these forces
combine and
contribute to the overall stability of the folded protein where the degree to
which ideal
packing occurs determines the degree of relative stability of the protein. The
result of
maximum packing is to produce a center of residues or hydrophobic core that
has
maximum shielding from solvent.
1o Since it is likely that lipophilic drugs would reside in the hydrophobic
core of a
peptide, it would require energy to unfold the peptide before the drug can be
released.
The unfolding process requires overcoming the hydrophobic effect by hydrating
the
amino acids or achieving the melting temperature of the protein. The heat of
hydration is
a destabilization of a protein. Typically, the folded state of a protein is
favored by only
15 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at
neutral pH and
at room temperature requires chemical reagents. In fact, partial unfolding of
a protein is
often observed prior to the onset of irreversible chemical or conformation
processes.
Moreover, protein conformation generally controls the rate and extent of
deleterious
chemical reactions.
2o Conformational protection of active agents by proteins depends on the
stability of
the protein's folded state and the thermodynamics associated with the agent's
decomposition. Conditions necessary for the agent's decomposition should be
different
than for protein unfolding.
Selection of the amino acids will depend on the physical properties desired.
For
25 instance, if increase in bulk or lipophilicity is desired, then the carrier
polypeptide will be
enriched in the amino acids in the table provided below. Polar amino acids, on
the other
hand, can be selected to increase the hydrophilicity of the polypeptide.
Ionizing amino acids can be selected for pH controlled peptide unfolding.
Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the
stomach, but will
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ionize upon entry into the intestine. Conversely, basic amino acids, such as
histidine,
lysine and arginine, ionize in the stomach and are neutral in an alkaline
environment.
Other factors such as ~-~ interactions between aromatic residues, kinking of
the
peptide chain by addition of proline, disulfide crosslinking and hydrogen
bonding can all
be used to select the optimum amino acid sequence for a given application.
Ordering of
the linear sequence can influence how these interactions can be maximized and
is
important in directing the secondary and tertiary structures of the
polypeptide.
Furthermore, amino acids with reactive side chains (e.g., glutamic acid,
lysine,
aspartic acid, serine, threonine and cysteine) can be incorporated for
attaching multiple
active agents or adjuvants to the same carrier peptide. This is particularly
useful if a
synergistic effect between two or more active agents is desired.
As stated above, variable molecular weights of the carrier compound can have
profound effects on the active agent release kinetics. As a result, low
molecular weight
active agent delivery systems are preferred. An advantage of this invention is
that chain
length and molecular weight of the polypeptide can be optimized depending on
the level
of conformational protection desired. This property can be optimized in
concert with the
kinetics of the first order release mechanism. Thus, another advantage of this
invention is
that prolonged release time can be imparted by increasing the molecular weight
of the
carrier polypeptide. Another, significant advantage of the invention is that
the kinetics of
2o active agent release is primarily controlled by the enzymatic hydrolysis of
the key bond
between the carrier peptide and the active agent.
Dextran is the only polysaccharide known that has been explored as a
macromolecular carrier for the covalent binding of drug for colon specific
drug delivery.
Generally, it was only possible to load up to 1/10 of the total drug-dextran
conjugate
weight with drug. As stated earlier, polysaccharides are digested mainly in
the colon and
drug absorption is mainly limited to the colon. As compared to dextran, this
invention
has two major advantages. First, peptides are hydrolyzed by any one of several
aminopeptidases found in the intestinal lumen or associated with the brush-
border
membrane and so active agent release and subsequent absorption can occur in
the
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jejunum or the ileum. Second, the molecular weight of the carrier molecule can
be
controlled and, thus, active agent loading can also be controlled.
As a practical example, the following table lists the molecular weights of
lipophilic amino acids (less one water molecule) and selected analgesics and
vitamins.
TABLE
Amino acid MW Active ate' MW
Glycine 57 Acetaminophen 151
Alanine 71 Vitamin B6 (Pyroxidine) 169
Valine 99 Vitamin C (Ascorbic acid) 176
Leucine 113 Aspirin 180
Isoleucine 113 Ibuprofen 206
Phenylalanine147 Retinoic acid 300
Tyrosine 163 Vitamin B2 (Riboflavin) 376
Vitamin D2 397
Vitamin E (Tocopherol) 431
Lipophilic amino acids are preferred because conformational protection through
the
stomach is important for the selected active agents, which were selected based
on ease of
covalent attachment to an oligopeptide. Eighteen was subtracted from the amino
acid's
molecular weight so that their condensation into a polypeptide is considered.
For
example, a decamer of glycine (MW=588) linked to aspirin would have a total
molecular
weight of 750 and aspirin would represent 24% of the total weight of the
active agent
delivery composition or over two times the maximum drug loading for dextran.
This is
only for an N- or C- terminus application, for those active agents attached to
pendant
groups of decaglutamic acid, for instance, a drug with a molecular weight of
180 could
t5 conceivably have a loading of 58%, although this may not be entirely
practical.
The alcohol, amine or carboxylic acid group of an active agent may be
covalently
attached to the N-terminus, the C-terminus or the side chain of the
oligopeptide or
polypeptide. The location of attachment depends somewhat on the functional
group
selection. For instance, if the active drug is a carboxylic acid (e.g.,
aspirin) then the N-
terminus of the oligopeptide is the preferred point of attachment. If the
active agent is an
amine (e.g., ampicillin), then the C-terminus is the preferred point of
attachment in order
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to achieve a stable peptide linked active agent. In both, the C- and N-
terminus examples,
the peptide is, in essence, extended by one monomeric unit forming a new
peptide bond.
If the active agent is an alcohol, then either the C-terminus or the N-
terminus is the
preferred point of attachment in order to achieve a stable composition. As in
the example
above where the alcohol, norethindrone, was covalently attached to
poly(hydroxypropylglutamine), an alcohol can be converted into an
alkylchlorofonmate
with phosgene. This invention, then, pertains to the reaction of this key
intermediate
with the N-terminus of the peptide carrier. The active ingredient can be
released from
the peptide carrier by intestinal peptidases.
to The alcohol can be selectively bound to the gamma carboxylate of glutamic
acid
and then this conjugate covalently attached to the C-terminus of the peptide
Garner.
Because the glutamic acid-drug conjugate can be considered a dimer, this
product adds
two monomeric units to the C-terminus of the peptide carrier where the
glutamic acid
moiety serves as a spacer between the peptide and the drug as shown in Fig. 4.
Intestinal
15 enzymatic hydrolysis of the key peptide bond releases the glutamic acid-
drug moiety
from the peptide Garner. The newly formed free amine of the glutamic acid
residue will
then undergo an intramolecular transamination reaction, thereby, releasing the
active
agent with coincident formation of pyroglutamic acid as shown in Fig. 5.
Alternatively,
the glutamic acid-drug dimer can be converted into the gamma ester of glutamic
acid N-
20 carboxyanhydride. This intermediate can then be polymerized, as described
above, using
any suitable initiator as shown in Fig. 4. The product of this polymerization
is
polyglutamic acid with active ingredients attached to multiple pendant groups.
Hence,
maximum drug loading of the carrier peptide can be achieved. In addition,
other amino
acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to
impart
25 specific properties to the drug delivery system.
The invention also provides a method of imparting the same mechanism of action
for other polypeptides containing functional side chains. Examples include,
but are not
limited to, polylysine, polyasparagine, polyarginine, polyserine,
polycysteine,
polytyrosine, polythreonine and polyglutamine. The mechanism can translate to
these
30 polypeptides through a spacer or linker on the pendant group, which is
terminated,
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preferably, by the glutamic acid-drug dimer. This carrier peptide-drug
conjugate is
distinguished from the prior art by virtue of the fact that the primary
release of the drug
moiety relies on peptidases and not on esterases. Alternatively, the active
agent can be
attached directly to the pendant group where some other indigenous enzymes in
the
alimentary tract can affect release.
The active agent can be covalently attached to the N-terminus, the C-terminus
or
the side chain of the polypeptide using known techniques. Examples of linking
organic
compounds to the N-terminus type of a peptide include, but are not limited to,
the
attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides
and 1,3-
to dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another
example, there are
known techniques for forming peptide linked biotin and peptide linked
acridine.
In the present invention, acetaminophen is covalently attached to the
polypeptide
via its hydroxyl group.
The polypeptide carrier can be prepared using conventional techniques. A
15 preferred technique is copolymerization of mixtures of amino acid N-
carboxyanhydrides.
Alternatively, if a specific sequence is desired, a solid state automated
peptide synthesizer
can be used.
The addition of stabilizers to the composition has the potential of
stabilizing the
polypeptide further. Stabilizers such as sugar, amino acids, polyethylene
glycol (PEG)
2o and salts have been shown to prevent protein unfolding. In another
embodiment of the
invention, a pre-first order release of the active agent is imparted by
microencapsulating
the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid
complex,
PEG or salts.
There is evidence that hydrophilic compounds are absorbed through the
intestinal
25 epithelia efficiently via specialized transporters. The entire membrane
transport system is
intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one
can expect
that excitation of the membrane transport system will involve some sort of
specialized
adjuvant resulting in localized delivery of active agents. There are seven
known
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intestinal transport systems classified according to the physical properties
of the
transported substrate. They include the amino acid, oligopeptide, glucose,
monocarboxic
acid, phosphate, bile acid and the P-glycoprotein transport systems and each
has its own
associated mechanism of transport. The mechanisms can depend on hydrogen ions,
5 sodium ions, binding sites or other cofactors. The invention also allows
targeting the
mechanisms for intestinal epithelial transport systems to facilitate
absorption of active
agents.
In another embodiment of the invention, the composition includes one or more
adjuvants to enhance the bioavailability of the active agent. Addition of an
adjuvant is
1o particularly preferred when using an otherwise poorly absorbed active
agent. Suitable
adjuvants, for example, include: papain, which is a potent enzyme for
releasing the
catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which
activate
enzymes in the BBM; and bile acids, which have been attached to peptides to
enhance
absorption of the peptides.
15 Preferably, the resultant peptide-acetaminophen conjugate is formulated
into a
tablet using suitable excipients and can either be wet granulated or dry
compressed.
Compositions of the invention are, in essence, the formation of amides from
acids
and amines and can be prepared by the following examples.
Acid/N-terminus conjugation
2o An acid bioactive agent can be dissolved in DMF under nitrogen and cooled
to
0°C. The solution can then be treated with diisopropylcarbodiimide and
hydroxybenzotriazole followed by the amine peptide carrier. The reaction can
then be
stirred for several hours at room temperature, the urea by-product filtered
off, the product
precipitated out in ether and purified using gel permeation chromatography
(GPC) or
25 dialysis.
AmineJC-terminus conjugation
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The peptide carrier can be dissolved in DMF under nitrogen and cooled to
0°C.
The solution can then be treated with diisopropylcarbodiimide and
hydroxybenzotriazole
followed by the amine bioactive agent. The reaction can then be stirred for
several hours
at room temperature, the urea by-product filtered off, the product
precipitated out in ether
and purified using GPC or dialysis.
Alcohol/N-Terminus Coqjugation
In the following example the combination of the alcohol with triphosgene
produces a chloroformate, which when reacted with the N-terminus of the
peptide
produces a carbamate. Pursuant to this, an alcohol bioactive agent can be
treated with
triphosgene in dry DMF under nitrogen. The suitably protected peptide Garner
is then
added slowly and the solution stirred at room temperature for several hours.
The product
is then precipitated out in ether. The crude product is suitably deprotected
and purified
using GPC.
Other solvents, activating agents, cocatalysts and bases can be used. Examples
of
other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or
chlorinated
solvents such as chloroform. Examples of other activating agents include
dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst
is N-
hydroxysuccinimide. Examples of bases include pyrrolidinopyridine,
dimethylaminopyridine, triethylamine or tributylamine.
2o Preparation of Alkyl Glutamate
There have been over 30 different y alkyl glutamates prepared any one of which
may be suitable for the drug alcohol of choice. For example, a suspension of
glutamic
acid, the alcohol and concentrated hydrochloric acid can be prepared and
heated for
several hours. The 'y alkyl glutamate product can be precipitated out in
acetone, filtered,
dried and recrystallized from hot water.
Alkyl Glutamate/C-Terminus Conjugation
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The peptide carrier can be dissolved in DMF under nitrogen and cooled to
0°C.
The solution can then be treated with diisopropylcarbodiimide and
hydroxybenzotriazole
followed by the y alkyl glutamate bioactive agent. The reaction can then be
stirred for
several hours at room temperature, the urea by-product filtered off, the
product
precipitated out in ether and purified using GPC or dialysis.
Preparation of Alkyl Glutamate-NCA
y Alkyl glutamate can be suspended in dry THF where triphosgene is added and
the mixture refluxed under a nitrogen atmosphere until the mixture becomes
homogenous. The solution can be poured into heptane to precipitate the NCA
product,
which is filtered, dried and recrystallized from a suitable solvent.
Preparation of Poly[y Alkyl Glutamate]
y Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of
a primary amine can be added to the solution until it becomes viscous
(typically
overnight). The product can be isolated from the solution by pouring it into
water and
IS filtering. The product can be purified using GPC or dialysis.
Although illustrated and described above with reference to specific
embodiments,
the invention is nevertheless not intended to be limited to the details shown.
Rather,
various modifications may be made in the details within the scope and range of
equivalents of the claims and without departing from the spirit of the
invention.
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CLAIMS
What is claimed is:
1. A pharmaceutical composition comprising:
a polypeptide; and
acetaminophen covalently attached to said polypeptide.
2. The composition of claim 1 wherein said polypeptide is an oligopeptide.
3. The composition of claim 1 wherein said polypeptide is a homopolymer of a
naturally occurring amino acid.
4. The composition of claim 1 wherein said polypeptide is a heteropolymer of
to two or more naturally occurnng amino acids.
5. The composition of claim 1 wherein said polypeptide is a homopolymer of a
synthetic amino acid.
6. The composition of claim 1 wherein said polypeptide is a heteropolymer of
two or more synthetic amino acids.
7. The composition of claim 1 wherein said polypeptide is a heteropolymer of
one or more naturally occurring amino acids and one or more synthetic amino
acids.
8. The composition of claim 1 wherein acetaminophen is covalently attached to
a
side chain, the N-terminus or the C-terminus of said polypeptide.
9. The composition of claim 1 further comprising a microencapsulating agent.
10. The composition of claim 9 wherein said microencapsulating agent is
selected from the group consisting of polyethylene glycol (PEG), an amino
acid, a sugar
and a salt.
11. The composition of claim 1 further comprising an adjuvant.
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12. The composition of claim 11 wherein said adjuvant activates an intestinal
transporter.
13. The composition of claim 1 further comprising a pharmaceutically
acceptable
excipient.
14. The composition of claim 1 wherein said composition is in the form of an
ingestable tablet.
15. The composition of claim 1 wherein said composition is in the form of an
intravenous preparation.
16. The composition of claim 1 wherein said composition is in the form of an
oral suspension.
17. The composition of claim 1 wherein acetaminophen is conformationally
protected by folding of said polypeptide about said active agent.
18. The composition of claim 1 wherein said polypeptide is capable of
releasing
acetaminophen from said composition in a pH-dependent manner.
19. A method for protecting acetaminophen from degradation comprising
covalently attaching said active agent to a polypeptide.
20. A method for controlling release of acetaminophen from a composition
wherein said composition comprises a polypeptide, said method comprising
covalently
attaching acetaminophen to said polypeptide.
21. A method for delivering acetaminophen to a patient comprising
administering
to said patient a composition comprising:
a polypeptide; and
acetaminophen covalently attached to said polypeptide.
22. The method of claim 21 wherein acetaminophen is released from said
composition by an enzyme-catalyzed release.
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23. The method of claim 21 wherein acetaminophen is released from said
composition by a pH-dependent unfolding of said polypeptide.
24. The method of claim 21 wherein said active agent is released from said
composition in a sustained release.
25. The method of claim 21 wherein said composition further comprises an
adjuvant covalently attached to said polypeptide and wherein release of said
adjuvant
from said composition is controlled by said polypeptide.
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A NOVEL PHARMACEUTICAL COMPOUND CONTAINING
ACETAMINOPHEN AND CODEINE AND METHODS OF MAKING AND
USING SAME
FIELD OF THE INVENTION
The present invention relates to a novel pharmaceutical compound that
comprises
a polypeptide that is preferably covalently attached to both acetaminophen and
codeine,
as well as methods for protecting and administering acetaminophen and codeine
together.
This novel compound, referred to as a CARRIERWAVETN' Molecular Analogue (CMA),
to has the benefit of taking a known effective pharmaceutical agent that is
both well studied
and occupies a known segment of the pharmaceutical market, and combining it
with a
carrier compound that enhances the usefulness of the pharmaceutical agent
without
compromising its pharmaceutical effectiveness.
BACKGROUND OF THE INVENTION
15 Acetaminophen is a known pharmaceutical agent that is used in the treatment
of
minor aches and pains. Its chemical name is N-acetyl-p-aminophenol. It is
often used
in combination with codeine, whose chemical name is 7,8-didehydro-4,5-a-epoxy-
3-
methoxy-17-methylmephorninan-6a-ol. Both are commercially available and
readily
manufactured using published synthetic schemes by those of ordinary skill in
the art.
2o The novel pharmaceutical compound of the present invention is useful in
accomplishing one or more of the following goals: enhancement of the chemical
stability
of the original compound; alteration of the release profile of an orally
administered
product; enhanced digestion or absorbtion; targeted delivery to particular
tissue/cell type;
and provision for an oral dosage form when none exists. The novel
pharmaceutical
25 compound may contain one or more of the following: another active
pharmaceutical
agent, an adjuvant, or an inhibitor.
Active agent delivery systems are often critical for the effective delivery of
a
biologically active agent (active agent) to the appropriate target. The
importance of these
systems becomes magnified when patient compliance and active agent stability
are taken
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under consideration. For instance, one would expect patient compliance to
increase
markedly if an active agent is administered orally in lieu of an injection or
another
invasive technique. Increasing the stability of the active agent, such as
prolonging shelf
life or survival in the stomach, will assure dosage reproducibility and
perhaps even
reduce the number of dosages required which could improve patient compliance.
Absorption of an orally administered active agent is often blocked by the
harshly
acidic stomach milieu, powerful digestive enzymes in the GT tract,
permeability of
cellular membranes and transport across lipid bilayers. Incorporating
adjuvants such as
resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance
permeability of
cellular membranes. Microencapsulating active agents using protenoid
microspheres,
liposomes or polysaccharides have been effective in abating enzyme degradation
of the
active agent. Enzyme inhibiting adjuvants have also been used to prevent
enzyme
degradation. Enteric coatings have been used as a protector of pharmaceuticals
in the
stomach.
Active agent delivery systems also provide the ability to control the release
of the
active agent. For example, formulating diazepam with a copolymer of glutamic
acid and
aspartic acid enables a sustained release of the active agent. As another
example,
copolymers of lactic acid and glutaric acid are used to provide timed release
of human
growth hormone. A wide range of pharmaceuticals purportedly provide sustained
release
through microencapsulation of the active agent in amides of dicarboxylic
acids, modified
amino acids or thermally condensed amino acids. Slow release rendering
additives can
also be intermixed with a large array of active agents in tablet formulations.
Each of these technologies imparts enhanced stability and time-release
properties
to active agent substances. Unfortunately, these technologies suffer from
several
shortcomings. Incorporation of the active agent is often dependent on
diffusion into the
microencapsulating matrix, which may not be quantitative and may complicate
dosage
reproducibility. In addition, encapsulated drugs rely on diffusion out of the
matrix, which
is highly dependant on the water solubility of the active agent. Conversely,
water-soluble
microspheres swell by an infinite degree and, unfortunately, may release the
active agent
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in bursts with little active agent available for sustained release.
Furthermore, in some
technologies, control of the degradation process required for active agent
release is
unreliable. For example, an enterically coated active agent depends on pH to
release the
active agent and, as such, is difficult to control the rate of release.
In the past, use has been made of amino acid side chains of polypeptides as
pendant groups to which active agents can be attached. These technologies
typically
require the use of spacer groups between the amino acid pendant group and the
active
agent. The peptide-drug conjugates of this class of drug delivery system rely
on enzymes
in the bloodstream for the release of the drug and, as such, are not used for
oral
administration. Examples of timed and targeted release of injectable or
subcutaneous
pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer,
to the
gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a
peptide
spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been
covalently
attached directly to the beta carboxylate of polyaspartic acid without a
spacer group.
This prodrug formulation was designed as a colon-specific drug delivery system
where
the drug is released by bacterial hydrolytic enzymes residing in the large
intestines. The
released dexamethasone active agent, in turn, was targeted to treat large
bowel disorders
and was not intended to be absorbed into the bloodstream. Yet another
technology
combines the advantages of covalent drug attachment with liposome formation
where the
2o active ingredient is attached to highly ordered lipid films (known as HARs)
via a peptide
linker. Thus, there has been no drug delivery system, heretofore reported,
that
incorporates the concept of attaching an active ingredient to a polypeptide
pendant group
with its targeted delivery into the bloodstream via oral administration.
It is also important to control the molecular weight, molecular size and
particle
size of the active agent delivery system. Variable molecular weights have
unpredictable
diffusion rates and pharmacokinetics. High molecular weight Garners are
digested slowly
or late, as in the case of naproxen-linked dextran, which is digested almost
exclusively in
the colon by bacterial enzymes. High molecular weight microspheres usually
have high
moisture content which may present a problem with water labile active
ingredients.
Particle size not only becomes a problem with injectable drugs, as in the HAR
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application, but absorption through the brush-border membrane of the
intestines is limited
to less than 5 microns.
SUMMARY OF THE INVENTION
The present invention provides covalent attachment of the active agent
(acetaminophen and codeine) to a polymer of peptides or amino acids. The
invention is
distinguished from the above mentioned technologies by virtue of covalently
attaching
acetaminophen and codeine to the N-terminus, the C-terminus or directly to the
amino
acid side chain of an oligopeptide or polypeptide, also referred to herein as
a carrier
peptide. In certain applications, the polypeptide will stabilize the active
agent, primarily
in the stomach, through conformational protection. In these applications,
delivery of the
active agent is controlled, in part, by the kinetics of unfolding of the
carrier peptide.
Upon entry into the upper intestinal tract, indigenous enzymes release the
active
ingredient for absorption by the body by selectively hydrolyzing the peptide
bonds of the
carrier peptide. This enzymatic action introduces a second order sustained
release
mechanism.
Alternatively, the present invention provides a pharmaceutical composition
comprising acetaminophen and codeine microencapsulated by a polypeptide.
The invention provides a composition comprising a polypeptide and
acetaminophen and codeine covalently attached to the polypeptide. Preferably,
the
2o polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty
naturally
occurring amino acids, (iii) a heteropolymer of two or more naturally occurnng
amino
acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of
two or more
synthetic amino acids or (vi) a heteropolymer of one or more naturally
occurnng amino
acids and one or more synthetic amino acids.
acetaminophen and codeine preferably is covalently attached to a side chain,
the
N-terminus or the C-terminus of the polypeptide. In a preferred embodiment,
the active
agent is a carboxylic acid and is covalently attached to the N-terminus of the
polypeptide.
In another preferred embodiment, the active agent is an amine and is
covalently attached
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to the C-terminus of the polypeptide. In another preferred embodiment, the
active agent
is an alcohol and is covalently attached to the C-terminus of the polypeptide.
In yet
another preferred embodiment, the active agent is an alcohol and is covalently
attached to
the N-terminus of the polypeptide.
The composition of the invention can also include one or more of a
microencapsulating agent, an adjuvant and a pharmaceutically acceptable
excipient. The
microencapsulating agent can be selected from polyethylene glycol (PEG), an
amino
acid, a sugar and a salt. When an adjuvant is included in the composition, the
adjuvant
preferably activates an intestinal transporter.
to Preferably, the composition of the invention is in the fonm of an
ingestable tablet,
an intravenous preparation or an oral suspension. The active agent can be
conformationally protected by folding of the polypeptide about the active
agent. In
another embodiment, the polypeptide is capable of releasing the active agent
from the
composition in a pH-dependent manner.
15 The invention also provides a method for protecting acetaminophen and
codeine
from degradation comprising covalently attaching it to a polypeptide.
The invention also provides a method for delivering acetaminophen and codeine
to a patient, the patient being a human or a non-human animal, comprising
administering
to the patient a composition comprising a polypeptide and an active agent
covalently
2o attached to the polypeptide. In a preferred embodiment, acetaminophen and
codeine are
released from the composition by an enzyme-catalyzed release. In another
preferred
embodiment, acetaminophen and codeine are released in a time-dependent manner
based
on the pharmacokinetics of the enzyme-catalyzed release. In another preferred
embodiment, the composition further comprises a microencapsulating agent and
25 acetaminophen and codeine are released from the composition by dissolution
of the
microencapsulating agent. In another preferred embodiment, acetaminophen and
codeine
are released from the composition by a pH-dependent unfolding of the
polypeptide. In
another preferred embodiment, acetaminophen and codeine are released from the
composition in a sustained release. In yet another preferred embodiment, the
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composition further comprises an adjuvant covalently attached to the
polypeptide and
release of the adjuvant from the composition is controlled by the polypeptide.
The
adjuvant can be microencapsulated into a carrier peptide-drug conjugate for
biphasic
release of active ingredients.
The invention also provides a method for preparing a composition comprising a
polypeptide and an active agent covalently attached to the polypeptide. The
method
comprises the steps of:
(a) attaching acetaminophen to a side chain of an amino acid to form an active
agent/amino acid complex;
(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA)
from the active agent/amino acid complex; and
(c) polymerizing the active agendamino acid complex N-carboxyanhydride
(NCA).
In a preferred embodiment, steps (a) and (b) are repeated prior to step (c)
with a
second active agent. When steps (a) and (b) are repeated prior to step (c)
with a second
agent, acetaminophen and a second active agent can be copolymerized in step
(c). In
another preferred embodiment, the amino acid is glutamic acid and the active
agent is
released from the glutamic acid as a dimer upon a hydrolysis of the
polypeptide and
wherein the active agent is released from the glutamic acid by coincident
intramolecular
2o transamination. In another preferred embodiment, the glutamic acid is
replaced by an
amino acid selected from the group consisting of aspartic acid, arginine,
asparagine,
cysteine, lysine, threonine, and serine, and wherein the active agent is
attached to the side
chain of the amino acid to form an amide, a thioester, an ester, an ether, a
urethane, a
carbonate, an anhydride or a carbamate. In yet another preferred embodiment,
the
glutamic acid is replaced by a synthetic amino acid with a pendant group
comprising an
amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.
It is to be understood that both the foregoing general description and the
following detailed description are exemplary, but are not restrictive, of the
invention.
The general applications of this invention to other active pharmaceutical
agents is
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described in U.S. Patent Application Serial Number 09/642,820, filed August
22, 2000,
incorporated herein by reference.
DETAILED DESCRIPTION OF INVENTION
The present invention provides several benefits for active agent delivery.
First,
the invention can stabilize acetaminophen and prevent its digestion in the
stomach. In
addition, the pharmacologic effect can be prolonged by delayed release of
acetaminophen. Furthermore, active agents can be combined to produce
synergistic
effects. Also, absorption of the active agent in the intestinal tract can be
enhanced. The
invention also allows targeted delivery of active agents to specifics sites of
action.
to The composition of the invention comprises acetaminophen covalently
attached to
a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a
homopolymer of
one of the twenty naturally occurring amino acids, (iii) a heteropolymer of
two or more
naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid,
(v) a
heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of
one or
t5 more naturally occurring amino acids and one or more synthetic amino acids.
Proteins, oligopeptides and polypeptides are polymers of amino acids that have
primary, secondary and tertiary structures. The secondary structure of the
protein is the
local conformation of the polypeptide chain and consists of helices, pleated
sheets and
turns. The protein's amino acid sequence and the structural constraints on the
20 conformations of the chain determine the spatial arrangement of the
molecule. The
folding of the secondary structure and the spatial arrangement of the side
chains
constitute the tertiary structure.
Proteins fold because of the dynamics associated between neighboring atoms on
the protein and solvent molecules. The thermodynamics of protein folding and
unfolding
25 are defined by the free energy of a particular condition of the protein
that relies on a
particular model. The process of protein folding involves, amongst other
things, amino
acid residues packing into a hydrophobic core. The amino acid side chains
inside the
protein core occupy the same volume as they do in amino acid crystals. The
folded
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protein interior is therefore more like a crystalline solid than an oil drop
and so the best
model for determining forces contributing to protein stability is the solid
reference state.
The major forces contributing to the thermodynamics of protein folding are Van
der Waals interactions, hydrogen bonds, electrostatic interactions,
configurational
entropy and the hydrophobic effect. Considering protein stability, the
hydrophobic effect
refers to the energetic consequences of removing apolar groups from the
protein interior
and exposing them to water. Comparing the energy of amino acid hydrolysis with
protein unfolding in the solid reference state, the hydrophobic effect is the
dominant
force. Hydrogen bonds are established during the protein fold process and
intramolecular
1o bonds are formed at the expense of hydrogen bonds with water. Water
molecules are
"pushed out" of the packed, hydrophobic protein core. All of these forces
combine and
contribute to the overall stability of the folded protein where the degree to
which ideal
packing occurs determines the degree of relative stability of the protein. The
result of
maximum packing is to produce a center of residues or hydrophobic core that
has
maximum shielding from solvent.
Since it is likely that lipophilic drugs would reside in the hydrophobic core
of a
peptide, it would require energy to unfold the peptide before the drug can be
released.
The unfolding process requires overcoming the hydrophobic effect by hydrating
the
amino acids or achieving the melting temperature of the protein. The heat of
hydration is
2o a destabilization of a protein. Typically, the folded state of a protein is
favored by only
5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at
neutral pH and
at room temperature requires chemical reagents. In fact, partial unfolding of
a protein is
often observed prior to the onset of irreversible chemical or conformation
processes.
Moreover, protein conformation generally controls the rate and extent of
deleterious
chemical reactions.
Conformational protection of active agents by proteins depends on the
stability of
the protein's folded state and the thermodynamics associated with the agent's
decomposition. Conditions necessary for the agent's decomposition should be
different
than for protein unfolding.
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Selection of the amino acids will depend on the physical properties desired.
For
instance, if increase in bulk or lipophilicity is desired, then the Garner
polypeptide will be
enriched in the amino acids in the table provided below. Polar amino acids, on
the other
hand, can be selected to increase the hydrophilicity of the polypeptide.
Ionizing amino acids can be selected for pH controlled peptide unfolding.
Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the
stomach, but will
ionize upon entry into the intestine. Conversely, basic amino acids, such as
histidine,
lysine and arginine, ionize in the stomach and are neutral in an alkaline
environment.
Other factors such as n-~ interactions between aromatic residues, kinking of
the
peptide chain by addition of proline, disulfide crosslinking and hydrogen
bonding can all
be used to select the optimum amino acid sequence for a given application.
Ordering of
the linear sequence can influence how these interactions can be maximized and
is
important in directing the secondary and tertiary structures of the
polypeptide.
Furthermore, amino acids with reactive side chains (e.g., glutamic acid,
lysine,
~5 aspartic acid, serine, threonine and cysteine) can be incorporated for
attaching multiple
active agents or adjuvants to the same carrier peptide. This is particularly
useful if a
synergistic effect between two or more active agents is desired.
As stated above, variable molecular weights of the carrier compound can have
profound effects on the active agent release kinetics. As a result, low
molecular weight
2o active agent delivery systems are preferred. An advantage of this invention
is that chain
length and molecular weight of the polypeptide can be optimized depending on
the level
of conformational protection desired. This property can be optimized in
concert with the
kinetics of the first order release mechanism. Thus, another advantage of this
invention is
that prolonged release time can be imparted by increasing the molecular weight
of the
25 carrier polypeptide. Another, significant advantage of the invention is
that the kinetics of
active agent release is primarily controlled by the enzymatic hydrolysis of
the key bond
between the carrier peptide and the active agent.
9
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Dextran is the only polysaccharide known that has been explored as a
macromolecular carrier for the covalent binding of drug for colon specific
drug delivery.
Generally, it was only possible to load up to 1/10 of the total drug-dextran
conjugate
weight with drug. As stated earlier, polysaccharides are digested mainly in
the colon and
5 drug absorption is mainly limited to the colon. As compared to dextran, this
invention
has two major advantages. First, peptides are hydrolyzed by any one of several
aminopeptidases found in the intestinal lumen or associated with the brush-
border
membrane and so active agent release and subsequent absorption can occur in
the
jejunum or the ileum. Second, the molecular weight of the carrier molecule can
be
to controlled and, thus, active agent loading can also be controlled.
As a practical example, the following table lists the molecular weights of
lipophilic amino acids (less one water molecule) and selected analgesics and
vitamins.
TABLE
Amino acid MW Active agent MW
Glycine 57 Acetaminophen 151
Alanine 71 Vitamin B6 (Pyroxidine) 169
Valine 99 Vitamin C (Ascorbic acid) 176
Leucine 113 Aspirin 180
Isoleucine 113 Ibuprofen 206
Phenylalanine147 Rednoic acid 300
Tyrosine 163 Vitamin B2 (Riboflavin) 376
Vitamin DZ 397
Vitamin E (Tocopherol) 431
Lipophilic amino acids are preferred because conformational protection through
the
15 stomach is important for the selected active agents, which were selected
based on ease of
covalent attachment to an oligopeptide. Eighteen was subtracted from the amino
acid's
molecular weight so that their condensation into a polypeptide is considered.
For
example, a decamer of glycine (MW=588) linked to aspirin would have a total
molecular
weight of 750 and aspirin would represent 24% of the total weight of the
active agent
2o delivery composition or over two times the maximum drug loading for
dextran. This is
only for an N- or C- terminus application, for those active agents attached to
pendant
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groups of decaglutamic acid, for instance, a drug with a molecular weight of
180 could
conceivably have a loading of 58%, although this may not be entirely
practical.
The alcohol, amine or carboxylic acid group of an active agent may be
covalently
attached to the N-terminus, the C-terminus or the side chain of the
oligopeptide or
polypeptide. The location of attachment depends somewhat on the functional
group
selection. For instance, if the active drug is a carboxylic acid (e.g.,
aspirin) then the N-
terminus of the oligopeptide is the preferred point of attachment. If the
active agent is an
amine (e.g., ampicillin), then the C-terminus is the preferred point of
attachment in order
to achieve a stable peptide linked active agent. In both, the C- and N-
terminus examples,
the peptide is, in essence, extended by one monomeric unit forming a new
peptide bond.
If the active agent is an alcohol, then either the C-terminus or the N-
terminus is the
preferred point of attachment in order to achieve a stable composition. As in
the example
above where the alcohol, norethindrone, was covalently attached to
poly(hydroxypropylglutamine), an alcohol can be converted into an
alkylchloroformate
with phosgene. This invention, then, pertains to the reaction of this key
intermediate
with the N-terminus of the peptide carrier. The active ingredient can be
released from
the peptide carrier by intestinal peptidases.
The alcohol can be selectively bound to the gamma carboxylate of glutamic acid
and then this conjugate covalently attached to the C-terminus of the peptide
carrier.
Because the glutamic acid-drug conjugate can be considered a dimer, this
product adds
two monomeric units to the C-terminus of the peptide Garner where the glutamic
acid
moiety serves as a spacer between the peptide and the drug as shown in Fig. 4.
Intestinal
enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drug
moiety
from the peptide Garner. The newly formed free amine of the glutamic acid
residue will
then undergo an intramolecular transamination reaction, thereby, releasing the
active
agent with coincident formation of pyroglutamic acid as shown in Fig. 5.
Alternatively,
the glutamic acid-drug dimer can be converted into the gamma ester of glutamic
acid N-
carboxyanhydride. This intermediate can then be polymerized, as described
above, using
any suitable initiator as shown in Fig. 4. The product of this polymerization
is
polyglutamic acid with active ingredients attached to multiple pendant groups.
Hence,
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maximum drug loading of the carrier peptide can be achieved. In addition,
other amino
acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to
impart
specific properties to the drug delivery system.
The invention also provides a method of imparting the same mechanism of action
for other polypeptides containing functional side chains. Examples include,
but are not
limited to, polylysine, polyasparagine, polyarginine, polyserine,
polycysteine,
polytyrosine, polythreonine and polyglutamine. The mechanism can translate to
these
polypeptides through a spacer or linker on the pendant group, which is
terminated,
preferably, by the glutamic acid-drug dimer. This carrier peptide-drug
conjugate is
to distinguished from the prior art by virtue of the fact that the primary
release of the drug
moiety relies on peptidases and not on esterases. Alternatively, the active
agent can be
attached directly to the pendant group where some other indigenous enzymes in
the
alimentary tract can affect release.
The active agent can be covalently attached to the N-terminus, the C-terminus
or
15 the side chain of the polypeptide using known techniques. Examples of
linking organic
compounds to the N-terminus type of a peptide include, but are not limited to,
the
attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides
and 1,3-
dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example,
there are
known techniques for forming peptide linked biotin and peptide linked
acridine.
2o In the present invention, acetaminophen and codeine are covalently attached
to
the polypeptide via its hydroxyl group.
The polypeptide Garner can be prepared using conventional techniques. A
preferred technique is copolymerization of mixtures of amino acid N-
carboxyanhydrides.
Alternatively, if a specific sequence is desired, a solid state automated
peptide synthesizer
25 can be used.
The addition of stabilizers to the composition has the potential of
stabilizing the
polypeptide further. Stabilizers such as sugar, amino acids, polyethylene
glycol (PEG)
and salts have been shown to prevent protein unfolding. In another embodiment
of the
t2
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invention, a pre-first order release of the active agent is imparted by
microencapsulating
the Garner polypeptide-active agent conjugate in a polysaccharide, amino acid
complex,
PEG or salts.
There is evidence that hydrophilic compounds are absorbed through the
intestinal
epithelia efficiently via specialized transporters. The entire membrane
transport system is
intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one
can expect
that excitation of the membrane transport system will involve some sort of
specialized
adjuvant resulting in localized delivery of active agents. There are seven
known
intestinal transport systems classified according to the physical properties
of the
l0 transported substrate. They include the amino acid, oligopeptide, glucose,
monocarboxic
acid, phosphate, bile acid and the P-glycoprotein transport systems and each
has its own
associated mechanism of transport. The mechanisms can depend on hydrogen ions,
sodium ions, binding sites or other cofactors. The invention also allows
targeting the
mechanisms for intestinal epithelial transport systems to facilitate
absorption of active
15 agents.
In another embodiment of the invention, the composition includes one or more
adjuvants to enhance the bioavailability of the active agent. Addition of an
adjuvant is
particularly preferred when using an otherwise poorly absorbed active agent.
Suitable
adjuvants, for example, include: papain, which is a potent enzyme for
releasing the
2o catalytic domain of aminopeptidase-N into the lumen; glycorecognizers,
which activate
enzymes in the BBM; and bile acids, which have been attached to peptides to
enhance
absorption of the peptides.
Preferably, the resultant peptide-acetaminophen conjugate is formulated into a
tablet using suitable excipients and can either be wet granulated or dry
compressed.
25 Compositions of the invention are, in essence, the formation of amides from
acids
and amines and can be prepared by the following examples.
13
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Acid/N-terminus conjugation
An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to
0°C. The solution can then be treated with diisopropylcarbodiimide and
hydroxybenzotriazole followed by the amine peptide carrier. The reaction can
then be
stirred for several hours at room temperature, the urea by-product filtered
off, the product
precipitated out in ether and purified using gel permeation chromatography
(GPC) or
dialysis.
AminelC-terminus conjugation
The peptide carrier can be dissolved in DMF under nitrogen and cooled to
0°C.
The solution can then be treated with diisopropylcarbodiimide and
hydroxybenzotriazole
followed by the amine bioactive agent. The reaction can then be stirred for
several hours
at room temperature, the urea by-product filtered off, the product
precipitated out in ether
and purified using GPC or dialysis.
Atcohol/N-Terminus Conjugation
In the following example the combination of the alcohol with triphosgene
produces a chloroformate, which when reacted with the N-terminus of the
peptide
produces a carbamate. Pursuant to this, an alcohol bioactive agent can be
treated with
triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier
is then
added slowly and the solution stirred at room temperature for several hours.
The product
is then precipitated out in ether. The crude product is suitably deprotected
and purified
using GPC.
Other solvents, activating agents, cocatalysts and bases can be used. Examples
of
other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or
chlorinated
solvents such as chloroform. Examples of other activating agents include
dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst
is N-
hydroxysuccinimide. Examples of bases include pyrrolidinopyridine,
dimethylaminopyridine, triethylamine or tributylamine.
14
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Preparation of Alkyl Glutamate
There have been over 30 different y alkyl glutamates prepared any one of which
may be suitable for the drug alcohol of choice. For example, a suspension of
glutamic
acid, the alcohol and concentrated hydrochloric acid can be prepared and
heated for
several hours. The y alkyl glutamate product can be precipitated out in
acetone, filtered,
dried and recrystallized from hot water.
~-Alkyl GlutamatelC-Terminus Conjugation
The peptide Garner can be dissolved in DMF under nitrogen and cooled to
0°C.
The solution can then be treated with diisopropylcarbodiimide and
hydroxybenzotriazole
1o followed by the y alkyl glutamate bioactive agent. The reaction can then be
stirred for
several hours at room temperature, the urea by-product filtered off, the
product
precipitated out in ether and purified using GPC or dialysis.
Preparation of Alkyl Glutamate-NCA
y Alkyl glutamate can be suspended in dry THF where triphosgene is added and
15 the mixture refluxed under a nitrogen atmosphere until the mixture becomes
homogenous. The solution can be poured into heptane to precipitate the NCA
product,
which is filtered, dried and recrystallized from a suitable solvent.
Preparation of Poly[~ Alkyl Glutamate]
'y Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of
2o a primary amine can be added to the solution until it becomes viscous
(typically
overnight). The product can be isolated from the solution by pouring it into
water and
filtering. The product can be purified using GPC or dialysis.
Although illustrated and described above with reference to specific
embodiments,
the invention is nevertheless not intended to be limited to the details shown.
Rather,
25 various modifications may be made in the details within the scope and range
of
equivalents of the claims and without departing from the spirit of the
invention.
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CLAIMS
What is claimed is:
1. A pharmaceutical composition comprising:
a polypeptide; and
acetaminophen covalently attached to said polypeptide.
2. The composition of claim 1 wherein said polypeptide is an oligopeptide.
3. The composition of claim 1 wherein said polypeptide is a homopolymer of a
naturally occurring amino acid.
4. The composition of claim 1 wherein said polypeptide is a heteropolymer of
l0 two or more naturally occurring amino acids.
5. The composition of claim 1 wherein said polypeptide is a homopolymer of a
synthetic amino acid.
6. The composition of claim 1 wherein said polypeptide is a heteropolymer of
two or more synthetic amino acids.
7. The composition of claim 1 wherein said polypeptide is a heteropolymer of
one or more naturally occurnng amino acids and one or more synthetic amino
acids.
8. The composition of claim 1 wherein acetaminophen and codeine are
covalently attached to a side chain, the N-terminus or the C-terminus of said
polypeptide.
9. The composition of claim 1 further comprising a microencapsulating agent.
2o 10. The composition of claim 9 wherein said microencapsulating agent is
selected from the group consisting of polyethylene glycol (PEG), an amino
acid, a sugar
and a salt.
11. The composition of claim 1 further comprising an adjuvant.
16
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12. The composition of claim 11 wherein said adjuvant activates an intestinal
transporter.
13. The composition of claim 1 further comprising a pharmaceutically
acceptable
excipient.
14. The composition of claim 1 wherein said composition is in the form of an
ingestable tablet.
15. The composition of claim 1 wherein said composition is in the form of an
intravenous preparation.
16. The composition of claim 1 wherein said composition is in the form of an
oral suspension.
17. The composition of claim 1 wherein acetaminophen and codeine are
conformationally protected by folding of said polypeptide about said active
agent.
18. The composition of claim 1 wherein said polypeptide is capable of
releasing
acetaminophen from said composition in a pH-dependent manner.
19. A method for protecting acetaminophen from degradation comprising
covalently attaching said active agent to a polypeptide.
20. A method for controlling release of acetaminophen from a composition
wherein said composition comprises a polypeptide, said method comprising
covalently
attaching acetaminophen to said polypeptide.
21. A method for delivering acetaminophen to a patient comprising
administering
to said patient a composition comprising:
a polypeptide; and
acetaminophen covalently attached to said polypeptide.
22. The method of claim 21 wherein acetaminophen and codeine are released
from said composition by an enzyme-catalyzed release.
m
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23. The method of claim 21 wherein acetaminophen and codeine are released
from said composition by a pH-dependent unfolding of said polypeptide.
24. The method of claim 21 wherein said active agent is released from said
composition in a sustained release.
25. The method of claim 21 wherein said composition further comprises an
adjuvant covalently attached to said polypeptide and wherein release of said
adjuvant
from said composition is controlled by said polypeptide.
~8
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Abstract
A composition comprising a polypeptide and acetaminophen covalently attached
to the polypeptide. Also provided is a method for delivery of acetaminophen to
a patient
comprising administering to the patient a composition comprising a polypeptide
and
acetaminophen covalently attached to the polypeptide. Also provided is a
method for
protecting acetaminophen from degradation comprising covalently attaching it
to a
polypeptide. Also provided is a method for controlling release of
acetaminophen from a
composition comprising covalently attaching it to the polypeptide.
19
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A NOVEL PHARMACEUTICAL COMPOUND CONTAINING
PROPOXYPHENE AND METHODS OF MAKING AND USING SAME
FIELD OF THE INVENTION
5 The present invention relates to a novel pharmaceutical compound that
comprises
a polypeptide that is preferably covalently attached to propoxyphene, as well
as methods
for protecting and administering propoxyphene. This novel compound, referred
to as a
CARRIERWAVET"' Molecular Analogue (CMA), has the benefit of taking a known
effective pharmaceutical agent that is both well studied and occupies a known
segment of
10 the pharmaceutical market, and combining it with a Garner compound that
enhances the
usefulness of the pharmaceutical agent without compromising its pharmaceutical
effectiveness.
BACKGROUND OF THE INVENTION
Propoxyphene is a known pharmaceutical agent that is used in the treatment of
15 pain. It is a mild narcotic analgesic. It is both commercially available
and readily
manufactured using published synthetic schemes by those of ordinary skill in
the art. Its
structure is as follows:
a N ~ GH3
H3C:~~ f"~ ~I"~1
The novel pharmaceutical compound of the present invention is useful in
20 accomplishing one or more of the following goals: enhancement of the
chemical stability
of the original compound; alteration of the release profile of an orally
administered
product; enhanced digestion or absorption; targeted delivery to particular
tissue/cell type;
and provision for an oral dosage form when none exists. The novel
pharmaceutical
compound may contain one or more of the following: another active
pharmaceutical
25 agent, an adjuvant, or an inhibitor.
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Active agent delivery systems are often critical for the effective delivery of
a
biologically active agent (active agent) to the appropriate target. The
importance of these
systems becomes magnified when patient compliance and active agent stability
are taken
under consideration. For instance, one would expect patient compliance to
increase
markedly if an active agent is administered orally in lieu of an injection or
another
invasive technique. Increasing the stability of the active agent, such as
prolonging shelf
life or survival in the stomach, will assure dosage reproducibility and
perhaps even
reduce the number of dosages required which could improve patient compliance.
Absorption of an orally administered active agent is often blocked by the
harshly
1o acidic stomach milieu, powerful digestive enzymes in the GI tract,
permeability of
cellular membranes and transport across lipid bilayers. Incorporating
adjuvants such as
resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance
permeability of
cellular membranes. Microencapsulating active agents using protenoid
microspheres,
liposomes or polysaccharides have been effective in abating enzyme degradation
of the
15 active agent. Enzyme inhibiting adjuvants have also been used to prevent
enzyme
degradation. Enteric coatings have been used as a protector of pharmaceuticals
in the
stomach.
Active agent delivery systems also provide the ability to control the release
of the
active agent. For example, formulating diazepam with a copolymer of glutamic
acid and
2o aspartic acid enables a sustained release of the active agent. As another
example,
copolymers of lactic acid and glutaric acid are used to provide timed release
of human
growth hormone. A wide range of pharmaceuticals purportedly provide sustained
release
through microencapsulation of the active agent in amides of dicarboxylic
acids, modified
amino acids or thermally condensed amino acids. Slow release rendering
additives can
25 also be intermixed with a large array of active agents in tablet
formulations.
Each of these technologies imparts enhanced stability and time-release
properties
to active agent substances. Unfortunately, these technologies suffer from
several
shortcomings. Incorporation of the active agent is often dependent on
diffusion into the
microencapsulating matrix, which may not be quantitative and may complicate
dosage
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reproducibility. In addition, encapsulated drugs rely on diffusion out of the
matrix, which
is highly dependant on the water solubility of the active agent. Conversely,
water-soluble
microspheres swell by an infinite degree and, unfortunately, may release the
active agent
in bursts with little active agent available for sustained release.
Furthermore, in some
technologies, control of the degradation process required for active agent
release is
unreliable. For example, an enterically coated active agent depends on pH to
release the
active agent and, as such, is difficult to control the rate of release.
In the past, use has been made of amino acid side chains of polypeptides as
pendant groups to which active agents can be attached. These technologies
typically
1o require the use of spacer groups between the amino acid pendant group and
the active
agent. The peptide-drug conjugates of this class of drug delivery system rely
on enzymes
in the bloodstream for the release of the drug and, as such, are not used for
oral
administration. Examples of timed and targeted release of injectable or
subcutaneous
pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer,
to the
15 gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard,
via a peptide
spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been
covalently
attached directly to the beta carboxylate of polyaspartic acid without a
spacer group.
This prodrug formulation was designed as a colon-specific drug delivery system
where
the drug is released by bacterial hydrolytic enzymes residing in the large
intestines. The
20 released dexamethasone active agent, in turn, was targeted to treat large
bowel disorders
and was not intended to be absorbed into the bloodstream. Yet another
technology
combines the advantages of covalent drug attachment with liposome formation
where the
active ingredient is attached to highly ordered lipid films (known as HARs)
via a peptide
linker. Thus, there has been no drug delivery system, heretofore reported,
that
25 incorporates the concept of attaching an active ingredient to a polypeptide
pendant group
with its targeted delivery into the bloodstream via oral administration.
It is also important to control the molecular weight, molecular size and
particle
size of the active agent delivery system. Variable molecular weights have
unpredictable
diffusion rates and pharmacokinetics. High molecular weight Garners are
digested slowly
30 or late, as in the case of naproxen-linked dextran, which is digested
almost exclusively in
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the colon by bacterial enzymes. High molecular weight microspheres usually
have high
moisture content which may present a problem with water labile active
ingredients.
Particle size not only becomes a problem with injectable drugs, as in the HAR
application, but absorption through the brush-border membrane of the
intestines is limited
to less than S microns.
SUMMARY OF THE INVENTION
The present invention provides covalent attachment of the active agent
(propoxyphene) to a polymer of peptides or amino acids. The invention is
distinguished
from the above mentioned technologies by virtue of covalently attaching
propoxyphene
1o to the N-terminus, the C-terminus or directly to the amino acid side chain
of an
oligopeptide or polypeptide, also referred to herein as a carrier peptide. In
certain
applications, the polypeptide will stabilize the active agent, primarily in
the stomach,
through conformational protection. In these applications, delivery of the
active agent is
controlled, in part, by the kinetics of unfolding of the Garner peptide. Upon
entry into the
upper intestinal tract, indigenous enzymes release the active ingredient for
absorption by
the body by selectively hydrolyzing the peptide bonds of the carrier peptide.
This
enzymatic action introduces a second order sustained release mechanism.
Alternatively, the present invention provides a pharmaceutical composition
comprising propoxyphene microencapsulated by a polypeptide.
The invention provides a composition comprising a polypeptide and
propoxyphene covalently attached to the polypeptide. Preferably, the
polypeptide is (i)
an oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring
amino acids,
(iii) a heteropolymer of two or more naturally occurring amino acids, (iv) a
homopolymer
of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino
acids or
(vi) a heteropolymer of one or more naturally occurnng amino acids and one or
more
synthetic amino acids.
propoxyphene preferably is covalently attached to a side chain, the N-terminus
or
the.C-terminus of the polypeptide. In a preferred embodiment, the active agent
is a
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carboxylic acid and is covalently attached to the N-terminus of the
polypeptide. In
another preferred embodiment, the active agent is an amine and is covalently
attached to
the C-terminus of the polypeptide. In another preferred embodiment, the active
agent is
an alcohol and is covalently attached to the C-terminus of the polypeptide. In
yet another
preferred embodiment, the active agent is an alcohol and is covalently
attached to the N-
terminus of the polypeptide.
The composition of the invention can also include one or more of a
microencapsulating agent, an adjuvant and a pharmaceutically acceptable
excipient. The
microencapsulating agent can be selected from polyethylene glycol (PEG), an
amino
acid, a sugar and a salt. When an adjuvant is included in the composition, the
adjuvant
preferably activates an intestinal transporter.
Preferably, the composition of the invention is in the form of an ingestable
tablet,
an intravenous preparation or an oral suspension. The active agent can be
conformationally protected by folding of the polypeptide about the active
agent. In
another embodiment, the polypeptide is capable of releasing the active agent
from the
composition in a pH-dependent manner.
The invention also provides a method for protecting propoxyphene from
degradation comprising covalently attaching it to a polypeptide.
The invention also provides a method for delivering propoxyphene to a patient,
2o the patient being a human or a non-human animal, comprising administering
to the
patient a composition comprising a polypeptide and an active agent covalently
attached to
the polypeptide. In a preferred embodiment, propoxyphene is released from the
composition by an enzyme-catalyzed release. In another preferred embodiment,
propoxyphene is released in a time-dependent manner based on the
pharmacokinetics of
the enzyme-catalyzed release. In another preferred embodiment, the composition
further
comprises a microencapsulating agent and propoxyphene is released from the
composition by dissolution of the microencapsulating agent. In another
preferred
embodiment, propoxyphene is released from the composition by a pH-dependent
unfolding of the polypeptide. In another preferred embodiment, propoxyphene is
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released from the composition in a sustained release. In yet another preferred
embodiment, the composition further comprises an adjuvant covalently attached
to the
polypeptide and release of the adjuvant from the composition is controlled by
the
polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drug
conjugate for biphasic release of active ingredients.
The invention also provides a method for preparing a composition comprising a
polypeptide and an active agent covalently attached to the polypeptide. The
method
comprises the steps of:
(a) attaching propoxyphene to a side chain of an amino acid to form an active
agent/amino acid complex;
(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA)
from the active agendamino acid complex; and
(c) polymerizing the active agent/amino acid complex N-carboxyanhydride
(NCA).
In a preferred embodiment, steps (a) and (b) are repeated prior to step (c)
with a
second active agent. When steps (a) and (b) are repeated prior to step (c)
with a second
agent, propoxyphene and a second active agent can be copolymerized in step
(c). In
another preferred embodiment, the amino acid is glutamic acid and the active
agent is
released from the glutamic acid as a dimer upon a hydrolysis of the
polypeptide and
2o wherein the active agent is released from the glutamic acid by coincident
intramolecular
transamination. In another preferred embodiment, the glutamic acid is replaced
by an
amino acid selected from the group consisting of aspartic acid, arginine,
asparagine,
cysteine, lysine, threonine, and serine, and wherein the active agent is
attached to the side
chain of the amino acid to form an amide, a thioester, an ester, an ether, a
urethane, a
carbonate, an anhydride or a carbamate. In yet another preferred embodiment,
the
glutamic acid is replaced by a synthetic amino acid with a pendant group
comprising an
amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.
It is to be understood that both the foregoing general description and the
following detailed description are exemplary, but are not restrictive, of the
invention.
The general applications of this invention to other active pharmaceutical
agents is
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described in U.S. Patent Application Serial Number 09/642,820, filed August
22, 2000,
incorporated herein by reference.
DETAILED DESCRIPTION OF INVENTION
The present invention provides several benefits for active agent delivery.
First,
the invention can stabilize propoxyphene and prevent its digestion in the
stomach. In
addition, the pharmacologic effect can be prolonged by delayed release of
propoxyphene.
Furthermore, active agents can be combined to produce synergistic effects.
Also,
absorption of the active agent in the intestinal tract can be enhanced. The
invention also
allows targeted delivery of active agents to specifics sites of action.
1o The composition of the invention comprises propoxyphene covalently attached
to
a polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a
homopolymer of
one of the twenty naturally occurring amino acids, (iii) a heteropolymer of
two or more
naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid,
(v) a
heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of
one or
15 more naturally occurring amino acids and one or more synthetic amino acids.
Proteins, oligopeptides and polypeptides are polymers of amino acids that have
primary, secondary and tertiary structures. The secondary structure of the
protein is the
local conformation of the polypeptide chain and consists of helices, pleated
sheets and
turns. The protein's amino acid sequence and the structural constraints on the
2o conformations of the chain determine the spatial arrangement of the
molecule. The
folding of the secondary structure and the spatial arrangement of the side
chains
constitute the tertiary structure.
Proteins fold because of the dynamics associated between neighboring atoms on
the protein and solvent molecules. The thermodynamics of protein folding and
unfolding
25. are defined by the free energy of a particular condition of the protein
that relies on a
particular model. The process of protein folding involves, amongst other
things, amino
acid residues packing into a hydrophobic core. The amino acid side chains
inside the
protein core occupy the same volume as they do in amino acid crystals. The
folded
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protein interior is therefore more like a crystalline solid than an oil drop
and so the best
model for determining forces contributing to protein stability is the solid
reference state.
The major forces contributing to the thermodynamics of protein folding are Van
der Waals interactions, hydrogen bonds, electrostatic interactions,
configurational
entropy and the hydrophobic effect. Considering protein stability, the
hydrophobic effect
refers to the energetic consequences of removing apolar groups from the
protein interior
and exposing them to water. Comparing the energy of amino acid hydrolysis with
protein unfolding in the solid reference state, the hydrophobic effect is the
dominant
force. Hydrogen bonds are established during the protein fold process and
intramolecular
bonds are formed at the expense of hydrogen bonds with water. Water molecules
are
"pushed out" of the packed, hydrophobic protein core. All of these forces
combine and
contribute to the overall stability of the folded protein where the degree to
which ideal
packing occurs determines the degree of relative stability of the protein. The
result of
maximum packing is to produce a center of residues or hydrophobic core that
has
maximum shielding from solvent.
Since it is likely that lipophilic drugs would reside in the hydrophobic core
of a
peptide, it would require energy to unfold the peptide before the drug can be
released.
The unfolding process requires overcoming the hydrophobic effect by hydrating
the
amino acids or achieving the melting temperature of the protein. The heat of
hydration is
a destabilization of a protein. Typically, the folded state of a protein is
favored by only
5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at
neutral pH and
at room temperature requires chemical reagents. In fact, partial unfolding of
a protein is
often observed prior to the onset of irreversible chemical or conformation
processes.
Moreover, protein conformation generally controls the rate and extent of
deleterious
chemical reactions.
Conformational protection of active agents by proteins depends on the
stability of
the protein's folded state and the thermodynamics associated with the agent's
decomposition. Conditions necessary for the agent's decomposition should be
different
than for protein unfolding.
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Selection of the amino acids will depend on the physical properties desired.
For
instance, if increase in bulk or lipophilicity is desired, then the carrier
polypeptide will be
enriched in the amino acids in the table provided below. Polar amino acids, on
the other
hand, can be selected to increase the hydrophilicity of the polypeptide.
Ionizing amino acids can be selected for pH controlled peptide unfolding.
Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the
stomach, but will
ionize upon entry into the intestine. Conversely, basic amino acids, such as
histidine,
lysine and arginine, ionize in the stomach and are neutral in an alkaline
environment.
Other factors such as n-~ interactions between aromatic residues, kinking of
the
peptide chain by addition of proline, disulfide crosslinking and hydrogen
bonding can all
be used to select the optimum amino acid sequence for a given application.
Ordering of
the linear sequence can influence how these interactions can be maximized and
is
important in directing the secondary and tertiary structures of the
polypeptide.
Furthermore, amino acids with reactive side chains (e.g., glutamic acid,
lysine,
aspartic acid, serine, threonine and cysteine) can be incorporated for
attaching multiple
active agents or adjuvants to the same carrier peptide. This is particularly
useful if a
synergistic effect between two or more active agents is desired.
As stated above, variable molecular weights of the carrier compound can have
profound effects on the active agent release kinetics. As a result, low
molecular weight
2o active agent delivery systems are preferred. An advantage of this invention
is that chain
length and molecular weight of the polypeptide can be optimized depending on
the level
of conformational protection desired. This property can be optimized in
concert with the
kinetics of the first order release mechanism. Thus, another advantage of this
invention is
that prolonged release time can be imparted by increasing the molecular weight
of the
carrier polypeptide. Another, significant advantage of the invention is that
the kinetics of
active agent release is primarily controlled by the enzymatic hydrolysis of
the key bond
between the carrier peptide and the active agent.
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Dextran is the only polysaccharide known that has been explored as a
macromolecular Garner for the covalent binding of drug for colon specific drug
delivery.
Generally, it was only possible to load up to 1/10 of the total drug-dextran
conjugate
weight with drug. As stated earlier, polysaccharides are digested mainly in
the colon and
drug absorption is mainly limited to the colon. As compared to dextran, this
invention
has two major advantages. First, peptides are hydrolyzed by any one of several
aminopeptidases found in the intestinal lumen or associated with the brush-
border
membrane and so active agent release and subsequent absorption can occur in
the
jejunum or the ileum. Second, the molecular weight of the carrier molecule can
be
controlled and, thus, active agent loading can also be controlled.
As a practical example, the following table lists the molecular weights of
lipophilic amino acids (less one water molecule) and selected analgesics and
vitamins.
TABLE
Amino acid MW Active agent MW
Glycine 57 Acetaminophen 151
Alanine 71 Vitamin B6 (Pyroxidine) 169
Valine 99 Vitamin C (Ascorbic acid) 176
Leucine 113 Aspirin 180
Isoleucine 113 Ibuprofen 206
Phenylalanine 147 Retinoic acid 300
Tyrosine 163 Vitamin B2 (Riboflavin) 376
Vitamin D2 397
Vitamin E (Tocopherol) 431
Lipophilic amino acids are preferred because conformational protection through
the
stomach is important for the selected active agents, which were selected based
on ease of
covalent attachment to an oligopeptide. Eighteen was subtracted from the amino
acid's
molecular weight so that their condensation into a polypeptide is considered.
For
example, a decamer of glycine (MW=588) linked to aspirin would have a total
molecular
weight of 750 and aspirin would represent 24% of the total weight of the
active agent
delivery composition or over two times the maximum drug loading for dextran.
This is
only for an N- or C- terminus application, for those active agents attached to
pendant
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groups of decaglutamic acid, for instance, a drug with a molecular weight of
180 could
conceivably have a loading of 58%, although this may not be entirely
practical.
The alcohol, amine or carboxylic acid group of an active agent may be
covalently
attached to the N-terminus, the C-terminus or the side chain of the
oligopeptide or
polypeptide. The location of attachment depends somewhat on the functional
group
selection. For instance, if the active drug is a carboxylic acid (e.g.,
aspirin) then the N-
terminus of the oligopeptide is the preferred point of attachment. If the
active agent is an
amine (e.g., ampicillin), then the C-terminus is the preferred point of
attachment in order
to achieve a stable peptide linked active agent. In both, the C- and N-
terminus examples,
1o the peptide is, in essence, extended by one monomeric unit forming a new
peptide bond.
If the active agent is an alcohol, then either the C-terminus or the N-
terminus is the
preferred point of attachment in order to achieve a stable composition. As in
the example
above where the alcohol, norethindrone, was covalently attached to
poly(hydroxypropylglutamine), an alcohol can be converted into an
alkylchloroformate
with phosgene. This invention, then, pertains to the reaction of this key
intermediate
with the N-terminus of the peptide carrier. The active ingredient can be
released from
the peptide carrier by intestinal peptidases.
The alcohol can be selectively bound to the gamma carboxylate of glutamic acid
and then this conjugate covalently attached to the C-terminus of the peptide
carrier.
2o Because the glutamic acid-drug conjugate can be considered a dimer, this
product adds
two monomeric units to the C-terminus of the peptide carrier where the
glutamic acid
moiety serves as a spacer between the peptide and the drug as shown in Fig. 4.
Intestinal
enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drug
moiety
from the peptide Garner. The newly formed free amine of the glutamic acid
residue will
then undergo an intramolecular transamination reaction, thereby, releasing the
active
agent with coincident formation of pyroglutamic acid as shown in Fig. 5.
Alternatively,
the glutamic acid-drug dimer can be converted into the gamma ester of glutamic
acid N-
carboxyanhydride. This intermediate can then be polymerized, as described
above, using
any suitable initiator as shown in Fig. 4. The product of this polymerization
is
3o polyglutamic acid with active ingredients attached to multiple pendant
groups. Hence,
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maximum drug loading of the carrier peptide can be achieved. In addition,
other amino
acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to
impart
specific properties to the drug delivery system.
The invention also provides a method of imparting the same mechanism of action
for other polypeptides containing functional side chains. Examples include,
but are not
limited to, polylysine, polyasparagine, polyarginine, polyserine,
polycysteine,
polytyrosine, polythreonine and polyglutamine. The mechanism can translate to
these
polypeptides through a spacer or linker on the pendant group, which is
terminated,
preferably, by the glutamic acid-drug dimer. This carrier peptide-drug
conjugate is
to distinguished from the prior art by virtue of the fact that the primary
release of the drug
moiety relies on peptidases and not on esterases. Alternatively, the active
agent can be
attached directly to the pendant group where some other indigenous enzymes in
the
alimentary tract can affect release.
The active agent can be covalently attached to the N-terminus, the C-terminus
or
the side chain of the polypeptide using known techniques. Examples of linking
organic
compounds to the N-terminus type of a peptide include, but are not limited to,
the
attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides
and 1,3-
dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example,
there are
known techniques for forming peptide linked biotin and peptide linked
acridine.
2o In the present invention, propoxyphene is covalently attached to the
polypeptide
via a linker. This linker may be a small molecule containing 2-6 carbons and
one or more
functional groups (such as amines, amides, alcohols, or acids) or may be made
up of a
short chain of either amino acids or carbohydrates.
The polypeptide Garner can be prepared using conventional techniques. A
preferred technique is copolymerization of mixtures of amino acid N-
carboxyanhydrides.
Alternatively, if a specific sequence is desired, a solid state automated
peptide synthesizer
can be used.
12
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The addition of stabilizers to the composition has the potential of
stabilizing the
polypeptide further. Stabilizers such as sugar, amino acids, polyethylene
glycol (PEG)
and salts have been shown to prevent protein unfolding. In another embodiment
of the
invention, a pre-first order release of the active agent is imparted by
microencapsulating
the Garner polypeptide-active agent conjugate in a polysaccharide, amino acid
complex,
PEG or salts.
There is evidence that hydrophilic compounds are absorbed through the
intestinal
epithelia efficiently via specialized transporters. The entire membrane
transport system is
intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one
can expect
that excitation of the membrane transport system will involve some sort of
specialized
adjuvant resulting in localized delivery of active agents. There are seven
known
intestinal transport systems classified according to the physical properties
of the
transported substrate. They include the amino acid, oligopeptide, glucose,
monocarboxic
acid, phosphate, bile acid and the P-glycoprotein transport systems and each
has its own
associated mechanism of transport. The mechanisms can depend on hydrogen ions,
sodium ions, binding sites or other cofactors. The invention also allows
targeting the
mechanisms for intestinal epithelial transport systems to facilitate
absorption of active
agents.
In another embodiment of the invention, the composition includes one or more
adjuvants to enhance the bioavailability of the active agent. Addition of an
adjuvant is
particularly preferred when using an otherwise poorly absorbed active agent.
Suitable
adjuvants, for example, include: papain, which is a potent enzyme for
releasing the
catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which
activate
enzymes in the BBM; and bile acids, which have been attached to peptides to
enhance
absorption of the peptides.
Preferably, the resultant peptide-propoxyphene conjugate is formulated into a
tablet using suitable excipients and can either be wet granulated or dry
compressed.
Compositions of the invention are, in essence, the formation of amides from
acids
and amines and can be prepared by the following examples.
13
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Acid/N-terminus conjugation
An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to
0°C. The solution can then be treated with diisopropylcarbodiimide and
hydroxybenzotriazole followed by the amine peptide carrier. The reaction can
then be
stirred for several hours at room temperature, the urea by-product filtered
off, the product
precipitated out in ether and purified using gel permeation chromatography
(GPC) or
dialysis.
Amine/C-terminus conjugation
The peptide carrier can be dissolved in DMF under nitrogen and cooled to
0°C.
to The solution can then be treated with diisopropylcarbodiimide and
hydroxybenzotriazole
followed by the amine bioactive agent. The reaction can then be stirred for
several hours
at room temperature, the urea by-product filtered off, the product
precipitated out in ether
and purified using GPC or dialysis.
Alcohol/N-Terminus Conjugation
15 In the following example the combination of the alcohol with triphosgene
produces a chloroformate, which when reacted with the N-terminus of the
peptide
produces a carbamate. Pursuant to this, an alcohol bioactive agent can be
treated with
triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier
is then
added slowly and the solution stirred at room temperature for several hours.
The product
2o is then precipitated out in ether. The crude product is suitably
deprotected and purified
using GPC.
Other solvents, activating agents, cocatalysts and bases can be used. Examples
of
other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or
chlorinated
solvents such as chloroform. Examples of other activating agents include
25 dicyclohexylcarbodiimide or thionyl chloride. An example of another
cocatalyst is N-
hydroxysuccinimide. Examples of bases include pyrrolidinopyridine,
dimethylaminopyridine, triethylamine or tributylamine.
14
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Preparation of Alkyl Glutamate
There have been over 30 different y alkyl glutamates prepared any one of which
may be suitable for the drug alcohol of choice. For example, a suspension of
glutamic
acid, the alcohol and concentrated hydrochloric acid can be prepared and
heated for
several hours. The y alkyl glutamate product can be precipitated out in
acetone, filtered,
dried and recrystallized from hot water.
y~Alkyl Glutamate/C-Terminus Conjugation
The peptide carrier can be dissolved in DMF under nitrogen and cooled to
0°C.
The solution can then be treated with diisopropylcarbodiimide and
hydroxybenzotriazole
followed by the y alkyl glutamate bioactive agent. The reaction can then be
stirred for
several hours at room temperature, the urea by-product filtered off, the
product
precipitated out in ether and purified using GPC or dialysis.
Preparation of ~-Alkyl Glutamate-NCA
y Alkyl glutamate can be suspended in dry THF where triphosgene is added and
the mixture refluxed under a nitrogen atmosphere until the mixture becomes
homogenous. The solution can be poured into heptane to precipitate the NCA
product,
which is filtered, dried and recrystallized from a suitable solvent.
Preparation of Poly[fir Alkyl Glutamate]
y Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of
2o a primary amine can be added to the solution until it becomes viscous
(typically
overnight). The product can be isolated from the solution by pouring it into
water and
filtering. The product can be purified using GPC or dialysis.
Although illustrated and described above with reference to specific
embodiments,
the invention is nevertheless not intended to be limited to the details shown.
Rather,
various modifications may be made in the details within the scope and range of
equivalents of the claims and without departing from the spirit of the
invention.
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CLAIMS
What is claimed is:
1. A pharmaceutical composition comprising:
a polypeptide; and
propoxyphene covalently attached to said polypeptide.
2. The composition of claim 1 wherein said polypeptide is an oligopeptide.
3. The composition of claim 1 wherein said polypeptide is a homopolymer of a
naturally occurring amino acid.
4. The composition of claim 1 wherein said polypeptide is a heteropolymer of
two or more naturally occurring amino acids.
5. The composition of claim 1 wherein said polypeptide is a homopolymer of a
synthetic amino acid.
6. The composition of claim 1 wherein said polypeptide is a heteropolymer of
two or more synthetic amino acids.
7. The composition of claim 1 wherein said polypeptide is a heteropolymer of
one or more naturally occurring amino acids and one or more synthetic amino
acids.
8. The composition of claim 1 wherein propoxyphene is covalently attached to a
side chain, the N-terminus or the C-terminus of said polypeptide.
9. The composition of claim 1 further comprising a microencapsulating agent.
10. The composition of claim 9 wherein said microencapsulating agent is
selected from the group consisting of polyethylene glycol (PEG), an amino
acid, a sugar
and a salt.
11. The composition of claim 1 further comprising an adjuvant.
16
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12. The composition of claim 11 wherein said adjuvant activates an intestinal
transporter.
13. The composition of claim 1 further comprising a pharmaceutically
acceptable
excipient.
14. The composition of claim 1 wherein said composition is in the form of an
ingestable tablet.
15. The composition of claim 1 wherein said composition is in the form of an
intravenous preparation.
16. The composition of claim 1 wherein said composition is in the form of an
1o oral suspension.
17. The composition of claim 1 wherein propoxyphene is conformationally
protected by folding of said polypeptide about said active agent.
18. The composition of claim 1 wherein said polypeptide is capable of
releasing
propoxyphene from said composition in a pH-dependent manner.
19. A method for protecting propoxyphene from degradation comprising
covalently attaching said active agent to a polypeptide.
20. A method for controlling release of propoxyphene from a composition
wherein said composition comprises a polypeptide, said method comprising
covalently
attaching propoxyphene to said polypeptide.
21. A method for delivering propoxyphene to a patient comprising administering
to said patient a composition comprising:
a polypeptide; and
propoxyphene covalently attached to said polypeptide.
22. The method of claim 21 wherein propoxyphene is released from said
composition by an enzyme-catalyzed release.
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23. The method of claim 21 wherein propoxyphene is released from said
composition by a pH-dependent unfolding of said polypeptide.
24. The method of claim 21 wherein said active agent is released from said
composition in a sustained release.
25. The method of claim 21 wherein said composition further comprises an
adjuvant covalently attached to said polypeptide and wherein release of said
adjuvant
from said composition is controlled by said polypeptide.
t8
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Abstract
A composition comprising a polypeptide and propoxyphene covalently attached
to the polypeptide. Also provided is a method for delivery of propoxyphene to
a patient
comprising administering to the patient a composition comprising a polypeptide
and
propoxyphene covalently attached to the polypeptide: Also provided is a method
for
protecting propoxyphene from degradation comprising covalently attaching it to
a
polypeptide. Also provided is a method for controlling release of propoxyphene
from a
composition comprising covalently attaching it to the polypeptide.
19
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A NOVEL PHARMACEUTICAL COMPOUND CONTAI1~TING
ACETYLSALICYLIC ACID AND METHODS OF MAKING AND USING SAME
FIELD OF THE INVENTION
The present invention relates to a novel pharmaceutical compound that
comprises
a polypeptide that is preferably covalently attached to acetylsalicylic acid,
as well as
methods for protecting and administering acetylsalicylic acid. This novel
compound,
referred to as a CARRIERWAVETM Molecular Analogue (CMA), has the benefit of
taking a known effective pharmaceutical agent that is both well studied and
occupies a
1o known segment of the pharmaceutical market, and combining it with a carrier
compound
that enhances the usefulness of the pharmaceutical agent without compromising
its
pharmaceutical effectiveness.
BACKGROUND OF THE INVENTION
Acetylsalicylic acid is a known pharmaceutical agent that is used in the
treatment
of minor aches and pains. It is both commercially available and readily
manufactured
using published synthetic schemes by those of ordinary skill in the art.
The novel pharmaceutical compound of the present invention is useful in
accomplishing one or more of the following goals: enhancement of the chemical
stability
of the original compound; alteration of the release profile of an orally
administered
2o product; enhanced digestion or absorbtion; targeted delivery to particular
tissue/cell type;
and provision for an oral dosage form when none exists. The novel
pharmaceutical
compound may contain one or more of the following: another active
pharmaceutical
agent, an adjuvant, or an inhibitor.
Active agent delivery systems are often critical for the effective delivery of
a
biologically active agent (active agent) to the appropriate target. The
importance of these
systems becomes magnified when patient compliance and active agent stability
are taken
under consideration. For instance, one would expect patient compliance to
increase
markedly if an active agent is administered orally in lieu of an injection or
another
invasive technique. Increasing the stability of the active agent, such as
prolonging shelf
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life or survival in the stomach, will assure dosage reproducibility and
perhaps even
reduce the number of dosages required which could improve patient compliance.
Absorption of an orally administered active agent is often blocked by the
harshly
acidic stomach milieu, powerful digestive enzymes in the GI tract,
permeability of
cellular membranes and transport across lipid bilayers. Incorporating
adjuvants such as
resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance
permeability of
cellular membranes. Microencapsulating active agents using protenoid
microspheres,
liposomes or polysaccharides have been effective in abating enzyme degradation
of the
active agent. Enzyme inhibiting adjuvants have also been used to prevent
enzyme
degradation. Enteric coatings have been used as a protector of pharmaceuticals
in the
stomach.
Active agent delivery systems also provide the ability to control the release
of the
active agent. For example, formulating diazepam with a copolymer of glutamic
acid and
aspartic acid enables a sustained release of the active agent. As another
example,
copolymers of lactic acid and glutaric acid are used to provide timed release
of human
growth hormone. A wide range of pharmaceuticals purportedly provide sustained
release
through microencapsulation of the active agent in amides of dicarboxylic
acids, modified
amino acids or thermally condensed amino acids. Slow release rendering
additives can
also be intermixed with a large array of active agents in tablet formulations.
Each of these technologies imparts enhanced stability and time-release
properties
to active agent substances. Unfortunately, these technologies suffer from
several
shortcomings. Incorporation of the active agent is often dependent on
diffusion into the
microencapsulating matrix, which may not be quantitative and may complicate
dosage
reproducibility. In addition, encapsulated drugs rely on diffusion out of the
matrix, which
is highly dependant on the water solubility of the active agent. Conversely,
water-soluble
microspheres swell by an infinite degree and, unfortunately, may release the
active agent
in bursts with little active agent available for sustained release.
Furthermore, in some
technologies, control of the degradation process required for active agent
release is
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unreliable. For example, an enterically coated active agent depends on pH to
release the
active agent and, as such, is difficult to control the rate of release.
In the past, use has been made of amino acid side chains of polypeptides as
pendant groups to which active agents can be attached. These technologies
typically
require the use of spacer groups between the amino acid pendant group and the
active
agent. The peptide-drug conjugates of this class of drug delivery system rely
on enzymes
in the bloodstream for the release of the drug and, as such, are not used for
oral
administration. Examples of timed and targeted release of injectable or
subcutaneous
pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer,
to the
1o gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard,
via a peptide
spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been
covalently
attached directly to the beta carboxylate of polyaspartic acid without a
spacer group.
This prodrug formulation was designed as a colon-specific drug delivery system
where
the drug is released by bacterial hydrolytic enzymes residing in the large
intestines. The
released dexamethasone active agent, in turn, was targeted to treat large
bowel disorders
and was not intended to be absorbed into the bloodstream. Yet another
technology
combines the advantages of covalent drug attachment with liposome formation
where the
active ingredient is attached to highly ordered lipid films (known as HARs)
via a peptide
linker. Thus, there has been no drug delivery system, heretofore reported,
that
2o incorporates the concept of attaching an active ingredient to a polypeptide
pendant group
with its targeted delivery into the bloodstream via oral administration.
It is also important to control the molecular weight, molecular size and
particle
size of the active agent delivery system. Variable molecular weights have
unpredictable
diffusion rates and pharmacokinetics. High molecular weight carriers are
digested slowly
or late, as in the case of naproxen-linked dextran, which is digested almost
exclusively in
the colon by bacterial enzymes. High molecular weight microspheres usually
have high
moisture content which may present a problem with water labile active
ingredients.
Particle size not only becomes a problem with injectable drugs, as in the HAR
application, but absorption through the brush-border membrane of the
intestines is limited
to less than 5 microns.
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SUMMARY OF THE INVENTION
The present invention provides covalent attachment of the active agent
(acetylsalicylic acid) to a polymer of peptides or amino acids. The invention
is '
distinguished from the above mentioned technologies by virtue of covalently
attaching
acetylsalicylic acid to the N-terminus, the C-terminus or directly to the
amino acid side
chain of an oligopeptide or polypeptide, also referred to herein as a carrier
peptide. In
certain applications, the polypeptide will stabilize the active agent,
primarily in the
stomach, through conformational protection. In these applications, delivery of
the active
agent is controlled, in part, by the kinetics of unfolding of the carrier
peptide. Upon entry
into the upper intestinal tract, indigenous enzymes release the active
ingredient for
absorption by the body by selectively hydrolyzing the peptide bonds of the
carrier
peptide. This enzymatic action introduces a second order sustained release
mechanism.
Alternatively, the present invention provides a pharmaceutical composition
comprising acetylsalicylic acid microencapsulated by a polypeptide.
t 5 The invention provides a composition comprising a polypeptide and
acetylsalicylic acid covalently attached to the polypeptide. Preferably, the
polypeptide is
(i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally
occurring amino
acids, (iii) a heteropolymer of two or more naturally occurring amino acids,
(iv) a
homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more
synthetic
2o amino acids or (vi) a heteropolymer of one or more naturally occurring
amino acids and
one or more synthetic amino acids.
acetylsalicylic acid preferably is covalently attached to a side chain, the N-
terminus or the C-terminus of the polypeptide. In a preferred embodiment, the
active
agent is a carboxylic acid and is covalently attached to the N-terminus of the
polypeptide.
25 In another preferred embodiment, the active agent is an amine and is
covalently attached
to the C-terminus of the polypeptide. In another preferred embodiment, the
active agent
is an alcohol and is covalently attached to the C-terminus of the polypeptide.
In yet
another preferred embodiment, the active agent is an alcohol and is covalently
attached to
the N-terminus of the polypeptide.
4
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The composition of the invention can also include one or more of a
microencapsulating agent, an adjuvant and a pharmaceutically acceptable
excipient. The
microencapsulating agent can be selected from polyethylene glycol (PEG), an
amino
acid, a sugar and a salt. When an adjuvant is included in the composition, the
adjuvant
preferably activates an intestinal transporter.
Preferably, the composition of the invention is in the form of an ingestable
tablet,
an intravenous preparation or an oral suspension. The active agent can be
conformationally protected by folding of the polypeptide about the active
agent. In
another embodiment, the polypeptide is capable of releasing the active agent
from the
composition in a pH-dependent manner.
The invention also provides a method for protecting acetylsalicylic acid from
degradation comprising covalently attaching it to a polypeptide.
The invention also provides a method for delivering acetylsalicylic acid to a
patient, the patient being a human or a non-human animal, comprising
administering to
the patient a composition comprising a polypeptide and an active agent
covalently
attached to the polypeptide. In a preferred embodiment, acetylsalicylic acid
is released
from the composition by an enzyme-catalyzed release. In another preferred
embodiment,
acetylsalicylic acid is released in a time-dependent manner based on the
pharmacokinetics of the enzyme-catalyzed release. In another preferred
embodiment, the
2o composition further comprises a microencapsulating agent and
acetylsalicylic acid is
released from the composition by dissolution of the microencapsulating agent.
In another
preferred embodiment, acetylsalicylic acid is released from the composition by
a pH-
dependent unfolding of the polypeptide. In another preferred embodiment,
acetylsalicylic
acid is released from the composition in a sustained release. In yet another
preferred
embodiment, the composition further comprises an adjuvant covalently attached
to the
polypeptide and release of the adjuvant from the composition is controlled by
the
polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drug
conjugate for biphasic release of active ingredients.
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The invention also provides a method for preparing a composition comprising a
polypeptide and an active agent covalently attached to the polypeptide. The
method
comprises the steps of:
(a) attaching acetylsalicylic acid to a side chain of an amino acid to form an
active
agendamino acid complex;
(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA)
from the active agent/amino acid complex; and
(c) polymerizing the active agent/amino acid complex N-carboxyanhydride
(NCA).
In a preferred embodiment, steps (a) and (b) are repeated prior to step (c)
with a
second active agent. When steps (a) and (b) are repeated prior to step (c)
with a second
agent, acetylsalicylic acid and a second active agent can be copolymerized in
step (c). In
another preferred embodiment, the amino acid is glutamic acid and the active
agent is
released from the glutamic acid as a dimer upon a hydrolysis of the
polypeptide and
wherein the active agent is released from the glutamic acid by coincident
intramolecular
transamination. In another preferred embodiment, the glutamic acid is replaced
by an
amino acid selected from the group consisting of aspartic acid, arginine,
asparagine,
cysteine, lysine, threonine, and serine, and wherein the active agent is
attached to the side
chain of the amino acid to form an amide, a thioester, an ester, an ether, a
urethane, a
carbonate, an anhydride or a carbamate. In yet another preferred embodiment,
the
glutamic acid is replaced by a synthetic amino acid with a pendant group
comprising an
amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.
It is to be understood that both the foregoing general description and the
following detailed description are exemplary, but are not restrictive, of the
invention.
The general applications of this invention to other active pharmaceutical
agents is
described in U.S. Patent Application Serial Number 09/642,820, filed August
22, 2000,
incorporated herein by reference.
DETAILED DESCRIPTION OF INVENTION
The present invention provides several benefits for active agent delivery.
First,
the invention can stabilize acetylsalicylic acid and prevent its digestion in
the stomach.
6
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In addition, the pharmacologic effect can be prolonged by delayed release of
acetylsalicylic acid. Furthermore, active agents can be combined to produce
synergistic
effects. Also, absorption of the active agent in the intestinal tract can be
enhanced. The
invention also allows targeted delivery of active agents to specifics sites of
action.
The composition of the invention comprises acetylsalicylic acid covalently
attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide,
(ii) a
homopolymer of one of the twenty naturally occurring amino acids, (iii) a
heteropolymer
of two or more naturally occurnng amino acids, (iv) a homopolymer of a
synthetic amino
acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a
heteropolymer of
to one or more naturally occurring amino acids and one or more synthetic amino
acids.
Proteins, oligopeptides and polypeptides are polymers of amino acids that have
primary, secondary and tertiary structures. The secondary structure of the
protein is the
local conformation of the polypeptide chain and consists of helices, pleated
sheets and
turns. The protein's amino acid sequence and the structural constraints on the
conformations of the chain determine the spatial arrangement of the molecule.
The
folding of the secondary structure and the spatial arrangement of the side
chains
constitute the tertiary structure.
Proteins fold because of the dynamics associated between neighboring atoms on
the protein and solvent molecules. The thermodynamics of protein folding and
unfolding
2o are defined by the free energy of a particular condition of the protein
that relies on a
particular model. The process of protein folding involves, amongst other
things, amino
acid residues packing into a hydrophobic core. The amino acid side chains
inside the
protein core occupy the same volume as they do in amino acid crystals. The
folded
protein interior is therefore more like a crystalline solid than an oil drop
and so the best
model for determining forces contributing to protein stability is the solid
reference state.
The major forces contributing to the thermodynamics of protein folding are Van
der Waals interactions, hydrogen bonds, electrostatic interactions,
configurational
entropy and the hydrophobic effect. Considering protein stability, the
hydrophobic effect
refers to the energetic consequences of removing apolar groups from the
protein interior
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and exposing them to water. Comparing the energy of amino acid hydrolysis with
protein unfolding in the solid reference state, the hydrophobic effect is the
dominant
force. Hydrogen bonds are established during the protein fold process and
intramolecular
bonds are formed at the expense of hydrogen bonds with water. Water molecules
are
"pushed out" of the packed, hydrophobic protein core. All of these forces
combine and
contribute to the overall stability of the folded protein where the degree to
which ideal
packing occurs determines the degree of relative stability of the protein. The
result of
maximum packing is to produce a center of residues or hydrophobic core that
has
maximum shielding from solvent.
1o Since it is likely that lipophilic drugs would reside in the hydrophobic
core of a
peptide, it would require energy to unfold the peptide before the drug can be
released.
The unfolding process requires overcoming the hydrophobic effect by hydrating
the
amino acids or achieving the melting temperature of the protein. The heat of
hydration is
a destabilization of a protein. Typically, the folded state of a protein is
favored by only
15 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at
neutral pH and
at room temperature requires chemical reagents. In fact, partial unfolding of
a protein is
often observed prior to the onset of irreversible chemical or conformation
processes.
Moreover, protein conformation generally controls the rate and extent of
deleterious
chemical reactions.
20 Conformational protection of active agents by proteins depends on the
stability of
the protein's folded state and the thermodynamics associated with the agent's
decomposition. Conditions necessary for the agent's decomposition should be
different
than for protein unfolding.
Selection of the amino acids will depend on the physical properties desired.
For
25 instance, if increase in bulk or lipophilicity is desired, then the carrier
polypeptide will be
enriched in the amino acids in the table provided below. Polar amino acids, on
the other
hand, can be selected to increase the hydrophilicity of the polypeptide.
Ionizing amino acids can be selected for pH controlled peptide unfolding.
Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the
stomach, but will
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ionize upon entry into the intestine. Conversely, basic amino acids, such as
histidine,
lysine and arginine, ionize in the stomach and are neutral in an alkaline
environment.
Other factors such as n-n interactions between aromatic residues, kinking of
the
peptide chain by addition of proline, disulfide crosslinking and hydrogen
bonding can all
be used to select the optimum amino acid sequence for a given application.
Ordering of
the linear sequence can influence how these interactions can be maximized and
is
important in directing the secondary and tertiary structures of the
polypeptide.
Furthermore, amino acids with reactive side chains (e.g., glutamic acid,
lysine,
aspartic acid, serine, threonine and cysteine) can be incorporated for
attaching multiple
active agents or adjuvants to the same carrier peptide. This is particularly
useful if a
synergistic effect between two or more active agents is desired.
As stated above, variable molecular weights of the carrier compound can have
profound effects on the active agent release kinetics. As a result, low
molecular weight
active agent delivery systems are preferred. An advantage of this invention is
that chain
length and molecular weight of the polypeptide can be optimized depending on
the level
of conformational protection desired. This property can be optimized in
concert with the
kinetics of the first order release mechanism. Thus, another advantage of this
invention is
that prolonged release time can be imparted by increasing the molecular weight
of the
carrier polypeptide. Another, significant advantage of the invention is that
the kinetics of
active agent release is primarily controlled by the enzymatic hydrolysis of
the key bond
between the carrier peptide and the active agent.
Dextran is the only polysaccharide known that has been explored as a
macromolecular Garner for the covalent binding of dnug for colon specific drug
delivery.
Generally, it was only possible to load up to 1/10 of the total drug-dextran
conjugate
weight with drug. As stated earlier, polysaccharides are digested mainly in
the colon and
drug absorption is mainly limited to the colon. As compared to dextran, this
invention
has two major advantages. First, peptides are hydrolyzed by any one of several
aminopeptidases found in the intestinal lumen or associated with the brush-
border
membrane and so active agent release and subsequent absorption can occur in
the
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jejunum or the ileum. Second, the molecular weight of the carrier molecule can
be
controlled and, thus, active agent loading can also be controlled.
As a practical example, the following table lists the molecular weights of
lipophilic amino acids (less one water molecule) and selected analgesics and
vitamins.
TABLE
Amino acid MW Active agent MW
Glycine 57 Acetaminophen 151
Alanine 71 Vitamin B6 (Pyroxidine) 169
Valine 99 Vitamin C (Ascorbic acid) 176
Leucine 113 Aspirin 180
Isoleucine 113 Ibuprofen 206
Phenylalanine147 Retinoic acid 300
Tyrosine 163 Vitamin BZ (Riboflavin) 376
Vitamin D2 397
Vitamin E (Tocopherol) 431
Lipophilic amino acids are preferred because conformational protection through
the
stomach is important for the selected active agents, which were selected based
on ease of
covalent attachment to an oligopeptide. Eighteen was subtracted from the amino
acid's
molecular weight so that their condensation into a polypeptide is considered.
For
to example, a decamer of glycine (MW=588) linked to aspirin would have a total
molecular
weight of 750 and aspirin would represent 24% of the total weight of the
active agent
delivery composition or over two times the maximum drug loading for dextran.
This is
only for an N- or C- terminus application, for those active agents attached to
pendant
groups of decaglutamic acid, for instance, .a drug with a molecular weight of
180 could
conceivably have a loading of 58%, although this may not be entirely
practical.
The alcohol, amine or carboxylic acid group of an active agent may be
covalently
attached to the N-terminus, the C-terminus or the side chain of the
oligopeptide or
polypeptide. The location of attachment depends somewhat on the functional
group
selection. For instance, if the active drug is a carboxylic acid (e.g.,
aspirin) then the N-
2o terminus of the oligopeptide is the preferred point of attachment. If the
active agent is an
amine (e.g., ampicillin), then the C-terminus is the preferred point of
attachment in order
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to achieve a stable peptide linked active agent. In both, the C- and N-
terminus examples,
the peptide is, in essence, extended by one monomeric unit forming a new
peptide bond.
If the active agent is an alcohol, then either the C-terminus or the N-
terminus is the
preferred point of attachment in order to achieve a stable composition. As in
the example
above where the alcohol, norethindrone, was covalently attached to
poly(hydroxypropylglutamine), an alcohol can be converted into an
alkylchloroformate
with phosgene. This invention, then, pertains to the reaction of this key
intermediate
with the N-terminus of the peptide carrier. The active ingredient can be
released from
the peptide carrier by intestinal peptidases.
The alcohol can be selectively bound to the gamma carboxylate of glutamic acid
and then this conjugate covalently attached to the C-terminus of the peptide
Garner.
Because the glutamic acid-drug conjugate can be considered a dimer, this
product adds
two monomeric units to the C-terminus of the peptide Garner where the glutamic
acid
moiety serves as a spacer between the peptide and the drug as shown in Fig. 4.
Intestinal
enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drug
moiety
from the peptide carrier. The newly formed free amine of the glutamic acid
residue will
then undergo an intramolecular transamination reaction, thereby, releasing the
active
agent with coincident formation of pyroglutamic acid as shown in Fig. 5.
Alternatively,
the glutamic acid-drug dimer can be converted into the gamma ester of glutamic
acid N-
2o carboxyanhydride. This intermediate can then be polymerized, as described
above, using
any suitable initiator as shown in Fig. 4. The product of this polymerization
is
polyglutamic acid with active ingredients attached to multiple pendant groups.
Hence,
maximum drug loading of the carrier peptide can be achieved. In addition,
other amino
acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to
impart
specific properties to the drug delivery system.
The invention also provides a method of imparting the same mechanism of action
for other polypeptides containing functional side chains. Examples include,
but are not
limited to, polylysine, polyasparagine, polyarginine, polyserine,
polycysteine,
polytyrosine, polythreonine and polyglutamine. The mechanism can translate to
these
polypeptides through a spacer or linker on the pendant group, which is
terminated,
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preferably, by the glutamic acid-drug dimer. This Garner peptide-drug
conjugate is
distinguished from the prior art by virtue of the fact that the primary
release of the drug
moiety relies on peptidases and not on esterases. Alternatively, the active
agent can be
attached directly to the pendant group where some other indigenous enzymes in
the
alimentary tract can affect release.
The active agent can be covalently attached to the N-terminus, the C-terminus
or
the side chain of the polypeptide using known techniques. Examples of linking
organic
compounds to the N-terminus type of a peptide include, but are not limited to,
the
attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides
and 1,3-
dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example,
there are
known techniques for forming peptide linked biotin and peptide linked
acridine.
In the present invention, acetylsalicylic acid is covalently attached to the
polypeptide via the hydroxy group.
The polypeptide carrier can be prepared using conventional techniques. A
preferred technique is copolymerization of mixtures of amino acid N-
carboxyanhydrides.
Alternatively, if a specific sequence is desired, a solid state automated
peptide synthesizer
can be used.
The addition of stabilizers to the composition has the potential of
stabilizing the
polypeptide further. Stabilizers such as sugar, amino acids, polyethylene
glycol (PEG)
2o and salts have been shown to prevent protein unfolding. In another
embodiment of the
invention, a pre-first order release of the active agent is imparted by
microencapsulating
the Garner polypeptide-active agent conjugate in a polysaccharide, amino acid
complex,
PEG or salts.
There is evidence that hydrophilic compounds are absorbed through the
intestinal
epithelia efficiently via specialized transporters. The entire membrane
transport system is
intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one
can expect
that excitation of the membrane transport system will involve some sort of
specialized
adjuvant resulting in localized delivery of active agents. There are seven
known
12
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intestinal transport systems classified according to the physical properties
of the
transported substrate. They include the amino acid, oligopeptide, glucose,
monocarboxic
acid, phosphate, bile acid and the P-glycoprotein transport systems and each
has its own
associated mechanism of transport. The mechanisms can depend on hydrogen ions,
sodium ions, binding sites or other cofactors. The invention also allows
targeting the
mechanisms for intestinal epithelial transport systems to facilitate
absorption of active
agents.
In another embodiment of the invention, the composition includes one or more
adjuvants to enhance the bioavailability of the active agent. Addition of an
adjuvant is
particularly preferred when using an otherwise poorly absorbed active agent.
Suitable
adjuvants, for example, include: papain, which is a potent enzyme for
releasing the
catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which
activate
enzymes in the BBM; and bile acids, which have been attached to peptides to
enhance
absorption of the peptides.
Preferably, the resultant peptide-acetylsalicylic acid conjugate is formulated
into a
tablet using suitable excipients and can either be wet granulated or dry
compressed.
Compositions of the invention are, in essence, the formation of amides from
acids
and amines and can be prepared by the following examples.
Acid/N-terminus conjugation
An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to
0°C. The solution can then be treated with diisopropylcarbodiimide and
hydroxybenzotriazole followed by the amine peptide Garner. The reaction can
then be
stirred for several hours at room temperature, the urea by-product filtered
off, the product
precipitated out in ether and purified using gel permeation chromatography
(GPC) or
dialysis.
Amine/C-terminus conjugation
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The peptide carrier can be dissolved in DMF under nitrogen and cooled to
0°C.
The solution can then be treated with diisopropylcarbodiimide and
hydroxybenzotriazole
followed by the amine bioactive agent. The reaction can then be stirred for
several hours
at room temperature, the urea by-product filtered off, the product
precipitated out in ether
and purified using GPC or dialysis.
Alcohol/N-Terminus Conjugation
In the following example the combination of the alcohol with triphosgene
produces a chloroformate, which when reacted with the N-terminus of the
peptide
produces a carbamate. Pursuant to this, an alcohol bioactive agent can be
treated with
to triphosgene in dry DMF under nitrogen. The suitably protected peptide
carrier is then
added slowly and the solution stirred at room temperature for several hours.
The product
is then precipitated out in ether. The crude product is suitably deprotected
and purified
using GPC.
Other solvents, activating agents, cocatalysts and bases can be used. Examples
of
15 other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or
chlorinated
solvents such as chloroform. Examples of other activating agents include
dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst
is N-
hydroxysuccinimide. Examples of bases include pyrrolidinopyridine,
dimethylaminopyridine, triethylamine or tributylamine.
2o Preparation of Alkyl Glutamate
There have been over 30 different Y alkyl glutamates prepared any one of which
may be suitable for the drug alcohol of choice. For example, a suspension of
glutamic
acid, the alcohol and concentrated hydrochloric acid can be prepared and
heated for
several hours. The y alkyl glutamate product can be precipitated out in
acetone, filtered,
25 dried and recrystallized from hot water.
14
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y~Alkyl GlutamatelC-Terminus Conjugation
The peptide carrier can be dissolved in DMF under nitrogen and cooled to
0°C.
The solution can then be treated with diisopropylcarbodiimide and
hydroxybenzotriazole
followed by the y alkyl glutamate bioactive agent. The reaction can then be
stirred for
several hours at room temperature, the urea by-product filtered off, the
product
precipitated out in ether and purified using GPC or dialysis.
Preparation of Alkyl Glutamate-NCA
Y Alkyl glutamate can be suspended in dry THF where triphosgene is added and
the mixture refluxed under a nitrogen atmosphere until the mixture becomes
homogenous. The solution can be poured into heptane to precipitate the NCA
product,
which is filtered, dried and recrystallized from a suitable solvent.
Preparation of Poly[~-Alkyl Glutamate]
y Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of
a primary amine can be added to the solution until it becomes viscous
(typically
overnight). The product can be isolated from the solution by pouring it into
water and
filtering. The product can be purified using GPC or dialysis.
Although illustrated and described above with reference to specific
embodiments,
the invention is nevertheless not intended to be limited to the details shown.
Rather,
various modifications may be made in the details within the scope and range of
equivalents of the claims and without departing from the spirit of the
invention.
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CLAIMS
What is claimed is:
1. A pharmaceutical composition comprising:
a polypeptide; and
acetylsalicylic acid covalently attached to said polypeptide.
2. The composition of claim 1 wherein said polypeptide is an oligopeptide.
3. The composition of claim 1 wherein said polypeptide is a homopolymer of a
naturally occurring amino acid.
4. The composition of claim 1 wherein said polypeptide is a heteropolymer of
l0 two or more naturally occurring amino acids.
5. The composition of claim 1 wherein said polypeptide is a homopolymer of a
synthetic amino acid.
6. The composition of claim 1 wherein said polypeptide is a heteropolymer of
two or more synthetic amino acids.
7. The composition of claim 1 wherein said polypeptide is a heteropolymer of
one or more naturally occurring amino acids and one or more synthetic amino
acids.
8. The composition of claim 1 wherein acetylsalicylic acid is covalently
attached
to a side chain, the N-terminus or the C-terminus of said polypeptide.
9. The composition of claim 1 further comprising a microencapsulating agent.
10. The composition of claim 9 wherein said microencapsulating agent is
selected from the group consisting of polyethylene glycol (PEG), an amino
acid, a sugar
and a salt.
11. The composition of claim 1 further comprising an adjuvant.
16
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12. The composition of claim 11 wherein said adjuvant activates an intestinal
transporter.
13. The composition of claim 1 further comprising a pharmaceutically
acceptable
excipient.
14. The composition of claim 1 wherein said composition is in the form of an
ingestable tablet.
15. The composition of claim 1 wherein said composition is in the form of an
intravenous preparation.
16. The composition of claim 1 wherein said composition is in the form of an
oral suspension.
17. The composition of claim 1 wherein acetylsalicylic acid is
conformationally
protected by folding of said polypeptide about said active agent.
18. The composition of claim 1 wherein said polypeptide is capable of
releasing
acetylsalicylic acid from said composition in a pH-dependent manner.
19. A method for protecting acetylsalicylic acid from degradation comprising
covalently attaching said active agent to a polypeptide.
20. A method for controlling release of acetylsalicylic acid from a
composition
wherein said composition comprises a polypeptide, said method comprising
covalently
attaching acetylsalicylic acid to said polypeptide.
21. A method for delivering acetylsalicylic acid to a patient comprising
administering to said patient a composition comprising:
a polypeptide; and
acetylsalicylic acid covalently attached to said polypeptide.
22. The method of claim 21 wherein acetylsalicylic acid is released from said
composition by an enzyme-catalyzed release.
t7
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23. The method of claim 21 wherein acetylsalicylic acid is released from said
composition by a pH-dependent unfolding of said polypeptide.
24. The method of claim 21 wherein said active agent is released from said
composition in a sustained release.
25. The method of claim 21 wherein said composition further comprises an
adjuvant covalently attached to said polypeptide and wherein release of said
adjuvant
from said composition is controlled by said polypeptide.
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Abstract
A composition comprising a polypeptide and acetylsalicylic acid covalently
attached to the polypeptide. Also provided is a method for delivery of
acetylsalicylic
acid to a patient comprising administering to the patient a composition
comprising a
polypeptide and acetylsalicylic acid covalently attached to the polypeptide.
Also
provided is a method for protecting acetylsalicylic acid from degradation
comprising
covalently attaching it to a polypeptide. Also provided is a method for
controlling release
of acetylsalicylic acid from a composition comprising covalently attaching it
to the
polypeptide.
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A NOVEL PHARMACEUTICAL COMPOUND CONTAINING ACITRETIN
AND METHODS OF MAKING AND USING SAME
FIELD OF THE INVENTION
The present invention relates to a novel pharmaceutical compound that
comprises
a polypeptide that is preferably covalently attached to acitretin, as well as
methods for
protecting and administering acitretin. This novel compound, referred to as a
CARRIERWAVETM Molecular Analogue (CMA), has the benefit of taking a known
effective pharmaceutical agent that is both well studied and occupies a known
segment of
1o the pharmaceutical market, and combining it with a Garner compound that
enhances the
usefulness of the pharmaceutical agent without compromising its pharmaceutical
effectiveness.
BACKGROUND OF THE INVENTION
Acitretin is a known pharmaceutical agent that is used in the treatment of
15 psoriasis. Its chemical name is (all-E)-9-(4-methoxy-2,3,6-trimethylphenyl)-
3,7-
dimethyl-2,4,6,8-nonatetraenoic acid. It is both commercially available and
readily
manufactured using published synthetic schemes by those of ordinary skill in
the art. Its
structure is:
0
c0
0
2o The novel pharmaceutical compound of the present invention is useful in
accomplishing one or more of the following goals: enhancement of the chemical
stability
of the original compound; alteration of the release profile of an orally
administered
product; enhanced digestion or absorption; targeted delivery to particular
tissue/cell type;
and provision for an oral dosage form when none exists. The novel
pharmaceutical
25 compound may contain one or more of the following: another active
pharmaceutical
agent, an adjuvant, or an inhibitor.
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Active agent delivery systems are often critical for the effective delivery of
a
biologically active agent (active agent) to the appropriate target. The
importance of these
systems becomes magnified when patient compliance and active agent stability
are taken
under consideration. For instance, one would expect patient compliance to
increase
markedly if an active agent is administered orally in lieu of an injection or
another
invasive technique. Increasing the stability of the, active agent, such as
prolonging shelf
life or survival in the stomach, will assure dosage reproducibility and
perhaps even
reduce the number of dosages required which could improve patient compliance.
Absorption of an orally administered active agent is often blocked by the
harshly
1o acidic stomach milieu, powerful digestive enzymes in the GI tract,
permeability of
cellular membranes and transport across lipid bilayers. Incorporating
adjuvants such as
resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance
permeability of
cellular membranes. Microencapsulating active agents using protenoid
microspheres,
liposomes or polysaccharides have been effective in abating enzyme degradation
of the
15 active agent. Enzyme inhibiting adjuvants have also been used to prevent
enzyme
degradation. Enteric coatings have been used as a protector of pharmaceuticals
in the
stomach.
Active agent delivery systems also provide the ability to control the release
of the
active agent. For example, formulating diazepam with a copolymer of glutamic
acid and
2o aspartic acid enables a sustained release of the active agent. As another
example,
copolymers of lactic acid and glutaric acid are used to provide timed release
of human
growth hormone. A wide range of pharmaceuticals purportedly provide sustained
release
through microencapsulation of the active agent in amides of dicarboxylic
acids, modified
amino acids or thermally condensed amino acids. Slow release rendering
additives can
25 also be intermixed with a large array of active agents in tablet
formulations.
Each of these technologies imparts enhanced stability and time-release
properties
to active agent substances. Unfortunately, these technologies suffer from
several
shortcomings. Incorporation of the active agent is often dependent on
diffusion into the
microencapsulating matrix, which may not be quantitative and may complicate
dosage
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reproducibility. In addition, encapsulated drugs rely on diffusion out of the
matrix, which
is highly dependant on the water solubility of the active agent. Conversely,
water-soluble
microspheres swell by an infinite degree and, unfortunately, may release the
active agent
in bursts with little active agent available for sustained release.
Furthermore, in some
technologies, control of the degradation process required for active agent
release is
unreliable. For example, an enterically coated active agent depends on pH to
release the
active agent and, as such, is difficult to control the rate of release.
In the past, use has been made of amino acid side chains of polypeptides as
pendant groups to which active agents can be attached. These technologies
typically
require the use of spacer groups between the amino acid pendant group and the
active
agent. The peptide-drug conjugates of this class of drug delivery system rely
on enzymes
in the bloodstream for the release of the drug and, as such, are not used for
oral
administration. Examples of timed and targeted release of injectable or
subcutaneous
pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer,
to the
gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a
peptide
spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been
covalently
attached directly to the beta carboxylate of polyaspartic acid without a
spacer group.
This prodrug formulation was designed as a colon-specific drug delivery system
where
the drug is released by bacterial hydrolytic enzymes residing in the large
intestines. The
released dexamethasone active agent, in turn, was targeted to treat large
bowel disorders
and was not intended to be absorbed into the bloodstream. Yet another
technology
combines the advantages of covalent drug attachment with liposome formation
where the
active ingredient is attached to highly ordered lipid films (known as HARs)
via a peptide
linker. Thus, there has been no drug delivery system, heretofore reported,
that
incorporates the concept of attaching an active ingredient to a polypeptide
pendant group
with its targeted delivery into the bloodstream via oral administration.
It is also important to control the molecular weight, molecular size and
particle
size of the active agent delivery system. Variable molecular weights have
unpredictable
diffusion rates and pharmacokinetics. High molecular weight carriers are
digested slowly
or late, as in the case of naproxen-linked dextran, which is digested almost
exclusively in
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the colon by bacterial enzymes. High molecular weight microspheres usually
have high
moisture content which may present a problem with water labile active
ingredients.
Particle size not only becomes a problem with injectable drugs, as in the HAR
application, but absorption through the brush-border membrane of the
intestines is limited
to less than 5 microns.
SUMMARY OF THE INVENTION
The present invention provides covalent attachment of the active agent
(acitretin)
to a polymer of peptides or amino acids. The invention is distinguished from
the above-
mentioned technologies by virtue of covalently attaching acitretin to the N-
terminus, the
1o C-terminus or directly to the amino acid side chain of an oligopeptide or
polypeptide,
also referred to herein as a carrier peptide. In certain applications, the
polypeptide will
stabilize the active agent, primarily in the stomach, through conformational
protection.
In these applications, delivery of the active agent is controlled, in part, by
the kinetics of
unfolding of the carrier peptide. Upon entry into the upper intestinal tract,
indigenous
15 enzymes release the active ingredient for absorption by the body by
selectively
hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action
introduces a
second order sustained release mechanism.
Alternatively, the present invention provides a pharmaceutical composition
comprising acitretin microencapsulated by a polypeptide.
2o The invention provides a composition comprising a polypeptide and acitretin
covalently attached to the polypeptide. Preferably, the polypeptide is (i) an
oligopeptide,
(ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii)
a
heteropolymer of two or more naturally occurring amino acids, (iv) a
homopolymer of a
synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids
or (vi) a
25 heteropolymer of one or more naturally occurring amino acids and one or
more synthetic
amino acids.
Acitretin preferably is covalently attached to a side chain, the N-terminus or
the
C-terminus of the polypeptide. In a preferred embodiment, the active agent is
a
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carboxylic acid and is covalently attached to the N-terminus of the
polypeptide. In
another preferred embodiment, the active agent is an amine and is covalently
attached to
the C-terminus of the polypeptide. In another preferred embodiment, the active
agent is
an alcohol and is covalently attached to the C-terminus of the polypeptide. In
yet another
prefenred embodiment, the active agent is an alcohol and is covalently
attached to the N-
terminus of the,polypeptide.
The composition of the invention can also include one or more of a
microencapsulating agent, an adjuvant and a pharmaceutically acceptable
excipient. The
microencapsulating agent can be selected from polyethylene glycol (PEG), an
amino
acid, a sugar and a salt. When an adjuvant is included in the composition, the
adjuvant
preferably activates an intestinal transporter.
Preferably, the composition of the invention is in the form of an ingestable
tablet,
an intravenous preparation or an oral suspension. The active agent can be
conformationally protected by folding of the polypeptide about the active
agent. In
another embodiment, the polypeptide is capable of releasing the active agent
from the
composition in a pH-dependent manner.
The invention also provides a method for protecting acitretin from degradation
comprising covalently attaching it to a polypeptide.
The invention also provides a method for delivering acitretin to a patient,
the
2o patient being a human or a non-human animal, comprising administering to
the patient a
composition comprising a polypeptide and an active agent covalently attached
to the
polypeptide. In a preferred embodiment, acitretin is released from the
composition by an
enzyme-catalyzed release. In another preferred embodiment, acitretin is
released in a
time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed
release.
In another preferred embodiment, the composition further comprises a
microencapsulating agent and acitretin is released from the composition by
dissolution of
the microencapsulating agent. In another preferred embodiment, acitretin is
released
from the composition by a pH-dependent unfolding of the polypeptide. In
another
preferred embodiment, acitretin is released from the composition in a
sustained release.
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In yet another preferred embodiment, the composition further comprises an
adjuvant
covalently attached to the polypeptide and release of the adjuvant from the
composition is
controlled by the polypeptide. The adjuvant can be microencapsulated into a
carrier
peptide-drug conjugate for biphasic release of active ingredients.
The invention also provides a method for preparing a composition comprising a
polypeptide and an active agent covalently attached to the polypeptide. The
method
comprises the steps of:
(a) attaching acitretin to a side chain of an amino acid to form an active
agent/amino acid complex;
(b) forming an active agendamino acid complex N-carboxyanhydride (NCA)
from the active agent/amino acid complex; and
(c) polymerizing the active agent/amino acid complex N-carboxyanhydride
(NCA).
In a preferred embodiment, steps (a) and (b) are repeated prior to step (c)
with a
second active agent. When steps (a) and (b) are repeated prior to step (c)
with a second
agent, acitretin and a second active agent can be copolymerized in step (c).
In another
preferred embodiment, the amino acid is glutamic acid and the active agent is
released
from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and
wherein the
active agent is released from the glutamic acid by coincident intramolecular
transamination. In another preferred embodiment, the glutamic acid is replaced
by an
amino acid selected from the group consisting of aspartic acid, arginine,
asparagine,
cysteine, lysine, threonine, and serine, and wherein the active agent is
attached to the side
chain of the amino acid to form an amide, a thioester, an ester, an ether, a
urethane, a
carbonate, an anhydride or a carbamate. In yet another preferred embodiment,
the
glutamic acid is replaced by a synthetic amino acid with a pendant group
comprising an
amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.
It is to be understood that both the foregoing general description and the
following detailed description are exemplary, but are not restrictive, of the
invention.
The general applications of this invention to other active pharmaceutical
agents is
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described in U.S. Patent Application Serial Number 09/642,820, filed August
22, 2000,
incorporated herein by reference.
DETAILED DESCRIPTION OF INVENTION
The present invention provides several benefits for active agent delivery.
First,
the invention can stabilize acitretin and prevent its digestion in the
stomach. In~addition,
the pharmacologic effect can be prolonged by delayed release of acitretin.
Furthermore,
active agents can be combined to produce synergistic effects. Also, absorption
of the
active agent in the intestinal tract can be enhanced. The invention also
allows targeted
delivery of active agents to specifics sites of action.
1o The composition of the invention comprises acitretin covalently attached to
a
polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a
homopolymer of one
of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or
more
naturally occurnng amino acids, (iv) a homopolymer of a synthetic amino acid,
(v) a
heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of
one or
15 more naturally occurnng amino acids and one or more synthetic amino acids.
Proteins, oligopeptides and polypeptides are polymers of amino acids that have
primary, secondary and tertiary structures. The secondary structure of the
protein is the
local conformation of the polypeptide chain and consists of helices, pleated
sheets and
turns. The protein's amino acid sequence and the structural constraints on the
2o conformations of the chain determine the spatial arrangement of the
molecule. The
folding of the secondary structure and the spatial arrangement of the side
chains
constitute the tertiary structure.
Proteins fold because of the dynamics associated between neighboring atoms on
the protein and solvent molecules. The thermodynamics of protein folding and
unfolding
25 are defined by the free energy of a particular condition of the protein
that relies on a
particular model. The process of protein folding involves, amongst other
things, amino
acid residues packing into a hydrophobic core. The amino acid side chains
inside the
protein core occupy the same volume as they do in amino acid crystals. The
folded
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protein interior is therefore more like a crystalline solid than an oil drop
and so the best
model for determining forces contributing to protein stability is the solid
reference state.
The major forces contributing to the thermodynamics of protein folding are Van
der Waals interactions, hydrogen bonds, electrostatic interactions,
configurational
entropy and the hydrophobic effect. Considering protein stability, the
hydrophobic effect
refers to the energetic consequences of removing apolar groups from the
protein interior
and exposing them to water. Comparing the energy of amino acid hydrolysis with
protein unfolding in the solid reference state, the hydrophobic effect is the
dominant
force. Hydrogen bonds are established during the protein fold process and
intramolecular
1o bonds are formed at the expense of hydrogen bonds with water. Water
molecules are
"pushed out" of the packed, hydrophobic protein core. All of these forces
combine and
contribute to the overall stability of the folded protein where the degree to
which ideal
packing occurs determines the degree of relative stability of the protein. The
result of
maximum packing is to produce a center of residues or hydrophobic core that
has
15 maximum shielding from solvent.
Since it is likely that lipophilic drugs would reside in the hydrophobic core
of a
peptide, it would require energy to unfold the peptide before the drug can be
released.
The unfolding process requires overcoming the hydrophobic effect by hydrating
the
amino acids or achieving the melting temperature of the protein. The heat of
hydration is
2o a destabilization of a protein. Typically, the folded state of a protein is
favored by only
5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at
neutral pH and
at room temperature requires chemical reagents. In fact, partial unfolding of
a protein is
often observed prior to the onset of irreversible chemical or conformation
processes.
Moreover, protein conformation generally controls the rate and extent of
deleterious
25 chemical reactions.
Conformational protection of active agents by proteins depends on the
stability of
the protein's folded state and the thermodynamics associated with the agent's
decomposition. Conditions necessary for the agent's decomposition should be
different
than for protein unfolding.
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Selection of the amino acids will depend on the physical properties desired.
For
instance, if increase in bulk or lipophilicity is desired, then the carrier
polypeptide will be
enriched in the amino acids in the table provided below. Polar amino acids, on
the other
hand, can be selected to increase the hydrophilicity of the polypeptide.
Ionizing amino acids can be selected for pH controlled peptide unfolding.
Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the
stomach, but will
ionize upon entry into the intestine. Conversely, basic amino acids, such as
histidine,
lysine and arginine, ionize in the stomach and are neutral in an alkaline
environment.
Other factors such as n-n interactions between aromatic residues, kinking of
the
to peptide chain by addition of proline, disulfide crosslinking and hydrogen
bonding can all
be used to select the optimum amino acid sequence for a given application.
Ordering of
the linear sequence can influence how these interactions can be maximized and
is
important in directing the secondary and tertiary structures of the
polypeptide.
Furthermore, amino acids with reactive side chains (e.g., glutamic acid,
lysine,
15 aspartic acid, serine, threonine and cysteine) can be incorporated for
attaching multiple
active agents or adjuvants to the same carrier peptide. This is particularly
useful if a
synergistic effect between two or more active agents is desired.
As stated above, variable molecular weights of the carrier compound can have
profound effects on the active agent release kinetics. As a result, low
molecular weight
2o active agent delivery systems are preferred. An advantage of this invention
is that chain
length and molecular weight of the polypeptide can be optimized depending on
the level
of conformational protection desired. This property can be optimized in
concert with the
kinetics of the first order release mechanism. Thus, another advantage of this
invention is
that prolonged release time can be imparted by increasing the molecular weight
of the
25 carrier polypeptide. Another, significant advantage of the invention is
that the kinetics of
active agent,release is primarily controlled by the enzymatic hydrolysis of
the key bond
between the carrier peptide and the active agent.
9
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Dextran is the only polysaccharide known that has been explored as a
macromolecular carrier for the covalent binding of drug for colon specific
drug delivery.
Generally, it was only possible to load up to 1/10 of the total drug-dextran
conjugate
weight with drug. As stated earlier, polysaccharides are digested mainly in
the colon and
drug absorption is mainly limited to the colon. As compared to dextran, this
invention
has two major advantages. First, peptides are hydrolyzed by any one of several
aminopeptidases found in the intestinal lumen or associated with the brush-
border
membrane and so active agent release and subsequent absorption can occur in
the
jejunum or the ileum. Second, the molecular weight of the carrier molecule can
be
1o controlled and, thus, active agent loading can also be controlled.
As a practical example, the following table lists the molecular weights of
lipophilic amino acids (less one water molecule) and selected analgesics and
vitamins.
TABLE
Amino acid MW Active agent MW
Glycine 57 Acetaminophen 151
Alanine 71 Vitamin B6 (Pyroxidine) 169
Valine 99 Vitamin C (Ascorbic acid) 176
Leucine 113 Aspirin 180
Isoleucine 113 Ibuprofen 206
Phenylalanine147 Retinoic acid 300
Tyrosine 163 Vitamin B2 (Riboflavin) 376
Vitamin D2 397
Vitamin E (Tocopherol) 431
Lipophilic amino acids are preferred because conformational protection through
the
~5 stomach is important for the selected active agents, which were selected
based on ease of
covalent attachment to an oligopeptide. Eighteen was subtracted from the amino
acid's
molecular weight so that their condensation into a polypeptide is considered.
For
example, a decamer of glycine (MW=588) linked to aspirin would have a total
molecular
weight of 750 and aspirin would represent 24% of the total weight of the
active agent
2o delivery composition or over two times the maximum drug loading for
dextran. This is
only for an N- or C- terminus application, for those active agents attached to
pendant
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groups of decaglutamic acid, for instance, a drug with a molecular weight of
180 could
conceivably have a loading of 58%, although this may not be entirely
practical.
The alcohol, amine or carboxylic acid group of an active agent may be
covalently
attached to the N-terminus, the C-terminus or the side chain of the
oligopeptide or
polypeptide. The location of attachment depends somewhat on the functional
group
selection. For instance, if the active drug is a carboxylic acid (e.g.,
aspirin) then the N-
terminus of the oligopeptide is the preferred point of attachment. If the
active agent is an
amine (e.g., ampicillin), then the C-terminus is the preferred point of
attachment in order
to achieve a stable peptide linked active agent. In both, the C- and N-
terminus examples,
l0 the peptide is, in essence, extended by one monomeric unit forming a new
peptide bond.
If the active agent is an alcohol, then either the C-terminus or the N-
terminus is the
preferred point of attachment in order to achieve a stable composition. As in
the example
above where the alcohol, norethindrone, was covalently attached to
poly(hydroxypropylglutamine), an alcohol can be converted into an
alkylchloroformate
15 with phosgene. This invention, then, pertains to the reaction of this key
intermediate with
the N-terminus of the peptide carrier. The active ingredient can be released
from the
peptide carrier by intestinal peptidases.
The alcohol can be selectively bound to the gamma carboxylate of glutamic acid
and then this conjugate covalently attached to the C-terminus of the peptide
carrier.
2o Fecause the glutamic acid-drug conjugate can be considered a dimer, this
product adds
two monomeric units to the C-terminus of the peptide carrier where the
glutamic acid
moiety serves as a spacer between the peptide and the drug as shown in Fig. 4.
Intestinal
enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drug
moiety
from the peptide carrier. The newly formed free amine of the glutamic acid
residue will
25 then undergo an intramolecular transamination reaction, thereby, releasing
the active
agent with coincident formation of pyroglutamic acid as shown in Fig. 5.
Alternatively,
the glutamic acid-drug dimer can be converted into the gamma ester of glutamic
acid N-
carboxyanhydride. This intermediate can then be polymerized, as described
above, using
any suitable initiator as shown in Fig. 4. The product of this polymerization
is
3o polyglutamic acid with active ingredients attached to multiple pendant
groups. Hence,
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maximum drug loading of the carrier peptide can be achieved. In addition,
other amino
acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to
impart
specific properties to the drug delivery system.
The invention also provides a method of imparting the same mechanism of action
for other polypeptides containing functional side chains. Examples include,
but are not
limited to, polylysine, polyasparagine, polyarginine, polyserine,
polycysteine,
polytyrosine, polythreonine and polyglutamine. The mechanism can translate to
these
polypeptides through a spacer or linker on the pendant group, which is
terminated,
preferably, by the glutamic acid-drug dimer. This Garner peptide-drug
conjugate is
1o distinguished from the prior art by virtue of the fact that the primary
release of the drug
moiety relies on peptidases and not on esterases. Alternatively, the active
agent can be
attached directly to the pendant group where some other indigenous enzymes in
the
alimentary tract can affect release.
The active agent can be covalently attached to the N-terminus, the C-terminus
or
the side chain of the polypeptide using known techniques. Examples of linking
organic
compounds to the N-terminus type of a peptide include, but are not limited to,
the
attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides
and 1,3-
dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example,
there are
known techniques for forming peptide linked biotin and peptide linked
acridine.
2o In the present invention, acitretin is covalently attached to the
polypeptide via the
carboxylic acid group.
The polypeptide Garner can be prepared using conventional techniques. A
preferred technique is copolymerization of mixtures of amino acid N-
carboxyanhydrides.
Alternatively, if a specific sequence is desired, a solid state automated
peptide synthesizer
can be used.
The addition of stabilizers to the composition has the potential of
stabilizing the
polypeptide further. Stabilizers such as sugar, amino acids, polyethylene
glycol (PEG)
and salts have been shown to prevent protein unfolding. In another embodiment
of the
12
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invention, a pre-first order release of the active agent is imparted by
microencapsulating
the Garner polypeptide-active agent conjugate in a polysaccharide, amino acid
complex,
PEG or salts.
There is evidence that hydrophilic compounds are absorbed through the
intestinal
epithelia efficiently via specialized transporters. The entire membrane
transport system is
intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one
can expect
that excitation of the membrane transport system will involve some sort of
specialized
adjuvant resulting in localized delivery of active agents. There are seven
known
intestinal transport systems classified according to the physical properties
of the
to transported substrate. They include the amino acid, oligopeptide, glucose,
monocarboxic
acid, phosphate, bile acid and the P-glycoprotein transport systems and each
has its own
associated mechanism of transport. The mechanisms can depend on hydrogen ions,
sodium ions, binding sites or other cofactors. The invention also allows
targeting the
mechanisms for intestinal epithelial transport systems to facilitate
absorption of active
15 agents.
In another embodiment of the invention, the composition includes one or more
adjuvants to enhance the bioavailability of the active agent. Addition of an
adjuvant is
particularly preferred when using an otherwise poorly absorbed active agent.
Suitable
adjuvants, for example, include: papain, which is a potent enzyme for
releasing the
2o catalytic domain of aminopeptidase-N into the lumen; glycorecognizers,
which activate
enzymes in the BBM; and bile acids, which have been attached to peptides to
enhance
absorption of the peptides.
Preferably, the resultant peptide-acitretin conjugate is formulated into a
tablet
using suitable excipients and can either be wet granulated or dry compressed.
25 Compositions of the invention are, in essence, the formation of amides from
acids
and amines and can be prepared by the following examples.
13
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Acid/N-terminus conjugation
An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to
0°C. The solution can then be treated with diisopropylcarbodiimide and
hydroxybenzotriazole followed by the amine peptide Garner. The reaction can
then be
stirred for several hours at room temperature, the urea by-product filtered
off, the product
precipitated out in ether and purified using gel permeation chromatography
(GPC) or
dialysis.
AminelC-terminus conjugation
The peptide Garner can be dissolved in DNiF under nitrogen and cooled to
0°C.
~ o The solution can then be treated with diisopropylcarbodiimide and
hydroxybenzotriazole
followed by the amine bioactive agent. The reaction can then be stirred for
several hours
at room temperature, the urea by-product filtered off, and the product
precipitated out in
ether and purified using GPC or dialysis.
Alcohol/N-Terminus Conjugation
In the following example the combination of the alcohol with triphosgene
produces a chloroformate, which when reacted with the N-terminus of the
peptide
produces a carbamate. Pursuant to this, an alcohol bioactive agent can be
treated with
triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier
is then
added slowly and the solution stirred at room temperature for several hours.
The product
2o is then precipitated out in ether. The crude product is suitably
deprotected and purified
using GPC.
Other solvents, activating agents, cocatalysts and bases can be used. Examples
of
other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or
chlorinated
solvents such as chloroform. Examples of other activating agents include
dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst
is N-
hydroxysuccinimide. Examples of bases include pyrrolidinopyridine,
dimethylaminopyridine, triethylamine or tributylamine.
14
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Preparation of Alkyl Glutamate
There have been over 30 different y alkyl glutamates prepared any one of which
may be suitable for the drug alcohol of choice. For example, a suspension of
glutamic
acid, the alcohol and concentrated hydrochloric acid can be prepared and
heated for
several hours. The y alkyl glutamate product can be precipitated out in
acetone, filtered,
dried and recrystallized from hot water.
~y~-Alkyl Glutamate/C-Terminus Conjugation
The peptide carrier can be dissolved in DMF under nitrogen and cooled to
0°C.
The solution can then be treated with diisopropylcarbodiimide and
hydroxybenzotriazole
followed by the y alkyl glutamate bioactive agent. The reaction can then be
stirred for
several hours at room temperature, the urea by-product filtered off, and the
product
precipitated out in ether and purified using GPC or dialysis.
Preparation of y~Alkyl Glutamate-NCA
y Alkyl glutamate can be suspended in dry TI-iF where triphosgene is added and
the mixture refluxed under a nitrogen atmosphere until the mixture becomes
homogenous. The solution can be poured into heptane to precipitate the NCA
product,
which is filtered, dried and recrystallized from a suitable solvent.
Preparation of Poly['y Alkyl Glutamate]
y Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of
2o a primary amine can be added to the solution until it becomes viscous
(typically
overnight). The product can be isolated from the solution by pouring it into
water and
filtering. The product can be purified using GPC or dialysis.
Although illustrated and described above with reference to specific
embodiments,
the invention is nevertheless not intended to be limited to the details shown.
Rather,
various modifications may be made in the details within the scope and range of
equivalents of the claims and without departing from the spirit of the
invention.
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CLAIMS
What is claimed is:
1. A pharmaceutical composition comprising:
a polypeptide; and
acitretin covalently attached to said polypeptide.
2. The composition of claim 1 wherein said polypeptide is an oligopeptide.
3. The composition of claim 1 wherein said polypeptide is a homopolymer of a
naturally occurnng amino acid.
4. The composition of claim 1 wherein said polypeptide is a heteropolymer of
to two or more naturally occurring amino acids.
5. The composition of claim 1 wherein said polypeptide is a homopolymer of a
synthetic amino acid.
6. The composition of claim 1 wherein said polypeptide is a heteropolymer of
two or more synthetic amino acids.
7. The composition of claim 1 wherein said polypeptide is a heteropolymer of
one or more naturally occurring amino acids and one or more synthetic amino
acids.
8. The composition of claim 1 wherein acitretin is covalently attached to a
side
chain, the N-terminus or the C-terminus of said polypeptide.
9. The composition of claim 1 further comprising a microencapsulating agent.
10. The composition of claim 9 wherein said microencapsulating agent is
selected from the group consisting of polyethylene glycol (PEG), an amino
acid, a sugar
and a salt.
11. The composition of claim 1 further comprising an adjuvant.
16
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12. The composition of claim 11 wherein said adjuvant activates an intestinal
transporter.
13. The composition of claim 1 further comprising a pharmaceutically
acceptable
excipient.
14. The composition of claim 1 wherein said composition is in the form of an
ingestable tablet.
15. The composition of claim 1 wherein said composition is in the form of an
intravenous preparation.
16. The composition of claim 1 wherein said composition is in the form of an
l0 oral suspension.
17. The composition of claim 1 wherein acitretin is conformationally protected
by folding of said polypeptide about said active agent.
18. The composition of claim 1 wherein said polypeptide is capable of
releasing
acitretin from said composition in a pH-dependent manner.
19. A method for protecting acitretin from degradation comprising covalently
attaching said active agent to a polypeptide.
20. A method for controlling release of acitretin from a composition wherein
said
composition comprises a polypeptide, said method comprising covalently
attaching
acitretin to said polypeptide.
21. A method for delivering acitretin to a patient comprising administering to
said patient a composition comprising:
a polypeptide; and
acitretin covalently attached to said polypeptide.
22. The method of claim 21 wherein acitretin is released from said composition
by an enzyme-catalyzed release.
17
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23. The method of claim 21 wherein acitretin is released from said composition
by a pH-dependent unfolding of said polypeptide.
24. The method of claim 21 wherein said active agent is released from said
composition in a sustained release.
25. The method of claim 21 wherein said composition further comprises an
adjuvant covalently attached to said polypeptide and wherein release of said
adjuvant
from said composition is controlled by said polypeptide.
t8
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Abstract
A composition comprising a polypeptide and acitretin covalently attached to
the
polypeptide. Also provided is a method for delivery of acitretin to a patient
comprising
administering to the patient a composition comprising a polypeptide and
acitretin
covalently attached to the polypeptide. Also provided is a method for
protecting acitretin
from degradation comprising covalently attaching it to a polypeptide. Also
provided is a
method for controlling release of acitretin from a composition comprising
covalently
attaching it to the polypeptide.
19
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A NOVEL PHARMACEUTICAL COMPOUND CONTAIrIING
ACTIVATED PROTEIN C AND METHODS OF MAKING AND USING SAME
FIELD OF THE INVENTION
The present invention relates to a novel pharmaceutical compound that
comprises
a polypeptide that is preferably covalently attached to activated protein C,
as well as
methods for protecting and administering activated protein C. This novel
compound,
referred to as a CARRIERWAVETN' Molecular Analogue (CMA), has the benefit of
taking a known effective pharmaceutical agent that is both well studied and
occupies a
1o known segment of the pharmaceutical market, and combining it with a Garner
compound
that enhances the usefulness of the pharmaceutical agent without compromising
its
pharmaceutical effectiveness.
BACKGROUND OF THE INVENTION
Activated protein C is a known pharmaceutical agent that is used in the
treatment
15 of blood clots. Its structure is well known and it is both commercially
available and
readily manufactured using published synthetic schemes by those of ordinary
skill in the
art.
The novel pharmaceutical compound of the present invention is useful in
accomplishing one or more of the following goals: enhancement of the chemical
stability
2o of the original compound; alteration of the release profile of an orally
administered
product; enhanced digestion or absorption; targeted delivery to particular
tissue/cell type;
and provision for an oral dosage form when none exists. The novel
pharmaceutical
compound may contain one or more of the following: another active
pharmaceutical
agent, an adjuvant, or an inhibitor.
25 Active agent delivery systems are often critical for the effective delivery
of a
biologically active agent (active agent) to the appropriate target. The
importance of these
systems becomes magnified when patient compliance and active agent stability
are taken
under consideration. For instance, one would expect patient compliance to
increase
markedly if an active agent is administered orally in lieu of an injection or
another
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invasive technique. Increasing the stability of the active agent, such as
prolonging shelf
life or survival in the stomach, will assure dosage reproducibility and
perhaps even
reduce the number of dosages required which could improve patient compliance.
Absorption of an orally administered active agent is often blocked by the
harshly
acidic stomach milieu, powerful digestive enzymes in the GI tract,
permeability of
cellular membranes and transport across lipid bilayers. Incorporating
adjuvants such as
resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance
permeability of
cellular membranes. Microencapsulating active agents using protenoid
microspheres,
liposomes or polysaccharides have been effective in abating enzyme degradation
of the
to active agent. Enzyme inhibiting adjuvants have also been used to prevent
enzyme
degradation. Enteric coatings have been used as a protector of pharmaceuticals
in the
stomach.
Active agent delivery systems also provide the ability to control the release
of the
active agent. For example, formulating diazepam with a copolymer of glutamic
acid and
15 aspartic acid enables a sustained release of the active agent. As another
example,
copolymers of lactic acid and glutaric acid are used to provide timed release
of human
growth hormone. A wide range of pharmaceuticals purportedly provide sustained
release
through microencapsulation of the active agent in amides of dicarboxylic
acids, modified
amino acids or thermally condensed amino acids. Slow release rendering
additives can
20 also be intermixed with a large array of active agents in tablet
formulations.
Each of these technologies imparts enhanced stability and time-release
properties
to active agent substances. Unfortunately, these technologies suffer from
several
shortcomings. Incorporation of the active agent is often dependent on
diffusion into the
microencapsulating matrix, which may not be quantitative and may complicate
dosage
25 reproducibility. In addition, encapsulated drugs rely on diffusion out of
the matrix, which
is highly dependant on the water solubility of the active agent. Conversely,
water-soluble
microspheres swell by an infinite degree and, unfortunately, may release the
active agent
in bursts with little active agent available for sustained release.
Furthermore, in some
technologies, control of the degradation process required for active agent
release is
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unreliable. For example, an enterically coated active agent depends on pH to
release the
active agent and, as such, is difficult to control the rate of release.
In the past, use has been made of amino acid side chains of polypeptides as
pendant groups to which active agents can be attached. These technologies
typically
require the use of spacer groups between the amino acid pendant group and the
active
agent. The peptide-drug conjugates of this class of drug delivery system rely
on enzymes
in the bloodstream for the release of the drug and, as such, are not used for
oral
administration. Examples of timed and targeted release of injectable or
subcutaneous
pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer,
to the
to gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard,
via a peptide
spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been
covalently
attached directly to the beta carboxylate of polyaspartic acid without a
spacer group.
This prodrug formulation was designed as a colon-specific drug delivery system
where
the drug is released by bacterial hydrolytic enzymes residing in the large
intestines. The
15 released dexamethasone active agent, in turn, was targeted to treat large
bowel disorders
and was not intended to be absorbed into the bloodstream. Yet another
technology
combines the advantages of covalent drug attachment with liposome formation
where the
active ingredient is attached to highly ordered lipid films (known as HARs)
via a peptide
linker. Thus, there has been no drug delivery system, heretofore reported,
that
20 incorporates the concept of attaching an active ingredient to a polypeptide
pendant group
with its targeted delivery into the bloodstream via oral administration.
It is also important to control the molecular weight, molecular size and
particle
size of the active agent delivery system. Variable molecular weights have
unpredictable
diffusion rates and pharmacokinetics. High molecular weight carriers are
digested slowly
25 or late, as in the case of naproxen-linked dextran, which is digested
almost exclusively in
the colon by bacterial enzymes. High molecular weight microspheres usually
have high
moisture content which may present a problem with water labile active
ingredients.
Particle size not only becomes a problem with injectable drugs, as in the HAR
application, but absorption through the brush-border membrane of the
intestines is limited
30 to less than 5 microns.
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SUMMARY OF TIDE INVENTION
The present invention provides covalent attachment of the active agent
(activated
protein C) to a polymer of peptides or amino acids. The invention is
distinguished from
the above-mentioned technologies by virtue of covalently attaching activated
protein C to
the N-terminus, the C-terminus or directly to the amino acid side chain of an
oligopeptide
or polypeptide, also referred to herein as a Garner peptide. In certain
applications, the
polypeptide will stabilize the active agent, primarily in the stomach, through
conformational protection. In these applications, delivery of the active agent
is
controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon
entry into the
1o upper intestinal tract, indigenous enzymes release the active ingredient
for absorption by
the body by selectively hydrolyzing the peptide bonds of the carrier peptide.
This
enzymatic action introduces a second order sustained release mechanism.
Alternatively, the present invention provides a pharmaceutical composition
comprising activated protein C microencapsulated by a polypeptide.
The invention provides a composition comprising a polypeptide and activated
protein C covalently attached to the polypeptide. Preferably, the polypeptide
is (i) an
oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring
amino acids,
(iii) a heteropolymer of two or more naturally occurnng amino acids, (iv) a
homopolymer
of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino
acids or
(vi) a heteropolymer of one or more naturally occurring amino acids and one or
more
synthetic amino acids.
Activated protein C preferably is covalently attached to a side chain, the N-
terminus or the C-terminus of the polypeptide. In a preferred embodiment, the
active
agent is a carboxylic acid and is covalently attached to the N-terminus of the
polypeptide.
In another preferred embodiment, the active agent is an amine and is
covalently attached
to the C-terminus of the polypeptide. In another preferred embodiment, the
active agent
is an alcohol and is covalently attached to the C-terminus of the polypeptide.
In yet
another preferred embodiment, the active agent is an alcohol and is covalently
attached to
the N-terminus of the polypeptide.
4
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The composition of the invention can also include one or more of a
microencapsulating agent, an adjuvant and a pharmaceutically acceptable
excipient. The
microencapsulating agent can be selected from polyethylene glycol (PEG), an
amino
acid, a sugar and a salt. When an adjuvant is included in the composition, the
adjuvant
preferably activates an intestinal transporter.
Preferably, the composition of the invention is in the form of an ingestable
tablet,
an intravenous preparation or an oral suspension. The active agent can be
conformationally protected by folding of the polypeptide about the active
agent. In
another embodiment, the polypeptide is capable of releasing the active agent
from the
1o composition in a pH-dependent manner.
The invention also provides a method for protecting activated protein C from
degradation comprising covalently attaching it to a polypeptide.
The invention also provides a method for delivering activated protein C to a
patient, the patient being a human or a non-human animal, comprising
administering to
15 the patient a composition comprising a polypeptide and an active agent
covalently
attached to the polypeptide. In a preferred embodiment, activated protein C is
released
from the composition by an enzyme-catalyzed release. In another preferred
embodiment,
activated protein C is released in a time-dependent manner based on the
pharmacokinetics of the enzyme-catalyzed release. In another preferred
embodiment, the
2o composition further comprises a microencapsulating agent and activated
protein C is
released from the composition by dissolution of the microencapsulating agent.
In another
preferred embodiment, activated protein C is released from the composition by
a pH-
dependent unfolding of the polypeptide. In another preferred embodiment,
activated
protein C is released from the composition in a sustained release. In yet
another preferred
25 embodiment, the composition further comprises an adjuvant covalently
attached to the
polypeptide and release of the adjuvant from the composition is controlled by
the
polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drug
conjugate for biphasic release of active ingredients.
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The invention also provides a method for preparing a composition comprising a
polypeptide and an active agent covalently attached to the polypeptide. The
method
comprises the steps of:
(a) attaching activated protein C to a side chain of an amino acid to form an
active
agent/amino acid complex;
(b) forming an active agendamino acid complex N-carboxyanhydride (NCA)
from the active agent/amino acid complex; and
(c) polymerizing the active agendamino acid complex N-carboxyanhydride
(NCA).
t0 In a preferred embodiment, steps (a) and (b) are repeated prior to step (c)
with a
second active agent. When steps (a) and (b) are repeated prior to step (c)
with a second
agent, activated protein C and a second active agent can be copolymerized in
step (c). In
another preferred embodiment, the amino acid is glutamic acid and the active
agent is
released from the glutamic acid as a dimer upon a hydrolysis of the
polypeptide and
15 wherein the active agent is released from the glutamic acid by coincident
intramolecular
transamination. In another preferred embodiment, the glutamic acid is replaced
by an
amino acid selected from the group consisting of aspartic acid, arginine,
asparagine,
cysteine, lysine, threonine, and serine, and wherein the active agent is
attached to the side
chain of the amino acid to form an amide, a thioester, an ester, an ether, a
urethane, a
2o carbonate, an anhydride or a carbamate. In yet another preferred
embodiment, the
glutamic acid is replaced by a synthetic amino acid with a pendant group
comprising an
amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.
It is to be understood that both the foregoing general description and the
following detailed description are exemplary, but are not restrictive, of the
invention.
25 The general applications of this invention to other active pharmaceutical
agents is
described in U.S. Patent Application Serial Number 09/642,820, filed August
22, 2000,
incorporated herein by reference.
DETAILED DESCRIPTION OF INVENTION
The present invention provides several benefits for active agent delivery.
First,
30 the invention can stabilize activated protein C and prevent its digestion
in the stomach.
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In addition, the pharmacologic effect can be prolonged by delayed release of
activated
protein C. Furthermore, active agents can be combined to produce synergistic
effects.
Also, absorption of the active agent in the intestinal tract can be enhanced.
The invention
also allows targeted delivery of active agents to specifics sites of action.
The composition of the invention comprises activated protein C covalently
attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide,
(ii) a
homopolymer of one of the twenty naturally occurring amino acids, (iii) a
heteropolymer
of two or more naturally occurring amino acids, (iv) a homopolymer of a
synthetic amino
acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a
heteropolymer of
one or more naturally occurnng amino acids and one or more synthetic amino
acids.
Proteins, oligopeptides and polypeptides are polymers of amino acids that have
primary, secondary and tertiary structures. The secondary structure of the
protein is the
local conformation of the polypeptide chain and consists of helices, pleated
sheets and
turns. The protein's amino acid sequence and the structural constraints on the
conformations of the chain determine the spatial arrangement of the molecule.
The
folding of the secondary structure and the spatial arrangement of the side
chains
constitute the tertiary structure.
Proteins fold because of the dynamics associated between neighboring atoms on
the protein and solvent molecules. The thermodynamics of protein folding and
unfolding
2o are defined by the free energy of a particular condition of the protein
that relies on a
particular model. The process of protein folding involves, amongst other
things, amino
acid residues packing into a hydrophobic core. The amino acid side chains
inside the
protein core occupy the same volume as they do in amino acid crystals. The
folded
protein interior is therefore more like a crystalline solid than an oil drop
and so the best
model for determining forces contributing to protein stability is the solid
reference state.
The major forces contributing to the thermodynamics of protein folding are Van
der Waals interactions, hydrogen bonds, electrostatic interactions,
configurational
entropy and the hydrophobic effect. Considering protein stability, the
hydrophobic effect
refers to the energetic consequences of removing apolar groups from the
protein interior
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and exposing them to water. Comparing the energy of amino acid hydrolysis with
protein unfolding in the solid reference state, the hydrophobic effect is the
dominant
force. Hydrogen bonds are established during the protein fold process and
intramolecular
bonds are formed at the expense of hydrogen bonds with water. Water molecules
are
"pushed out" of the packed, hydrophobic protein core. All of these forces
combine and
contribute to the overall stability of the folded protein where the degree to
which ideal
packing occurs determines the degree of relative stability of the protein. The
result of
maximum packing is to produce a center of residues or hydrophobic core that
has
maximum shielding from solvent.
1o Since it is likely that lipophilic drugs would reside in the hydrophobic
core of a
peptide, it would require energy to unfold the peptide before the drug can be
released.
The unfolding process requires overcoming the hydrophobic effect by hydrating
the
amino acids or achieving the melting temperature of the protein. The heat of
hydration is
a destabilization of a protein. Typically, the folded state of a protein is
favored by only
~5 5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at
neutral pH and
at room temperature requires chemical reagents. In fact, partial unfolding of
a protein is
often observed prior to the onset of irreversible chemical or conformation
processes.
Moreover, protein conformation generally controls the rate and extent of
deleterious
chemical reactions.
2o Conformational protection of active agents by proteins depends on the
stability of
the protein's folded state and the thermodynamics associated with the agent's
decomposition. Conditions necessary for the agent's decomposition should be
different
than for protein unfolding.
Selection of the amino acids will depend on the physical properties desired.
For
25 instance, if increase in bulk or lipophilicity is desired, then the carrier
polypeptide will be
enriched in the amino acids in the table provided below. Polar amino acids, on
the other
hand, can be selected to increase the hydrophilicity of the polypeptide.
Ionizing amino acids can be selected for pH controlled peptide unfolding.
Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the
stomach, but will
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ionize upon entry into the intestine. Conversely, basic amino acids, such as
histidine,
lysine and arginine, ionize in the stomach and are neutral in an alkaline
environment.
Other factors such as n-n interactions between aromatic residues, kinking of
the
peptide chain by addition of proline, disulfide crosslinking and hydrogen
bonding can all
be used to select the optimum amino acid sequence for a given application.
Ordering of
the linear sequence can influence how these interactions can be maximized and
is
important in directing the secondary and tertiary structures of the
polypeptide.
Furthermore, amino acids with reactive side chains (e.g., glutamic acid,
lysine,
aspartic acid, serine, threonine and cysteine) can be incorporated for
attaching multiple
active agents or adjuvants to the same Garner peptide. This is particularly
useful if a
synergistic effect between two or more active agents is desired.
As stated above, variable molecular weights of the Garner compound can have
profound effects on the active agent release kinetics. As a result, low
molecular weight
active agent delivery systems are preferred. An advantage of this invention is
that chain
length and molecular weight of the polypeptide can be optimized depending on
the level
of conformational protection desired. This property can be optimized in
concert with the
kinetics of the first order release mechanism. Thus, another advantage of this
invention is
that prolonged release time can be imparted by increasing the molecular weight
of the
Garner polypeptide. Another, significant advantage of the invention is that
the kinetics of
2o active agent release is primarily controlled by the enzymatic hydrolysis of
the key bond
between the carrier peptide and the active agent.
Dextran is the only polysaccharide known that has been explored as a
macromolecular carrier for the covalent binding of drug for colon specific
drug delivery.
Generally, it was only possible to load up to 1/10 of the total drug-dextran
conjugate
weight with drug. As stated earlier, polysaccharides are digested mainly in
the colon and
drug absorption is mainly limited to the colon. As compared to dextran, this
invention
has two major advantages. First, peptides are hydrolyzed by any one of several
aminopeptidases found in the intestinal lumen or associated with the brush-
border
membrane and so active agent release and subsequent absorption can occur in
the
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jejunum or the ileum. Second, the molecular weight of the carrier molecule can
be
controlled and, thus, active agent loading can also be controlled.
As a practical example, the following table lists the molecular weights of
lipophilic amino acids (less one water molecule) and selected analgesics and
vitamins.
TABLE
Amino acid MW Active agent MW
Glycine 57 Acetaminophen 151
Alanine 71 Vitamin B6 (Pyroxidine) 169
Valine 99 Vitamin C (Ascorbic acid) 176
Leucine 113 Aspirin 180
Isoleucine 113 Ibuprofen 206
Phenylalanine147 Retinoic acid 300
Tyrosine 163 Vitamin B2 (Riboflavin) 376
Vitamin D2 397
Vitamin E (Tocopherol) 431
Lipophilic amino acids are preferred because conformational protection through
the
stomach is important for the selected active agents, which were selected based
on ease of
covalent attachment to an oligopeptide. Eighteen was subtracted from the amino
acid's
molecular weight so that their condensation into a polypeptide is considered.
For
1o example, a decamer of glycine (MW=588) linked to aspirin would have a total
molecular
weight of 750 and aspirin would represent 24% of the total weight of the
active agent
delivery composition or over two times the maximum drug loading for dextran.
This is
only for an N- or C- terminus application, for those active agents attached to
pendant
groups of decaglutamic acid, for instance, a drug with a molecular weight of
180 could
conceivably have a loading of 58%, although this may not be entirely
practical.
The alcohol, amine or carboxylic acid group of an active agent may be
covalently
attached to the N-terminus, the C-terminus or the side chain of the
oligopeptide or
polypeptide. The location of attachment depends somewhat on the functional
group
selection. For instance, if the active drug is a carboxylic acid (e.g.,
aspirin) then the N-
2o terminus of the oligopeptide is the preferred point of attachment. If the
active agent is an
amine (e.g., ampicillin), then the C-terminus is the preferred point of
attachment in order
to
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to achieve a stable peptide linked active agent. In both, the C- and N-
terminus examples,
the peptide is, in essence, extended by one monomeric unit forming a new
peptide bond.
If the active agent is an alcohol, then either the C-terminus or the N-
terminus is the
preferred point of attachment in order to achieve a stable composition. As in
the example
above where the alcohol, norethindrone, was covalently attached to
poly(hydroxypropylglutamine), an alcohol can be converted into an
alkylchloroformate
with phosgene. This invention, then, pertains to the reaction of this key
intermediate with
the N-terminus of the peptide Garner. The active ingredient can be released
from the
peptide Garner by intestinal peptidases.
The alcohol can be selectively bound to the gamma carboxylate of glutamic acid
and then this conjugate covalently attached to the C-terminus of the peptide
Garner.
Because the glutamic acid-drug conjugate can be considered a dimer, this
product adds
two monomeric units to the C-terminus of the peptide carrier where the
glutamic acid
moiety serves as a spacer between the peptide and the drug as shown in Fig. 4.
Intestinal
enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drug
moiety
from the peptide carrier. The newly formed free amine of the glutamic acid
residue will
then undergo an intramolecular transamination reaction, thereby, releasing the
active
agent with coincident formation of pyroglutamic acid as shown in Fig. 5.
Alternatively,
the glutamic acid-drug dimer can be converted into the gamma ester of glutamic
acid N-
2o carboxyanhydride. This intermediate can then be polymerized, as described
above, using
any suitable initiator as shown in Fig. 4. The product of this polymerization
is
polyglutamic acid with active ingredients attached to multiple pendant groups.
Hence,
maximum drug loading of the carrier peptide can be achieved. In addition,
other amino
acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to
impart
specific properties to the drug delivery system.
The invention also provides a method of imparting the same mechanism of action
for other polypeptides containing functional side chains. Examples include,
but are not
limited to, polylysine, polyasparagine, polyarginine, polyserine,
polycysteine,
polytyrosine, polythreonine and polyglutamine. The mechanism can translate to
these
polypeptides through a spacer or linker on the pendant group, which is
terminated,
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preferably, by the glutamic acid-drug dimer. This carrier peptide-drug
conjugate is
distinguished from the prior art by virtue of the fact that the primary
release of the drug
moiety relies on peptidases and not on esterases. Alternatively, the active
agent can be
attached directly to the pendant group where some other indigenous enzymes in
the
alimentary tract can affect release.
The active agent can be covalently attached to the N-terminus, the C-terminus
or
the side chain of the polypeptide using known techniques. Examples of linking
organic
compounds to the N-terminus type of a peptide include, but are not limited to,
the
attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides
and 1,3-
dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example,
there are
known techniques for forming peptide linked biotin and peptide linked
acridine.
In the present invention, activated protein C is covalently attached to the
polypeptide via a peptide bond.
The polypeptide Garner can be prepared using conventional techniques. A
preferred technique is copolymerization of mixtures of amino acid N-
carboxyanhydrides.
Alternatively, if a specific sequence is desired, a solid state automated
peptide synthesizer
can be used.
The addition of stabilizers to the composition has the potential of
stabilizing the
polypeptide further. Stabilizers such as sugar, amino acids, polyethylene
glycol (PEG)
2o and salts have been shown to prevent protein unfolding. In another
embodiment of the
invention, a pre-first order release of the active agent is imparted by
microencapsulating
the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid
complex,
PEG or salts.
There is evidence that hydrophilic compounds are absorbed through the
intestinal
epithelia efficiently via specialized transporters. The entire membrane
transport system is
intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one
can expect
that excitation of the membrane transport system will involve some sort of
specialized
adjuvant resulting in localized delivery of active agents. There are seven
known
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intestinal transport systems classified according to the physical properties
of the
transported substrate. They include the amino acid, oligopeptide, glucose,
monocarboxic
acid, phosphate, bile acid and the P-glycoprotein transport systems and each
has its own
associated mechanism of transport. The mechanisms can depend on hydrogen ions,
sodium ions, binding sites or other cofactors. The invention also allows
targeting the
mechanisms for intestinal epithelial transport systems to facilitate
absorption of active
agents.
In another embodiment of the invention, the composition includes one or more
adjuvants to enhance the bioavailability of the active agent. Addition of an
adjuvant is
1o particularly preferred when using an otherwise poorly absorbed active
agent. Suitable
adjuvants, for example, include: papain, which is a potent enzyme for
releasing the
catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which
activate
enzymes in the BBM; and bile acids, which have been attached to peptides to
enhance
absorption of the peptides.
15 Preferably, the resultant peptide-activated protein C conjugate is
formulated into a
tablet using suitable excipients and can either be wet granulated or dry
compressed.
Compositions of the invention are, in essence, the formation of amides from
acids
and amines and can be prepared by the following examples.
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Acid/N-terminus conjugation
An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to
0°C. The solution can then be treated with diisopropylcarbodiimide and
hydroxybenzotriazole followed by the amine peptide carrier. The reaction can
then be
stirred for several hours at room temperature, the urea by-product filtered
off, the product
precipitated out in ether and purified using gel permeation chromatography
(GPC) or
dialysis.
1o Amine/C-terminus conjugation
The peptide carrier can be dissolved in DMF under nitrogen and cooled to
0°C.
The solution can then be treated with diisopropylcarbodiimide and
hydroxybenzotriazole
followed by the amine bioactive agent. The reaction can then be stirred for
several hours
at room temperature, the urea by-product filtered off, and the product
precipitated out in
ether and purified using GPC or dialysis.
Alcohol/N-Terminus Conjugation
In the following example the combination of the alcohol with triphosgene
produces a chloroformate, which when reacted with the N-terminus of the
peptide
produces a carbamate. Pursuant to this, an alcohol bioactive agent can be
treated with
2o triphosgene in dry DMF under nitrogen. The suitably protected peptide
carrier is then
added slowly and the solution stirred at room temperature for several hours.
The product
is then precipitated out in ether. The crude product is suitably deprotected
and purified
using GPC.
Other solvents, activating agents, cocatalysts and bases can be used. Examples
of
other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or
chlorinated
solvents such as chloroform. Examples of other activating agents include
dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst
is N-
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hydroxysuccinimide. Examples of bases include pyrrolidinopyridine,
dimethylaminopyridine, triethylamine or tributylamine.
Preparation of Alkyl Glutamate
There have been over 30 different y alkyl glutamates prepared any one of which
may be suitable for the drug alcohol of choice. For example, a suspension of
glutamic
acid, the alcohol and concentrated hydrochloric acid can be prepared and
heated for
several hours. The y alkyl glutamate product can be precipitated out in
acetone, filtered,
dried and recrystallized from hot water.
Alkyl GlutamatelC-Terminus Coqjugation
The peptide Garner can be dissolved in DMF under nitrogen and cooled to
0°C.
The solution can then be treated with diisopropylcarbodiimide and
hydroxybenzotriazole
followed by the y alkyl glutamate bioactive agent. The reaction can then be
stirred for
several hours at room temperature, the urea by-product filtered off, and the
product
precipitated out in ether and purified using GPC or dialysis.
15 Preparation of Alkyl Glutamate-NCA
Y Alkyl glutamate can be suspended in dry THF where triphosgene is added and
the mixture refluxed under a nitrogen atmosphere until the mixture becomes
homogenous. The solution can be poured into heptane to precipitate the NCA
product,
which is filtered, dried and recrystallized from a suitable solvent.
2o Preparation of Poly['y Alkyl Glutamate]
y Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of
a primary amine can be added to the solution until it becomes viscous
(typically
overnight). The product can be isolated from the solution by pouring it into
water and
filtering. The product can be purified using GPC or dialysis.
25 Although illustrated and described above with reference to specific
embodiments,
the invention is nevertheless not intended to be limited to the details shown.
Rather,
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various modifications may be made in the details within the scope and range of
equivalents of the claims and without departing from the spirit of the
invention.
CLAIMS
What is claimed is:
1. A pharmaceutical composition comprising:
a polypeptide; and
activated protein C covalently attached to said polypeptide.
2. The composition of claim 1 wherein said polypeptide is an oligopeptide.
3. The composition of claim 1 wherein said polypeptide is a homopolymer of a
naturally occurnng amino acid.
4. The composition of claim 1 wherein said polypeptide is a heteropolymer of
two or more naturally occurring amino acids.
5. The composition of claim 1 wherein said polypeptide is a homopolymer of a
synthetic amino acid.
6. The composition of claim 1 wherein said polypeptide is a heteropolymer of
two or more synthetic amino acids.
7. The composition of claim 1 wherein said polypeptide is a heteropolymer of
one or more naturally occurnng amino acids and one or more synthetic amino
acids.
8. The composition of claim 1 wherein activated protein C is covalently
attached
to a side chain, the N-terminus or the C-terminus of said polypeptide.
9. The composition of claim 1 further comprising a microencapsulating agent.
16
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10. The composition of claim 9 wherein said microencapsulating agent is
selected from the group consisting of polyethylene glycol (PEG), an amino
acid, a sugar
and a salt.
11. The composition of claim 1 further comprising an adjuvant.
12. The composition of claim 11 wherein said adjuvant activates an intestinal
transporter.
13. The composition of claim 1 further comprising a pharmaceutically
acceptable
excipient.
14. The composition of claim 1 wherein said composition is in the form of an
ingestable tablet.
15. The composition of claim 1 wherein said composition is in the form of an
intravenous preparation.
16. The composition of claim 1 wherein said composition is in the form of an
oral suspension.
17. The composition of claim 1 wherein activated protein C is conformationally
protected by folding of said polypeptide about said active agent.
18. The composition of claim 1 wherein said polypeptide is capable of
releasing
activated protein C from said composition in a pH-dependent manner.
19. A method for protecting activated protein C from degradation comprising
covalently attaching said active agent to a polypeptide.
20. A method for controlling release of activated protein C from a composition
wherein said composition comprises a polypeptide, said method comprising
covalently
attaching activated protein C to said polypeptide.
21. A method for delivering activated protein C to a patient comprising
administering to said patient a composition comprising:
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a polypeptide; and
activated protein C covalently attached to said polypeptide.
22. The method of claim 21 wherein activated protein C is released from said
composition by an enzyme-catalyzed release.
23. The method of claim 21 wherein activated protein C is released from said
composition by a pH-dependent unfolding of said polypeptide.
24. The method of claim 21 wherein said active agent is released from said
composition in a sustained release.
25. The method of claim 21 wherein said composition further comprises an
adjuvant covalently attached to said polypeptide and wherein release of said
adjuvant
from said composition is controlled by said polypeptide.
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Abstract
A composition comprising a polypeptide and activated protein C covalently
attached to the polypeptide. Also provided is a method for delivery of
activated protein C
to a patient comprising administering to the patient a composition comprising
a
polypeptide and activated protein C covalently attached to the polypeptide.
Also
provided is a method for protecting activated protein C from degradation
comprising
covalently attaching it to a polypeptide. Also provided is a method for
controlling release
of activated protein C from a composition comprising covalently attaching it
to the
polypeptide.
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A NOVEL PHARMACEUTICAL COMPOUND CONTAINING ACYCLOVIR
AND METHODS OF MAKING AND USING SAME
FIELD OF THE INVENTION
The present invention relates to a novel pharmaceutical compound that
comprises
a polypeptide that is preferably covalently attached to acyclovir, as well as
methods for
protecting and administering acyclovir. This novel compound, referred to as a
CARRIERWAVE''"s Molecular Analogue (CMA), has the benefit of taking a known
effective pharmaceutical agent that is both well studied and occupies a known
segment of
to the pharmaceutical market, and combining it with a carrier compound that
enhances the
usefulness of the pharmaceutical agent without compromising its pharmaceutical
effectiveness.
BACKGROUND OF THE INVENTION
Acyclovir is a known pharmaceutical agent that is an andviral drug used in the
15 treatment of herpes simplex viruses. Acyclovir is both commercially
available and
readily manufactured using public synthetic schemes by those of ordinary skill
in the art.
Its chemical name is 2-amino-1,9-dihydro-9-[(2-hydroxyethoxy)methyl]-6H-purin-
6-one.
Its structure is:
O
HIV
OH
~ N
HZN' 'N
O
The novel pharmaceutical compound of the present invention is useful in
accomplishing one or more of the following goals: enhancement of the chemical
stability
of the original compound; alteration of the release profile of an orally
administered
product; enhanced digestion or absorption; targeted delivery to particular
tissue/cell type;
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and provision for an oral dosage form when none exists. The novel
pharmaceutical
compound may contain one or more of the following: another active
pharmaceutical
agent, an adjuvant, or an inhibitor.
Active agent delivery systems are often critical for the effective delivery of
a
biologically active agent (active agent) to the appropriate target. The
importance of these
systems becomes magnified when patient compliance and active agent stability
are taken
under consideration. For instance, one would expect patient compliance to
increase
markedly if an active agent is administered orally in lieu of an injection or
another
invasive technique. Increasing the stability of the active agent, such as
prolonging shelf
life or survival in the stomach, will assure dosage reproducibility and
perhaps even
reduce the number of dosages required which could improve patient compliance.
Absorption of an orally administered active agent is often blocked by the
harshly
acidic stomach milieu, powerful digestive enzymes in the GI tract,
permeability of
cellular membranes and transport across lipid bilayers. Incorporating
adjuvants such as
resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance
permeability of
cellular membranes. Microencapsulating active agents using protenoid
microspheres,
liposomes or polysaccharides have been effective in abating enzyme degradation
of the
active agent. Enzyme inhibiting adjuvants have also been used to prevent
enzyme
degradation. Enteric coatings have been used as a protector of pharmaceuticals
in the
stomach.
Active agent delivery systems also provide the ability to control the release
of the
active agent. For example, formulating diazepam with a copolymer of glutamic
acid and
aspartic acid enables a sustained release of the active agent. As another
example,
copolymers of lactic acid and glutaric acid are used to provide timed release
of human
growth hormone. A wide range of pharmaceuticals purportedly provide sustained
release
through microencapsulation of the active agent in amides of dicarboxylic
acids, modified
amino acids or thermally condensed amino acids. Slow release rendering
additives can
also be intermixed with a large array of active agents in tablet formulations.
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Each of these technologies imparts enhanced stability and time-release
properties
to active agent substances. Unfortunately, these technologies suffer from
several
shortcomings. Incorporation of the active agent is often dependent on
diffusion into the
microencapsulating matrix, which may not be quantitative and may complicate
dosage
reproducibility. In addition, encapsulated drugs rely on diffusion out of the
matrix, which
is highly dependant on the water solubility of the active agent. Conversely,
water-soluble
microspheres swell by an infinite degree and, unfortunately, may release the
active agent
in bursts with little active agent available for sustained release.
Furthermore, in some
technologies, control of the degradation process required for active agent
release is
unreliable. For example, an enterically coated active agent depends on pH to
release the
active agent and, as such, is difficult to control the rate of release.
In the past, use has been made of amino acid side chains of polypeptides as
pendant groups to which active agents can be attached. These technologies
typically
require the use of spacer groups between the amino acid pendant group and the
active
agent. The peptide-drug conjugates of this class of drug delivery system rely
on enzymes
in the bloodstream for the release of the drug and, as such, are not used for
oral
administration. Examples of timed and targeted release of injectable or
subcutaneous
pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer,
to the
gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a
peptide
spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been
covalently
attached directly to the beta carboxylate of polyaspartic acid without a
spacer group.
This prodrug formulation was designed as a colon-specific drug delivery system
where
the drug is released by bacterial hydrolytic enzymes residing in the large
intestines. The
released dexamethasone active agent, in turn, was targeted to treat large
bowel disorders
and was not intended to be absorbed into the bloodstream. Yet another
technology
combines the advantages of covalent drug attachment with liposome formation
where the
active ingredient is attached to highly ordered lipid films (known as HARs)
via a peptide
linker. Thus, there has been no drug delivery system, heretofore reported,
that
incorporates the concept of attaching an active ingredient to a polypeptide
pendant group
with its targeted delivery into the bloodstream via oral administration.
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It is also important to control the molecular weight, molecular size and
particle
size of the active agent delivery system. Variable molecular weights have
unpredictable
diffusion rates and pharmacokinetics. High molecular weight carriers are
digested slowly
or late, as in the case of naproxen-linked dextran, which is digested almost
exclusively in
the colon by bacterial enzymes. High molecular weight microspheres usually
have high
moisture content which may present a problem with water labile active
ingredients.
Particle size not only becomes a problem with injectable drugs, as in the HAR
application, but absorption through the brush-border membrane of the
intestines is limited
to less than 5 microns.
SUMMARY OF THE INVENTION
The present invention provides covalent attachment of the active agent
(acyclovir)
to a polymer of peptides or amino acids. The invention is distinguished from
the above-
mentioned technologies by virtue of covalently attaching acyclovir to the N-
terminus, the
C-terminus or directly to the amino acid side chain of an oligopeptide or
polypeptide,
also referred to herein as a carrier peptide. In certain applications, the
polypeptide will
stabilize the active agent, primarily in the stomach, through conformational
protection.
In these applications, delivery of the active agent is controlled, in part, by
the kinetics of
unfolding of the carrier peptide. Upon entry into the upper intestinal tract,
indigenous
enzymes release the active ingredient for absorption by the body by
selectively
hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action
introduces a
second order sustained release mechanism.
Alternatively, the present invention provides a pharmaceutical composition
comprising acyclovir microencapsulated by a polypeptide.
The invention provides a composition comprising a polypeptide and acyclovir
covalently attached to the polypeptide. Preferably, the polypeptide is (i) an
oligopeptide,
(ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii)
a
heteropolymer of two or more naturally occurring amino acids, (iv) a
homopolymer of a
synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids
or (vi) a
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heteropolymer of one or more naturally occurring amino acids and one or more
synthetic
amino acids.
Acyclovir preferably is covalently attached to a side chain, the N-terminus or
the
C-terminus of the polypeptide. In a preferred embodiment, the active agent is
a
carboxylic acid and is covalently attached to the N-terminus of the
polypeptide. In
another preferred embodiment, the active agent is an amine and is covalently
attached to
the C-terminus of the polypeptide. In another preferred embodiment, the active
agent is
an alcohol and is covalently attached to the C-terminus of the polypeptide. In
yet another
preferred embodiment, the active agent is an alcohol and is covalently
attached to the N-
terminus of the polypeptide.
The composition of the invention can also include one or more of a
microencapsulating agent, an adjuvant and a pharmaceutically acceptable
excipient. The
microencapsulating agent can be selected from polyethylene glycol (PEG), an
amino
acid, a sugar and a salt. When an adjuvant is included in the composition, the
adjuvant
preferably activates an intestinal transporter.
Preferably, the composition of the invention is in the form of an ingestable
tablet,
an intravenous preparation or an oral suspension. The active agent can be
conformationally protected by folding of the polypeptide about the active
agent. In
another embodiment, the polypeptide is capable of releasing the active agent
from the
composition in a pH-dependent manner.
The invention also provides a method for protecting acyclovir from degradation
comprising covalently attaching it to a polypeptide.
The invention also provides a method for delivering acyclovir to a patient,
the
patient being a human or a non-human animal, comprising administering to the
patient a
composition comprising a polypeptide and an active agent covalently attached
to the
polypeptide. In a preferred embodiment, acyclovir is released from the
composition by
an enzyme-catalyzed release. In another preferred embodiment, acyclovir is
released in a
time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed
release.
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In another preferred embodiment, the composition further comprises a
microencapsulating agent and acyclovir is released from the composition by
dissolution
of the microencapsulating agent. In another preferred embodiment, acyclovir is
released
from the composition by a pH-dependent unfolding of the polypeptide. In
another
preferred embodiment, acyclovir is released from the composition in a
sustained release.
In yet another preferred embodiment, the composition further comprises an
adjuvant
covalently attached to the polypeptide and release of the adjuvant from the
composition is
controlled by the polypeptide. The adjuvant can be microencapsulated into a
Garner
peptide-drug conjugate for biphasic release of active ingredients.
to The invention also provides a method for preparing a composition comprising
a
polypeptide and an active agent covalently attached to the polypeptide. The
method
comprises the steps of:
(a) attaching acyciovir to a side chain of an amino acid to form an active
agent/amino acid complex;
t5 (b) forming an active agent/amino acid complex N-carboxyanhydride (NCA)
from the active agent/amino acid complex; and
(c) polymerizing the active agent/amino acid complex N-carboxyanhydride
(NCA).
In a preferred embodiment, steps (a) and (b) are repeated prior to step (c)
with a
2o second active agent. When steps (a) and (b) are repeated prior to step (c)
with a second
agent, acyclovir and a second active agent can be copolymerized in step (c).
In another
preferred embodiment, the amino acid is glutamic acid and the active agent is
released
from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and
wherein the
active agent is released from the glutamic acid by coincident intramolecular
25 transamination. In another preferred embodiment, the glutamic acid is
replaced by an
amino acid selected from the group consisting of aspartic acid, arginine,
asparagine,
cysteine, lysine, threonine, and serine, and wherein the active agent is
attached to the side
chain of the amino acid to form an amide, a thioester, an ester, an ether, a
urethane, a
carbonate, an anhydride or a carbamate. In yet another preferred embodiment,
the
30 glutamic acid is replaced by a synthetic amino acid with a pendant group
comprising an
amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.
6
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It is to be understood that both the foregoing general description and the
following detailed description are exemplary, but are not restrictive, of the
invention.
The general applications of this invention to other active pharmaceutical
agents is
described in U.S. Patent Application Serial Number 09/642,820, filed August
22, 2000,
incorporated herein by reference.
DETAILED DESCRIPTION OF INVENTION
The present invention provides several benefits for active agent delivery.
First,
the invention can stabilize acyclovir and prevent its digestion in the
stomach. In addition,
the pharmacologic effect can be prolonged by delayed release of acyclovir.
Furthermore,
to active agents can be combined to produce synergistic effects. Also,
absorption of the
active agent in the intestinal tract can be enhanced. The invention also
allows targeted
delivery of active agents to specifics sites of action.
The composition of the invention comprises acyclovir covalently attached to a
polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a
homopolymer of one
15 of the twenty naturally occurring amino acids, (iii) a heteropolymer of two
or more
naturally occurnng amino acids, (iv) a homopolymer of a synthetic amino acid,
(v) a
heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of
one or
more naturally occurring amino acids and one or more synthetic amino acids.
Proteins, oligopeptides and polypeptides are polymers of amino acids that have
20 primary, secondary and tertiary structures. The secondary structure of the
protein is the
local conformation of the polypeptide chain and consists of helices, pleated
sheets and
turns. The protein's amino acid sequence and the structural constraints on the
conformations of the chain determine the spatial arrangement of the molecule.
The
folding of the secondary structure and the spatial arrangement of the side
chains
25 constitute the tertiary structure.
Proteins fold because of the dynamics associated between neighboring atoms on
the protein and solvent molecules. The thermodynamics of protein folding and
unfolding
are defined by the free energy of a particular condition of the protein that
relies on a
particular model. The process of protein folding involves, amongst other
things, amino
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acid residues packing into a hydrophobic core. The amino acid side chains
inside the
protein core occupy the same volume as they do in amino acid crystals. The
folded
protein interior is therefore more like a crystalline solid than an oil drop
and so the best
model for determining forces contributing to protein stability is the solid
reference state.
The major forces contributing to the thermodynamics of protein folding are Van
der Waals interactions, hydrogen bonds, electrostatic interactions,
configurational
entropy and the hydrophobic effect. Considering protein stability, the
hydrophobic effect
refers to the energetic consequences of removing apolar groups from the
protein interior
and exposing them to water. Comparing the energy of amino acid hydrolysis with
1o protein unfolding in the solid reference state, the hydrophobic effect is
the dominant
force. Hydrogen bonds are established during the protein fold process and
intramolecular
bonds are formed at the expense of hydrogen bonds with water. Water molecules
are
"pushed out" of the packed, hydrophobic protein core. All of these forces
combine and
contribute to the overall stability of the folded protein where the degree to
which ideal
15 packing occurs determines the degree of relative stability of the protein.
The result of
maximum packing is to produce a center of residues or hydrophobic core that
has
maximum shielding from solvent.
Since it is likely that lipophilic drugs would reside in the hydrophobic core
of a
peptide, it would require energy to unfold the peptide before the drug can be
released.
20 The unfolding process requires overcoming the hydrophobic effect by
hydrating the
amino acids or achieving the melting temperature of the protein. The heat of
hydration is
a destabilization of a protein. Typically, the folded state of a protein is
favored by only
5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at
neutral pH and
at room temperature requires chemical reagents. In fact, partial unfolding of
a protein is
25 often observed prior to the onset of irreversible chemical or conformation
processes.
Moreover, protein conformation generally controls the rate and extent of
deleterious
chemical reactions.
Conformational protection of active agents by proteins depends on the
stability of
the protein's folded state and the thermodynamics associated with the agent's
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decomposition. Conditions necessary for the agent's decomposition should be
different
than for protein unfolding.
Selection of the amino acids will depend on the physical properties desired.
For
instance, if increase in bulk or lipophilicity is desired, then the carrier
polypeptide will be
enriched in the amino acids in the table provided below. Polar amino acids, on
the other
hand, can be selected to increase the hydrophilicity of the polypeptide.
Ionizing amino acids can be selected for pH controlled peptide unfolding.
Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the
stomach, but will
ionize upon entry into the intestine. Conversely, basic amino acids, such as
histidine,
1o lysine and arginine, ionize in the stomach and are neutral in an alkaline
environment.
Other factors such as ~-~ interactions between aromatic residues, kinking of
the
peptide chain by addition of proline, disulfide crosslinking and hydrogen
bonding can all
be used to select the optimum amino acid sequence for a given application.
Ordering of
the linear sequence can influence how these interactions can be maximized and
is
15 important in directing the secondary and tertiary structures of the
polypeptide.
Furthermore, amino acids with reactive side chains (e.g., glutamic acid,
lysine,
aspartic acid, serine, threonine and cysteine) can be incorporated for
attaching multiple
active agents or adjuvants to the same Garner peptide. This is particularly
useful if a
synergistic effect between two or more active agents is desired.
2o As stated above, variable molecular weights of the carrier compound can
have
profound effects on the active agent release kinetics. As a result, low
molecular weight
active agent delivery systems are preferred. An advantage of this invention is
that chain
length and molecular weight of the polypeptide can be optimized depending on
the level
of conformational protection desired. This property can be optimized in
concert with the
25 kinetics of the first order release mechanism. Thus, another advantage of
this invention is
that prolonged release time can be imparted by increasing the molecular weight
of the
carrier polypeptide. Another, significant advantage of the invention is that
the kinetics of
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active agent release is primarily controlled by the enzymatic hydrolysis of
the key bond
between the carrier peptide and the active agent.
Dextran is the only polysaccharide known that has been explored as a
macromolecular carrier for the covalent binding of drug for colon specific
drug delivery.
Generally, it was only possible to load up to 1/10 of the total drug-dextran
conjugate
weight with drug. As stated earlier, polysaccharides are digested mainly in
the colon and
drug absorption is mainly limited to the colon. As compared to dextran, this
invention
has two major advantages. First, peptides are hydrolyzed by any one of several
aminopeptidases found in the intestinal lumen or associated with the brush-
border
t0 membrane and so active agent release and subsequent absorption can occur in
the
jejunum or the ileum. Second, the molecular weight of the carrier molecule can
be
controlled and, thus, active agent loading can also be controlled.
As a practical example, the following table lists the molecular weights of
lipophilic amino acids (less one water molecule) and selected analgesics and
vitamins.
TABLE
Amino acid MW Active agent MW
Glycine 57 Acetaminophen 151
Alanine 71 Vitamin B6 (Pyroxidine) 169
Valine 99 Vitamin C (Ascorbic acid) 176
Leucine 113 Aspirin 180
Isoleucine 113 Ibuprofen 206
Phenylalanine147 Retinoic acid 300
Tyrosine 163 Vitamin B2 (Riboflavin) 376
Vitamin Dz 397
Vitamin E (Tocopherol) 431
Lipophilic amino acids are preferred because conformational protection through
the
stomach is important for the selected active agents, which were selected based
on ease of
covalent attachment to an oligopeptide. Eighteen was subtracted from the amino
acid's
molecular weight so that their condensation into a polypeptide is considered.
For
example, a decamer of glycine (MW=588) linked to aspirin would have a total
molecular
weight of 750 and aspirin would represent 24% of the total weight of the
active agent
to
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delivery composition or over two times the maximum drug loading for dextran.
This is
only for an N- or C- terminus application, for those active agents attached to
pendant
groups of decaglutamic acid, for instance, a drug with a molecular weight of
180 could
conceivably have a loading of 58%, although this may not be entirely
practical.
The alcohol, amine or carboxylic acid group of an active agent may be
covalently
attached to the N-terminus, the C-terminus or the side chain of the
oligopeptide or
polypeptide. The location of attachment depends somewhat on the functional
group
selection. For instance, if the active drug is a carboxylic acid (e.g.,
aspirin) then the N-
terminus of the oligopeptide is the preferred point of attachment. If the
active agent is an
to amine (e.g., ampicillin), then the C-terminus is the preferred point of
attachment in order
to achieve a stable peptide linked active agent. In both, the C- and N-
terminus examples,
the peptide is, in essence, extended by one monomeric unit forming a new
peptide bond.
If the active agent is an alcohol, then either the C-terminus or the N-
terminus is the
preferred point of attachment in order to achieve a stable composition. As in
the example
is above where the alcohol, norethindrone, was covalently attached to
poly(hydroxypropylglutamine), an alcohol can be converted into an
alkylchloroformate
with phosgene. This invention, then, pertains to the reaction of this key
intermediate with
the N-terminus of the peptide carrier. The active ingredient can be released
from the
peptide Garner by intestinal peptidases.
2o The alcohol can be selectively bound to the gamma carboxylate of glutamic
acid
and then this conjugate covalently attached to the C-terminus of the peptide
Garner.
Because the glutamic acid-drug conjugate can be considered a dimer, this
product adds
two monomeric units to the C-terminus of the peptide carrier where the
glutamic acid
moiety serves as a spacer between the peptide and the drug as shown in Fig. 4.
Intestinal
25 enzymatic hydrolysis of the key peptide bond releases the glutamic acid-
drug moiety
from the peptide carrier. The newly formed free amine of the glutamic acid
residue will
then undergo an intramolecular transamination reaction, thereby, releasing the
active
agent with coincident formation of pyroglutamic acid as shown in Fig. 5.
Alternatively,
the glutamic acid-drug dimer can be converted into the gamma ester of glutamic
acid N-
3o carboxyanhydride. This intermediate can then be polymerized, as described
above, using
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any suitable initiator as shown in Fig. 4. The product of this polymerization
is
polyglutamic acid with active ingredients attached to multiple pendant groups.
Hence,
maximum drug loading of the carrier peptide can be achieved. In addition,
other amino
acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to
impart
specific properties to the drug delivery system.
The invention also provides a method of imparting the same mechanism of action
for other polypeptides containing functional side chains. Examples include,
but are not
limited to, polylysine, polyasparagine, polyarginine, polyserine,
polycysteine,
polytyrosine, polythreonine and polyglutamine. The mechanism can translate to
these
polypeptides through a spacer or linker on the pendant group, which is
terminated,
preferably, by the glutamic acid-drug dimer. This carrier peptide-drug
conjugate is
distinguished from the prior art by virtue of the fact that the primary
release of the drug
moiety relies on peptidases and not on esterases. Alternatively, the active
agent can be
attached directly to the pendant group where some other indigenous enzymes in
the
alimentary tract can affect release.
The active agent can be covalently attached to the N-terminus, the C-terminus
or
the side chain of the polypeptide using known techniques. Examples of linking
organic
compounds to the N-terminus type of a peptide include, but are not limited to,
the
attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides
and 1,3-
dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example,
there are
known techniques for forming peptide linked biotin and peptide linked
acridine.
In the present invention, acyclovir is covalently attached to the polypeptide
via
the hydroxyl group.
The polypeptide carrier can be prepared using conventional techniques. A
preferred technique is copolymerization of mixtures of amino acid N-
carboxyanhydrides.
Alternatively, if a specific sequence is desired, a solid state automated
peptide synthesizer
can be used.
12
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The addition of stabilizers to the composition has the potential of
stabilizing the
polypeptide further. Stabilizers such as sugar, amino acids, polyethylene
glycol (PEG)
and salts have been shown to prevent protein unfolding. In another embodiment
of the
invention, a pre-first order release of the active agent is imparted by
microencapsulating
the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid
complex,
PEG or salts.
There is evidence that hydrophilic compounds are absorbed through the
intestinal
epithelia efficiently via specialized transporters. The entire membrane
transport system is
intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one
can expect
1o that excitation of the membrane transport system will involve some sort of
specialized
adjuvant resulting in localized delivery of active agents. There are seven
known
intestinal transport systems classified according to the physical properties
of the
transported substrate. They include the amino acid, oligopeptide, glucose,
monocarboxic
acid, phosphate, bile acid and the P-glycoprotein transport systems and each
has its own
15 associated mechanism of transport. The mechanisms can depend on hydrogen
ions,
sodium ions, binding sites or other cofactors. The invention also allows
targeting the
mechanisms for intestinal epithelial transport systems to facilitate
absorption of active
agents.
In another embodiment of the invention, the composition includes one or more
20 adjuvants to enhance the bioavailability of the active agent. Addition of
an adjuvant is
particularly preferred when using an otherwise poorly absorbed active agent.
Suitable
adjuvants, for example, include: papain, which is a potent enzyme for
releasing the
catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which
activate
enzymes in the BBM; and bile acids, which have been attached to peptides to
enhance
25 absorption of the peptides.
Preferably, the resultant peptide-acyclovir conjugate is formulated into a
tablet
using suitable excipients and can either be wet granulated or dry compressed.
Compositions of the invention are, in essence, the formation of amides from
acids
and amines and can be prepared by the following examples.
13
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Acid/N-terminus conjugation
An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to
0°C. The solution can then be treated with diisopropylcarbodiimide and
hydroxybenzotriazole followed by the amine peptide carrier. The reaction can
then be
stirred for several hours at room temperature, the urea by-product filtered
off, the product
precipitated out in ether and purified using gel permeation chromatography
(GPC) or
dialysis.
AminelC-terminus conjugation
The peptide Garner can be dissolved in DMF under nitrogen and cooled to
0°C.
The solution can then be treated with diisopropylcarbodiimide and
hydroxybenzotriazole
followed by the amine bioactive agent. The reaction can then be stirred for
several hours
at room temperature, the urea by-product filtered off, and the product
precipitated out in
ether and purified using GPC or dialysis.
Alcohol/N-Terminus Conjugation
In the following example the combination of the alcohol with triphosgene
produces a chloroformate, which when reacted with the N-terminus of the
peptide
produces a carbamate. Pursuant to this, an alcohol bioactive agent can be
treated with
triphosgene in dry DMF under nitrogen. The suitably protected peptide Garner
is then
added slowly and the solution stirred at room temperature for several hours.
The product
is then precipitated out in ether. The crude product is suitably deprotected
and purified
using GPC.
Other solvents, activating agents, cocatalysts and bases can be used. Examples
of
other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or
chlorinated
solvents such as chloroform. Examples of other activating agents include
dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst
is N-
hydroxysuccinimide. Examples of bases include pyrrolidinopyridine,
dimethylaminopyridine, triethylamine or tributylamine.
14
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Preparation of ~-Alkyl Glutamate
There have been over 30 different y alkyl glutamates prepared any one of which
may be suitable for the drug alcohol of choice. For example, a suspension of
glutamic
acid, the alcohol and concentrated hydrochloric acid can be prepared and
heated for
several hours. The y alkyl glutamate product can be precipitated out in
acetone, filtered,
dried and recrystallized from hot water.
~Alkyt Glutamate/C-Terminus Conjugation
The peptide carrier can be dissolved in DMF under nitrogen and cooled to
0°C.
The solution can then be treated with diisopropylcarbodiimide and
hydroxybenzotriazole
~o followed by the y alkyl glutamate bioactive agent. The reaction can then be
stirred for
several hours at room temperature, the urea by-product filtered off, and the
product
precipitated out in ether and purified using GPC or dialysis.
Preparation of ~y~.Alkyl Glutamate-NCA
y Alkyl glutamate can be suspended in dry THF where triphosgene is added and
15 the mixture refluxed under a nitrogen atmosphere until the mixture becomes
homogenous. The solution can be poured into heptane to precipitate the NCA
product,
which is filtered, dried and recrystallized from a suitable solvent.
Preparation of Poly(Y Alkyl Glutamate]
y Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of
2o a primary amine can be added to the solution until it becomes viscous
(typically
overnight). The product can be isolated from the solution by pouring it into
water and
filtering. The product can be purified using GPC or dialysis.
Although illustrated and described above with reference to specific
embodiments,
the invention is nevertheless not intended to be limited to the details shown.
Rather,
25 various modifications may be made in the details within the scope and range
of
equivalents of the claims and without departing from the spirit of the
invention.
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CLAIMS
What is claimed is:
1. A pharmaceutical composition comprising:
a polypeptide; and
acyclovir covalently attached to said polypeptide.
2. The composition of claim 1 wherein said polypeptide is an oligopeptide.
3. The composition of claim 1 wherein said polypeptide is a homopolymer of a
naturally occurring amino acid.
4. The composition of claim 1 wherein said polypeptide is a heteropolymer of
1o two or more naturally occurnng amino acids.
5. The composition of claim 1 wherein said polypeptide is a homopolymer of a
synthetic amino acid.
6. The composition of claim 1 wherein said polypeptide is a heteropolymer of
two or more synthetic amino acids.
7. The composition of claim 1 wherein said polypeptide is a heteropolymer of
one or more naturally occurring amino acids and one or more synthetic amino
acids.
8. The composition of claim 1 wherein acyclovir is covalently attached to a
side
chain, the N-terminus or the C-terminus of said polypeptide.
9. The composition of claim 1 further comprising a microencapsulating agent.
10. The composition of claim 9 wherein said microencapsulating agent is
selected from the group consisting of polyethylene glycol (PEG), an amino
acid, a sugar
and a salt.
11. The composition of claim 1 further comprising an adjuvant. '
16
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12. The composition of claim 11 wherein said adjuvant activates an intestinal
transporter.
13. The composition of claim 1 further comprising a pharmaceutically
acceptable
excipient.
14. The composition of claim 1 wherein said composition is in the form of an
ingestable tablet.
15. The composition of claim 1 wherein said composition is in the form of an
intravenous preparation.
16. The composition of claim 1 wherein said composition is in the form of an
oral suspension.
17. The composition of claim 1 wherein acyclovir is conformationally protected
by folding of said polypeptide about said active agent.
18. The composition of claim 1 wherein said polypeptide is capable of
releasing
acyclovir from said composition in a pH-dependent manner.
19. A method for protecting acyclovir from degradation comprising covalently
attaching said active agent to a polypeptide.
20. A method for controlling release of acyclovir from a composition wherein
said composition comprises a polypeptide, said method comprising covalently
attaching
acyclovir to said polypeptide.
21. A method for delivering acyclovir to a patient comprising administering to
said patient a composition comprising:
a polypeptide; and
acyclovir covalently attached to said polypeptide.
22. The method of claim 21 wherein acyclovir is released from said composition
by an enzyme-catalyzed release.
17
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23. The method of claim 21 wherein acyclovir is released from said composition
by a pH-dependent unfolding of said polypeptide.
24. The method of claim 21 wherein said active agent is released from said
composition in a sustained release.
25. The method of claim 21 wherein said composition further comprises an
adjuvant covalently attached to said polypeptide and wherein release of said
adjuvant
from said composition is controlled by said polypeptide.
18
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Abstract
A composition comprising a polypeptide and acyclovir covalently attached to
the
polypeptide. Also provided is a method for delivery of acyclovir to a patient
comprising
administering to the patient a composition comprising a polypeptide and
acyclovir
covalently attached to the polypeptide. Also provided is a method for
protecting
acyclovir from degradation comprising covalently attaching it to a
polypeptide. Also
provided is a method for controlling release of acyclovir from a composition
comprising
covalently attaching it to the polypeptide.
19
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A NOVEL PHARMACEUTICAL COMPOUND CONTAINING
ADEFOVIR DIPIVOXIL AND METHODS OF MAKING AND USING SAME
FIELD OF THE INVENTION
The present invention relates to a novel pharmaceutical compound that
comprises
a polypeptide that is preferably covalently attached to adefovir dipivoxil, as
well as
methods for protecting and administering adefovir dipivoxil. This novel
compound,
referred to as a CARRIERWAVETM Molecular Analogue (CMA), has the benefit of
taking a known effective pharmaceutical agent that is both well studied and
occupies a
known segment of the pharmaceutical market, and combining it with a carrier
compound
that enhances the usefulness of the pharmaceutical agent without compromising
its
pharmaceutical effectiveness.
BACKGROUND OF THE INVENTION
Adefovir dipivoxil is a known pharmaceutical agent that is used in the
treatment
of AIDS. Its chemical name is [[[2-(6-amino-9H-purin-9-yl)ethoxy]methyl]
phosphinylidene]bis(oxymethylene)-2,2-dimethylpropanoic acid. Its structure
is:
N
N~ N
N O
N I O~ O
~O'~Pi
Oi~O
~O
O:!~
The novel pharmaceutical compound of the present invention is useful in
accomplishing one or more of the following goals: enhancement of the chemical
stability
of the original compound; alteration of the release profile of an orally
administered
product; enhanced digestion or absorption; targeted delivery to particular
tissue/cell type;
and provision for an oral dosage form when none exists. The novel
pharmaceutical
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compound may contain one or more of the following: another active
pharmaceutical
agent, an adjuvant, or an inhibitor.
Active agent delivery systems are often critical for the effective delivery of
a
biologically active agent (active agent) to the appropriate target. The
importance of these
systems becomes magnified when patient compliance and active agent stability
are taken
under consideration. For instance, one would expect patient compliance to
increase
markedly if an active agent is administered orally in lieu of an injection or
another
invasive technique. Increasing the stability of the active agent, such as
prolonging shelf
life or survival in the stomach, will assure dosage reproducibility and
perhaps even
reduce the number of dosages required which could improve patient compliance.
Absorption of an orally administered active agent is often blocked by the
harshly
acidic stomach milieu, powerful digestive enzymes in the GI tract,
permeability of
cellular membranes and transport across lipid bilayers. Incorporating
adjuvants such as
resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance
permeability of
cellular membranes. Microencapsulating active agents using protenoid
microspheres,
liposomes or polysaccharides have been effective in abating enzyme degradation
of the
active agent. Enzyme inhibiting adjuvants have also been used to prevent
enzyme
degradation. Enteric coatings have been used as a protector of pharmaceuticals
in the
stomach.
2o Active agent delivery systems also provide the ability to control the
release of the
active agent. For example, formulating diazepam with a copolymer of glutamic
acid and
aspartic acid enables a sustained release of the active agent. As another
example,
copolymers of lactic acid and glutaric acid are used to provide timed release
of human
growth hormone. A wide range of pharmaceuticals purportedly provide sustained
release
through microencapsulation of the active agent in amides of dicarboxylic
acids, modified
amino acids or thermally condensed amino acids. Slow release rendering
additives can
also be intermixed with a large array of active agents in tablet formulations.
Each of these technologies imparts enhanced stability and time-release
properties
to active agent substances. Unfortunately, these technologies suffer from
several
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shortcomings. Incorporation of the active agent is often dependent on
diffusion into the
microencapsulating matrix, which may not be quantitative and may complicate
dosage
reproducibility. In addition, encapsulated drugs rely on diffusion out of the
matrix, which
is highly dependant on the water solubility of the active agent. Conversely,
water-soluble
microspheres swell by an infinite degree and, unfortunately, may release the
active agent
in bursts with little active agent available for sustained release.
Furthermore, in some
technologies, control of the degradation process required for active agent
release is
unreliable. For example, an enterically coated active agent depends on pH to
release the
active agent and, as such, is difficult to control the rate of release.
t o In the past, use has been made of amino acid side chains of polypeptides
as
pendant groups to which active agents can be attached. These technologies
typically
require the use of spacer groups between the amino acid pendant group and the
active
agent. The peptide-drug conjugates of this class of drug delivery system rely
on enzymes
in the bloodstream for the release of the drug and, as such, are not used for
oral
administration. Examples of timed and targeted release of injectable or
subcutaneous
pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer,
to the
gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a
peptide
spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been
covalently
attached directly to the beta carboxylate of polyaspartic acid without a
spacer group.
2o This prodrug formulation was designed as a colon-specific drug delivery
system where
the drug is released by bacterial hydrolytic enzymes residing in the large
intestines. The
released dexamethasone active agent, in turn, was targeted to treat large
bowel disorders
and was not intended to be absorbed into the bloodstream. Yet another
technology
combines the advantages of covalent drug attachment with liposome formation
where the
active ingredient is attached to highly ordered lipid films (known as HARs)
via a peptide
linker. Thus, there has been no drug delivery system, heretofore reported,
that
incorporates the concept of attaching an active ingredient to a polypeptide
pendant group
with its targeted delivery into the bloodstream via oral administration.
It is also important to control the molecular weight, molecular size and
particle
size of the active agent delivery system. Variable molecular weights have
unpredictable
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diffusion rates and pharmacokinetics. High molecular weight carriers are
digested slowly
or late, as in the case of naproxen-linked dextran, which is digested almost
exclusively in
the colon by bacterial enzymes. High molecular weight microspheres usually
have high
moisture content which may present a problem with water labile active
ingredients.
Particle size not only becomes a problem with injectable drugs, as in the HAR
application, but absorption through the brush-border membrane of the
intestines is limited
to less than 5 microns.
SUMMARY OF THE INVENTION
The present invention provides covalent attachment of the active agent
(adefovir
1o dipivoxil) to a polymer of peptides or amino acids. The invention is
distinguished from
the above-mentioned technologies by virtue of covalently attaching adefovir
dipivoxil to
the N-terminus, the C-terminus or directly to the amino acid side chain of an
oligopeptide
or polypeptide, also referred to herein as a carrier peptide. In certain
applications, the
polypeptide will stabilize the active agent, primarily in the stomach, through
15 conformational protection. In these applications, delivery of the active
agent is
controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon
entry into the
upper intestinal tract, indigenous enzymes release the active ingredient for
absorption by
the body by selectively hydrolyzing the peptide bonds of the carnet peptide.
This
enzymatic action introduces a second order sustained release mechanism.
20 Alternatively, the present invention provides a pharmaceutical composition
comprising adefovir dipivoxil microencapsulated by a polypeptide.
The invention provides a composition comprising a polypeptide and adefovir
dipivoxil covalently attached to the polypeptide. Preferably, the polypeptide
is (i) an
oligopeptide, (ii) a homopolymer of one of the twenty naturally occurring
amino acids,
25 (iii) a heteropolymer of two or more naturally occurring amino acids, (iv)
a homopolymer
of a synthetic amino acid, (v) a heteropolymer of two or more synthetic amino
acids or
(vi) a heteropolymer of one or more naturally occurring amino acids and one or
more
synthetic amino acids.
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Adefovir dipivoxil preferably is covalently attached to a side chain, the N-
terminus or the C-terminus of the polypeptide. In a preferred embodiment, the
active
agent is a carboxylic acid and is covalently attached to the N-terminus of the
polypeptide.
In another preferred embodiment, the active agent is an amine and is
covalently attached
to the C-terminus of the polypeptide. In another preferred embodiment, the
active agent
is an alcohol and is eovalently attached to the C-terminus of the polypeptide.
In yet
another preferred embodiment, the active agent is an alcohol and is covalently
attached to
the N-terminus of the polypeptide.
The composition of the invention can also include one or more of a
to microencapsulating agent, an adjuvant and a pharmaceutically acceptable
excipient. The
microencapsulating agent can be selected from polyethylene glycol (PEG), an
amino
acid, a sugar and a salt. When an adjuvant is included in the composition, the
adjuvant
preferably activates an intestinal transporter.
Preferably, the composition of the invention is in the form of an ingestable
tablet,
15 an intravenous preparation or an oral suspension. The active agent can be
conformationally protected by folding of the polypeptide about the active
agent. In
another embodiment, the polypeptide is capable of releasing the active agent
from the
composition in a pH-dependent manner.
The invention also provides a method for protecting adefovir dipivoxil from
20 degradation comprising covalently attaching it to a polypeptide.
The invention also provides a method for delivering adefovir dipivoxil to a
patient, the patient being a human or a non-human animal, comprising
administering to
the patient a composition comprising a polypeptide and an active agent
covalently
attached to the polypeptide. In a preferred embodiment, adefovir dipivoxil is
released
25 from the composition by an enzyme-catalyzed release. In another preferred
embodiment,
adefovir dipivoxil is released in a time-dependent manner based on the
pharmacokinetics
of the enzyme-catalyzed release. In another preferred embodiment, the
composition
further comprises a microencapsulating agent and adefovir dipivoxil is
released from the
composition by dissolution of the microencapsulating agent. In another
preferred
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embodiment, adefovir dipivoxil is released from the composition by a pH-
dependent
unfolding of the polypeptide. In another preferred embodiment, adefovir
dipivoxil is
released from the composition in a sustained release. In yet another preferred
embodiment, the composition further comprises an adjuvant covalently attached
to the
polypeptide and release of the adjuvant from the composition is controlled by
the
polypeptide. The adjuvant can be microencapsulated into a carrier peptide-drug
conjugate for biphasic release of active ingredients.
The invention also provides a method for preparing a composition comprising a
polypeptide and an active agent covalently attached to the polypeptide. The
method
to comprises the steps of:
(a) attaching adefovir dipivoxi~ to a side chain of an amino acid to form an
active
agent/amino acid complex;
(b) forming an active agentlamino acid complex N-carboxyanhydride (NCA)
from the active agent/amino acid complex; and
15 (c) polymerizing the active agent/amino acid complex N-carboxyanhydride
(NCA).
In a preferred embodiment, steps (a) and (b) are repeated prior to step (c)
with a
second active agent. When steps (a) and (b) are repeated prior to step (c)
with a second
agent, adefovir dipivoxil and a second active agent can be copolymerized in
step (c). In
2o another preferred embodiment, the amino acid is glutamic acid and the
active agent is
released from the glutamic acid as a dimer upon a hydrolysis of the
polypeptide and
wherein the active agent is released from the glutamic acid by coincident
intramolecular
transamination. In another preferred embodiment, the glutamic acid is replaced
by an
amino acid selected from the group consisting of aspartic acid, arginine,
asparagine,
25 cysteine, lysine, threonine, and serine, and wherein the active agent is
attached to the side
chain of the amino acid to form an amide, a thioester, an ester, an ether, a
urethane, a
carbonate, an anhydride or a carbamate. In yet another preferred embodiment,
the
glutamic acid is replaced by a synthetic amino acid with a pendant group
comprising an
amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.
6
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It is to be understood that both the foregoing general description and the
following detailed description are exemplary, but are not restrictive, of the
invention.
The general applications of this invention to other active pharmaceutical
agents is
described in U.S. Patent Application Serial Number 09!642,820, filed August
22, 2000,
incorporated herein by reference.
DETAILED DESCRIPTION OF INDENTION
The present invention provides several benefits for active agent delivery.
First,
the invention can stabilize adefovir dipivoxil and prevent its digestion in
the stomach. In
addition, the pharmacologic effect can be prolonged by delayed release of
adefovir
dipivoxil. Furthermore, active agents can be combined to produce synergistic
effects.
Also, absorption of the active agent in the intestinal tract can be enhanced.
The invention
also allows targeted delivery of active agents to specifics sites of action.
Adefovir dipivoxil is both commercially available and readily manufactured
using
published synthetic schemes by those of ordinary skill in the art.
The composition of the invention comprises adefovir dipivoxil covalently
attached to a polypeptide. Preferably, the polypeptide is (i) an oligopeptide,
(ii) a
homopolymer of one of the twenty naturally occurnng amino acids, (iii) a
heteropolymer
of two or more naturally occurring amino acids, (iv) a homopolymer of a
synthetic amino
acid, (v) a heteropolymer of two or more synthetic amino acids or (vi) a
heteropolymer of
2o one or more naturally occurnng amino acids and one or more synthetic amino
acids.
Proteins, oligopeptides and polypeptides are polymers of amino acids that have
primary, secondary and tertiary structures. The secondary structure of the
protein is the
local conformation of the polypeptide chain and consists of helices, pleated
sheets and
turns. The protein's amino acid sequence and the structural constraints on the
2s conformations of the chain determine the spatial arrangement of the
molecule. The
folding of the secondary structure and the spatial arrangement of the side
chains
constitute the tertiary structure.
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Proteins fold because of the dynamics associated between neighboring atoms on
the protein and solvent molecules. The thermodynamics of protein folding and
unfolding
are defined by the free energy of a particular condition of the protein that
relies on a
particular model. The process of protein folding involves, amongst other
things, amino
acid residues packing into a hydrophobic core. The amino acid side chains
inside the
protein core occupy the same volume as they do in amino acid crystals. The
folded
protein interior is therefore more like a crystalline solid than an oil drop
and so the best
model for determining forces contributing to protein stability is the solid
reference state.
The major forces contributing to the thermodynamics of protein folding are Van
1o der Waals interactions, hydrogen bonds, electrostatic interactions,
configurational
entropy and the hydrophobic effect. Considering protein stability, the
hydrophobic effect
refers to the energetic consequences of removing apolar groups from the
protein interior
and exposing them to water. Comparing the energy of amino acid hydrolysis with
protein unfolding in the solid reference state, the hydrophobic effect is the
dominant
force. Hydrogen bonds are established during the protein fold process and
intramolecular
bonds are formed at the expense of hydrogen bonds with water. Water molecules
are
"pushed out" of the packed, hydrophobic protein core. All of these forces
combine and
contribute to the overall stability of the folded protein where the degree to
which ideal
packing occurs determines the degree of relative stability of the protein. The
result of
2o maximum packing is to produce a center of residues or hydrophobic core that
has
maximum shielding from solvent.
Since it is likely that lipophilic drugs would reside in the hydrophobic core
of a
peptide, it would require energy to unfold the peptide before the drug can be
released.
The unfolding process requires overcoming the hydrophobic effect by hydrating
the
amino acids or achieving the melting temperature of the protein. The heat of
hydration is
a destabilization of a protein. Typically, the folded state of a protein is
favored by only
5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at
neutral pH and
at room temperature requires chemical reagents. In fact, partial unfolding of
a protein is
often observed prior to the onset of irreversible chemical or conformation
processes.
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Moreover, protein conformation generally controls the rate and extent of
deleterious
chemical reactions.
Conformational protection of active agents by proteins depends on the
stability of
the protein's folded state and the thermodynamics associated with the agent's
decomposition. Conditions necessary for the agent's decomposition should be
different
than for protein unfolding.
Selection of the amino acids will depend on the physical properties desired.
For
instance, if increase in bulk or lipophilicity is desired, then the Garner
polypeptide will be
enriched in the amino acids in the table provided below. Polar amino acids, on
the other
to hand, can be selected to increase the hydrophilicity of the polypeptide.
Ionizing amino acids can be selected for pH controlled peptide unfolding.
Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the
stomach, but will
ionize upon entry into the intestine. Conversely, basic amino acids, such as
histidine,
lysine and arginine, ionize in the stomach and are neutral in an alkaline
environment.
t5 Other factors such as n-~t interactions between aromatic residues, kinking
of the
peptide chain by addition of proline, disulfide crosslinking and hydrogen
bonding can all
be used to select the optimum amino acid sequence for a given application.
Ordering of
the linear sequence can influence how these interactions can be maximized and
is
important in directing the secondary and tertiary structures of the
polypeptide.
20 Furthermore, amino acids with reactive side chains (e.g., glutamic acid,
lysine,
aspartic acid, serine, threonine and cysteine) can be incorporated for
attaching multiple
active agents or adjuvants to the same carrier peptide. This is particularly
useful if a
synergistic effect between two or more active agents is desired.
As stated above, variable molecular weights of the carrier compound can have
25 profound effects on the active agent release kinetics. As a result, low
molecular weight
active agent delivery systems are preferred. An advantage of this invention is
that chain
length and molecular weight of the polypeptide can be optimized depending on
the level
of conformational protection desired. This property can be optimized in
concert with the
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lrinetics of the first order release mechanism. Thus, another advantage of
this invention is
that prolonged release time can be imparted by increasing the molecular weight
of the
Garner polypeptide. Another, significant advantage of the invention is that
the kinetics of
active agent release is primarily controlled by the enzymatic hydrolysis of
the key bond
between the Garner peptide and the active agent.
Dextran is the only polysaccharide known that has been explored as a
macromolecular carrier for the covalent binding of drug for colon specific
drug delivery.
Generally, it was only possible to load up to 1/10 of the total drug-dextran
conjugate
weight with drug. As stated earlier, polysaccharides are digested mainly in
the colon and
drug absorption is mainly limited to the colon. As compared to dextran, this
invention
has two major advantages. First, peptides are hydrolyzed by any one of several
aminopeptidases found in the intestinal lumen or associated with the brush-
border
membrane and so active agent release and subsequent absorption can occur in
the
jejunum or the ileum. Second, the molecular weight of the Garner molecule can
be
is controlled and, thus, active agent loading can also be controlled.
As a practical example, the following table lists the molecular weights of
lipophilic amino acids (less one water molecule) and selected analgesics and
vitamins.
TABLE
Amino acid NiW Active agent MW
Glycine 57 Acetaminophen 151
Alanine 71 Vitamin B6 (Pyroxidine)169
Valine 99 Vitamin C (Ascorbic 176
acid)
Leucine 113 Aspirin 180
Isoleucine 113 Ibuprofen 206
Phenylalanine 147 Retinoic acid 300
Tyrosine 163 Vitamin BZ (Riboflavin)376
Vitamin D2 397
Vitamin E (Tocopherol)431
Lipophilic amino acids are
preferred because conformational
protection through the
2o stomach is important for
the selected active agents,
which were selected based
on ease of
covalent attachment to an Eighteen was subtracted
oligopeptide. from the amino acid's
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molecular weight so that their condensation into a polypeptide is considered.
For
example, a decamer of glycine (MW=588) linked to aspirin would have a total
molecular
weight of 750 and aspirin would represent 24% of the total weight of the
active agent
delivery composition or over two times the maximum drug loading for dextran.
This is
only for an N- or C- terminus application, for those active agents attached to
pendant
groups of decaglutamic acid, for instance, a drug with a molecular weight of
180 could
conceivably have a loading of 58%, although this may not be entirely
practical.
The alcohol, amine or carboxylic acid group of an active agent may be
covalently
attached to the N-terminus, the C-terminus or the side chain of the
oligopeptide or
polypeptide. The location of attachment depends somewhat on the functional
group
selection. For instance, if the active drug is a carboxylic acid (e.g.,
aspirin) then the N-
terminus of the oligopeptide is the preferred point of attachment. If the
active agent is an
amine (e.g., ampicillin), then the C-terminus is the preferred point of
attachment in order
to achieve a stable peptide linked active agent. In both, the C- and N-
terminus examples,
the peptide is, in essence, extended by one monomeric unit forming a new
peptide bond.
If the active agent is an alcohol, then either the C-terminus or the N-
terminus is the
preferred point of attachment in order to achieve a stable composition. As in
the example
above where the alcohol, norethindrone, was covalently attached to
poly(hydroxypropylglutamine), an alcohol can be converted into an
alkylchloroformate
with phosgene. This invention, then, pertains to the reaction of this key
intermediate with
the N-terminus of the peptide carrier. The active ingredient can be released
from the
peptide Garner by intestinal peptidases.
The alcohol can be selectively bound to the gamma carboxylate of glutamic acid
and then this conjugate covalently attached to the C-terminus of the peptide
carrier.
Because the glutamic acid-drug conjugate can be considered a dimer, this
product adds
two monomeric units to the C-terminus of the peptide carrier where the
glutamic acid
moiety serves as a spacer between the peptide and the drug as shown in Fig. 4.
Intestinal
enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drug
moiety
from the peptide carrier. The newly formed free amine of the glutamic acid
residue will
then undergo an intramolecular transamination reaction, thereby, releasing the
active
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agent with coincident formation of pyroglutamic acid as shown in Fig. 5.
Alternatively,
the glutamic acid-drug dimer can be converted into the gamma ester of glutamic
acid N-
carboxyanhydride. This intermediate can then be polymerized, as described
above, using
any suitable initiator as shown in Fig. 4. The product of this polymerization
is
polyglutamic acid with active ingredients attached to multiple pendant groups.
Hence,
maximum drug loading of the Garner peptide can be achieved. 1n addition, other
amino
acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to
impart
specific properties to the drug delivery system.
The invention also provides a method of imparting the same mechanism of action
to for other polypeptides containing functional side chains. Examples include,
but are not
limited to, polylysine, polyasparagine, polyarginine, palyserine,
polycysteine,
polytyrosine, polythreonine and polyglutamine. The mechanism can translate to
these
polypeptides through a spacer or linker on the pendant group, which is
terminated,
preferably, by the glutamic acid-drug dimer. This carrier peptide-drug
conjugate is
15 distinguished from the prior art by virtue of the fact that the primary
release of the drug
moiety relies on peptidases and not on esterases. Alternatively, the active
agent can be
attached directly to the pendant group where some other indigenous enzymes in
the
alimentary tract can affect release.
The active agent can be covalently attached to the N-terminus, the C-terminus
or
2o the side chain of the polypeptide using known techniques. Examples of
linking organic
compounds to the N-terminus type of a peptide include, but are not limited to,
the
attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides
and 1,3-
dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example,
there are
known techniques for forming peptide linked biotin and peptide linked
acridine.
25 In the present invention, adefovir dipivoxil is covalently attached to the
polypeptide via the amino group.
The polypeptide carrier can be prepared using conventional techniques. A
preferred technique is copolymerization of mixtures of amino acid N-
carboxyanhydrides.
~2
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Alternatively, if a specific sequence is desired, a solid state automated
peptide synthesizer
can be used.
The addition of stabilizers to the composition has the potential of
stabilizing the
polypeptide further. Stabilizers such as sugar, amino acids, polyethylene
glycol (PEG)
and salts have been shown to prevent protein unfolding. In another embodiment
of the
invention, a pre-first order release of the active agent is imparted by
microencapsulating
the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid
complex,
PEG or salts.
There is evidence that hydrophilic compounds are absorbed through the
intestinal
epithelia efficiently via specialized transporters. The entire membrane
transport system is
intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one
can expect
that excitation of the membrane transport system will involve some sort of
specialized
adjuvant resulting in localized delivery of active agents. There are seven
known
intestinal transport systems classified according to the physical properties
of the
transported substrate. They include the amino acid, oligopeptide, glucose,
monocarboxic
acid, phosphate, bile acid and the P-glycoprotein transport systems and each
has its own
associated mechanism of transport. The mechanisms can depend on hydrogen ions,
sodium ions, binding sites or other cofactors. The invention also allows
targeting the
mechanisms for intestinal epithelial transport systems to facilitate
absorption of active
agents.
1n another embodiment of the invention, the composition includes one or more
adjuvants to enhance the bioavailability of the active agent. Addition of an
adjuvant is
particularly preferred when using an otherwise poorly absorbed active agent.
Suitable
adjuvants, for example, include: papain, which is a potent enzyme for
releasing the
catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which
activate
enzymes in the BBM; and bile acids, which have been attached to peptides to
enhance
absorption of the peptides.
Preferably, the resultant peptide-adefovir dipivoxil conjugate is formulated
into a
tablet using suitable excipients and can either be wet granulated or dry
compressed.
13
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Compositions of the invention are, in essence, the formation of amides from
acids
and amines and can be prepared by the following examples.
AcidJN-terminus conjugation
An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to
0°C. The solution can then be treated with diisopropylcarbodiimide and
hydroxybenzotriazole followed by the amine peptide carrier. The reaction can
then be
stirred for several hours at room temperature, the urea by-product filtered
off, the product
precipitated out in ether and purified using gel permeation chromatography
(GPC) or
dialysis.
1o AmineJC-terminus conjugation
The peptide carrier can be dissolved in DMF under nitrogen and cooled to
0°C.
The solution can then be treated with diisopropylcarbodiimide and
hydroxybenzotriazole
followed by the amine bioactive agent. The reaction can then be stirred for
several hours
at room temperature, the urea by-product filtered off, and the product
precipitated out in
15 ether and purified using GPC or dialysis.
AlcohollN-Terminus Conjugation
In the following example the combination of the alcohol with triphosgene
produces a chloroformate, which when reacted with the N-terminus of the
peptide
produces a carbamate. Pursuant to this, an alcohol bioactive agent can be
treated with
2o triphosgene in dry DMF under nitrogen. The suitably protected peptide
carrier is then
added slowly and the solution stirred at room temperature for several hours.
The product
is then precipitated out in ether. The crude product is suitably deprotected
and purified
using GPC.
Other solvents, activating agents, cocatalysts and bases can be used. Examples
of
25 other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or
chlorinated
solvents such as chloroform. Examples of other activating agents include
dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst
is N-
14
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hydroxysuccinimide. Examples of bases include pyrrolidinopyridine,
dimethylaminopyridine, triethylamine or tributylamine.
Preparation of y~Alkyl Glutamate
There have been over 30 different y alkyl glutamates prepared any one of which
may be suitable for the drug alcohol of choice. For example, a suspension of
glutamic
acid, the alcohol and concentrated hydrochloric acid can be prepared and
heated for
several hours. The y alkyl glutamate product can be precipitated out in
acetone, filtered,
dried and recrystallized from hot water.
Alkyl GlutamatelC-Terminus Conjugation
The peptide carrier can be dissolved in DMF under nitrogen and cooled to
0°C.
The solution can then be treated with diisopropylcarbodiimide and
hydroxybenzotriazole
followed by the y alkyl glutamate bioactive agent. The reaction can then be
stirred for
several hours at room temperature, the urea by-product filtered off, and the
product
precipitated out in ether and purified using GPC or dialysis.
Preparation of ~y~-Alkyl Glutamate-NCA
y Alkyl glutamate can be suspended in dry THF where triphosgene is added and
the mixture refluxed under a nitrogen atmosphere until the mixture becomes
homogenous. The solution can be poured into heptane to precipitate the NCA
product,
which is filtered, dried and recrystallized from a suitable solvent.
2o Preparation of Poly[y Alkyl Glutamate]
y Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of
a primary amine can be added to the solution until it becomes viscous
(typically
overnight). The product can be isolated from the solution by pouring it into
water and
filtering. The product can be purified using GPC or dialysis.
Although illustrated and described above with reference to specific
embodiments,
the invention is nevertheless not intended to be limited to the details shown.
Rather,
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various modifications may be made in the details within the scope and range of
equivalents of the claims and without departing from the spirit of the
invention.
16
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CLAIMS
What is claimed is:
1. A pharmaceutical composition comprising:
a polypeptide; and
adefovir dipivoxil covalently attached to said polypeptide.
2. The composition of claim 1 wherein said polypeptide is an oligopeptide.
3. The composition of claim 1 wherein said polypeptide is a homopolymer of a
naturally occurring amino acid.
4. The composition of claim 1 wherein said polypeptide is a heteropolymer of
two or more naturally occurring amino acids.
5. The composition of claim 1 wherein said polygeptide is a homopolymer of a
synthetic amino acid.
6. The composition of claim 1 wherein said polypeptide is a heteropolymer of
two or more synthetic amino acids.
7. The composition of claim 1 wherein said polypeptide is a heteropolymer of
one or more naturally occurnng amino acids and one or more synthetic amino
acids.
8. The composition of claim 1 wherein adefovir dipivoxil is covalently
attached
to a side chain, the N-terminus or the C-terminus of said polypeptide.
9. The composition of claim 1 further comprising a microencapsulating agent.
10. The composition of claim 9 wherein said microencapsulating agent is
selected from the group consisting of polyethylene glycol (PEG), an amino
acid, a sugar
and a salt.
11. The composition of claim 1 further comprising an adjuvant.
17
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12. The composition of claim 11 wherein said adjuvant activates an intestinal
transporter.
13. The composition of claim 1 further comprising a pharmaceutically
acceptable
excipient.
14. The composition of claim 1 wherein said composition is in the form of an
ingestable tablet.
15. The composition of claim 1 wherein said camgosition is in the form of an
intravenous preparation.
16. The composition of claim 1 wherein said composition is in the form of an
oral suspension.
17. The composition of claim 1 wherein adefovir dipivoxil is conformationally
protected by folding of said polypeptide about said active agent.
18. The composition of claim 1 wherein said polypeptide is capable of
releasing
adefovir dipivoxil from said composition in a pH-dependent manner.
19. A method for protecting adefovir dipivoxil from degradation comprising
covalently attaching said active agent to a polypeptide.
20. A method for controlling release of adefovir dipivoxil from a composition
wherein said composition comprises a polypeptide, said method comprising
covalently
attaching adefovir dipivoxil to said polypeptide.
21. A method for delivering adefovir dipivoxil to a patient comprising
administering to said patient a composition comprising:
a polypeptide; and
adefovir dipivoxil covalently attached to said polypeptide.
22. The method of claim 21 wherein adefovir dipivoxil is released from said
composition by an enzyme-catalyzed release.
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23. The method of claim 21 wherein adefovir dipivoxil is released from said
composition by a pH-dependent unfolding of said polypeptide.
24. The method of claim 21 wherein said active agent is released from said
composition in a sustained release.
25. The method of claim 21 wherein said composition further comprises an
adjuvant covalently attached to said polypeptide and wherein release of said
adjuvant
from said composition is controlled by said polypeptide.
Abstract
A composition comprising a polypeptide and adefovir dipivoxil covalently
attached to the polypeptide. Also provided is a method for delivery of
adefovir dipivoxil
to a patient comprising administering to the patient a composition comprising
a
polypeptide and adefovir dipivoxil covalently attached to the polypeptide.
Also provided
is a method for protecting adefovir dipivoxil from degradation comprising
covalently
attaching it to a polypeptide. Also provided is a method for controlling
release of
adefovir dipivoxil from a composition comprising covalently attaching it to
the
polypeptide.
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A NOVEL PHARMACEUTICAL COMPOUND CONTAINING ADENOSINE
AND METHODS OF MAKING AND USING SAME
FIELD OF THE INVENTION
The present invention relates to a novel pharmaceutical compound that
comprises
a polypeptide that is preferably covalently attached to adenosine, as well as
methods for
protecting and administering adenosine. This novel compound, referred to as a
CARRIERWAVETN' Molecular Analogue (CMA), has the benefit of taking a known
effective pharmaceutical agent that is both well studied and occupies a known
segment of
l0 the pharmaceutical market, and combining it with a carrier compound that
enhances the
usefulness of the pharmaceutical agent without compromising its pharmaceutical
effectiveness.
BACKGROUND OF THE INVENTION
Adenosine is a known pharmaceutical agent that is used as a coronary
vasodilator.
15 Its chemical name is 9-alpha-D-ribofuranosyl-9H-purin-6-amine. Its
structure is:
N
N ~ N
"N
O
O
O O
The novel pharmaceutical compound of the present invention is useful in
accomplishing one or more of the following goals: enhancement of the chemical
stability
of the original compound; alteration of the release profile of an orally
administered
2o product; enhanced digestion or absorption; targeted delivery to particular
tissuelcell type;
and provision for an oral dosage form when none exists. The novel
pharmaceutical
compound may contain one or more of the following: another active
pharmaceutical
agent, an adjuvant, or an inhibitor.
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Active agent delivery systems are often critical for the effective delivery of
a
biologically active agent (active agent) to the appropriate target. The
importance of these
systems becomes magnified when patient compliance and active agent stability
are taken
under consideration. For instance, one would expect patient compliance to
increase
markedly if an active agent is administered orally in lieu of an injection or
another
invasive technique. Increasing the stability of the active agent, such as
prolonging shelf
life or survival in the stomach, will assure dosage reproducibility and
perhaps even
reduce the number of dosages required which could improve patient compliance.
Absorption of an orally administered active agent is often blocked by the
harshly
acidic stomach milieu, powerful digestive enzymes in the GI tract,
permeability of
cellular membranes and transport across lipid bilayers. Incorporating
adjuvants such as
resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance
permeability of
cellular membranes. Microencapsulating active agents using protenoid
microspheres,
liposomes or polysaccharides have been effective in abating enzyme degradation
of the
active agent. Enzyme inhibiting adjuvants have also been used to prevent
enzyme
degradation. Enteric coatings have been used as a protector of pharmaceuticals
in the
stomach.
Active agent delivery systems also provide the ability to control the release
of the
active agent. For example, formulating diazepam with a copolymer of glutamic
acid and
aspartic acid enables a~sustained release of the active agent. As another
example,
copolymers of lactic acid and glutaric acid are used to provide timed release
of human
growth hormone. A wide range of pharmaceuticals purportedly provide sustained
release
through microencapsulation of the active agent in amides of dicarboxylic
acids, modified
amino acids or thermally condensed amino acids. Slow release rendering
additives can
also be intermixed with a large array of active agents in tablet formulations.
Each of these technologies imparts enhanced stability and time-release
properties
to active agent substances. Unfortunately, these technologies suffer from
several
shortcomings. Incorporation of the active agent is often dependent on
diffusion into the
microencapsulating matrix, which may not be quantitative and may complicate
dosage
2
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reproducibility. In addition, encapsulated drugs rely on diffusion out of the
matrix, which
is highly dependant on the water solubility of the active agent. Conversely,
water-soluble
microspheres swell by an infinite degree and, unfortunately, may release the
active agent
in bursts with little active agent available for sustained release.
Furthermore, in some
technologies, control of the degradation process required for active agent
release is
unreliable. For example, an enterically coated active agent depends on pH to
release the
active agent and, as such, is difficult to control the rate of release.
In the past, use has been made of amino acid side chains of polypeptides as
pendant groups to which active agents can be attached. These technologies
typically
require the use of spacer groups between the amino acid pendant group and the
active
agent. The peptide-drug conjugates of this class of drug delivery system rely
on enzymes
in the bloodstream for the release of the drug and, as such, are not used for
oral
administration. Examples of timed and targeted release of injectable or
subcutaneous
pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer,
to the
~ 5 gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard,
via a peptide
spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been
covalently
attached directly to the beta carboxylate of polyaspartic acid without a
spacer group.
This prodrug formulation was designed as a colon-specific drug delivery system
where
the drug is released by bacterial hydrolytic enzymes residing in the large
intestines. The
released dexamethasone active agent, in turn, was targeted to treat large
bowel disorders
and was not intended to be absorbed into the bloodstream. Yet another
technology
combines the advantages of covalent drug attachment with liposome formation
where the
active ingredient is attached to highly ordered lipid films (known as HARs)
via a peptide
linker. Thus, there has been no drug delivery system, heretofore reported,
that
incorporates the concept of attaching an active ingredient to a polypeptide
pendant group
with its targeted delivery into the bloodstream via oral administration.
It is also important to control the molecular weight, molecular size and
particle
size of the active agent delivery system. Variable molecular weights have
unpredictable
diffusion rates and pharmacokinetics. High molecular weight carriers are
digested slowly
or late, as in the case of naproxen-linked dextran, which is digested almost
exclusively in
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the colon by bacterial enzymes. High molecular weight microspheres usually
have high
moisture content which may present a problem with water labile active
ingredients.
Particle size not only becomes a problem with injectable drugs, as in the HAR
application, but absorption through the brush-border membrane of the
intestines is limited
to less than 5 microns.
SUMMARY OF THE INVENTION
The present invention provides covalent attachment of the active agent
(adenosine) to a polymer of peptides or amino acids. The invention is
distinguished from
the above-mentioned technologies by virtue of covalently attaching adenosine
to the N-
to terminus, the C-terminus or directly to the amino acid side chain of an
oligopepdde or
polypeptide, also referred to herein as a carrier peptide. In certain
applications, the
polypeptide will stabilize the active agent, primarily in the stomach, through
conformational protection. In these applications, delivery of the active agent
is
controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon
entry into the
15 upper intestinal tract, indigenous enzymes release the active ingredient
for absorption by
the body by selectively hydrolyzing the peptide bonds of the carrier peptide.
This
enzymatic action introduces a second order sustained release mechanism.
Alternatively, the present invention provides a pharmaceutical composition
comprising adenosine microencapsulated by a polypeptide.
2o The invention provides a composition comprising a polypeptide and adenosine
covalently attached to the polypeptide. Preferably, the polypeptide is (i) an
oligopeptide,
(ii) a homopolymer of one of the twenty naturally occurnng amino acids, (iii)
a
heteropolymer of two or more naturally occurring amino acids, (iv) a
homopolymer of a
synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids
or (vi) a
25 heteropolymer of one or more naturally occurring amino acids and one or
more synthetic
amino acids.
Adenosine preferably is covalently attached to a side chain, the N-terminus or
the
C-terminus of the polypeptide. In a preferred embodiment, the 'active agent is
a
4
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carboxylic acid and is covalently attached to the N-terminus of the
polypeptide. In
another preferred embodiment, the active agent is an amine and is covalently
attached to
the C-terminus of the polypeptide. In another preferred embodiment, the active
agent is
an alcohol and is covalently attached to the C-terminus of the polypeptide. In
yet another
preferred embodiment, the active agent is an alcohol and is covalently
attached to the N-
terminus of the polypeptide.
The composition of the invention can also include one or more of a
microencapsulating agent, an adjuvant and a pharmaceutically acceptable
excipient. The
microencapsulating agent can be selected from polyethylene glycol (PEG), an
amino
acid, a sugar and a salt. When an adjuvant is included in the composition, the
adjuvant
preferably activates an intestinal transporter.
Preferably, the composition of the invention is in the form of an ingestable
tablet,
an intravenous preparation or an oral suspension. The active agent can be
conformationally protected by folding of the polypeptide about the active
agent. In
another embodiment, the polypeptide is capable of releasing the active agent
from the
composition in a pH-dependent manner.
The invention also provides a method for protecting adenosine from degradation
comprising covalently attaching it to a polypeptide.
The invention also provides a method for delivering adenosine to a patient,
the
2o patient being a human or a non-human animal, comprising administering to
the patient a
composition comprising a polypeptide and an active agent covalently attached
to the
polypeptide. In a preferred embodiment, adenosine is released from the
composition by
an enzyme-catalyzed release. In another preferred embodiment, adenosine is
released in
a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed
release. In another preferred embodiment, the composition further comprises a
microencapsulating agent and adenosine is released from the composition by
dissolution
of the microencapsulating agent. In another preferred embodiment, adenosine is
released
from the composition by a pH-dependent unfolding of the polypeptide. In
another
preferred embodiment, adenosine is released from the composition in a
sustained release.
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In yet another preferred embodiment, the composition further comprises an
adjuvant
covalently attached to the polypeptide and release of the adjuvant from the
composition is
controlled by the polypeptide. The adjuvant can be microencapsulated into a
carrier
peptide-drug conjugate for biphasic release of active ingredients.
The invention also provides a method for preparing a composition comprising a
polypeptide and an active agent covalently attached to the polypeptide. The
method
comprises the steps of:
(a) attaching adenosine to a side chain of an amino acid to form an active
agent/amino acid complex;
(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA)
from the active agendamino acid complex; and
(c) polymerizing the active agendamino acid complex N-carboxyanhydride
(NCA).
In a preferred embodiment, steps (a) and (b) are repeated prior to step (c)
with a
second active agent. When steps (a) and (b) are repeated prior to step (c)
with a second
agent, adenosine and a second active agent can be copolymerized in step (c).
In another
preferred embodiment, the amino acid is glutamic acid and the active agent is
released
from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and
wherein the
active agent is released from the glutamic acid by coincident intramolecular
2o transamination. In another preferred embodiment, the glutamic acid is
replaced by an
amino acid selected from the group consisting of aspartic acid, arginine,
asparagine,
cysteine, lysine, threonine, and serine, and wherein the active agent is
attached to the side
chain of the amino acid to form an amide, a thioester, an ester, an ether, a
urethane, a
carbonate, an anhydride or a carbamate. In yet another preferred embodiment,
the
glutamic acid is replaced by a synthetic amino acid with a pendant group
comprising an
amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.
It is to be understood that both the foregoing general description and the
following detailed description are exemplary, but are not restrictive, of the
invention.
The general applications of this invention to other active pharmaceutical
agents is
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described in U.S: Patent Application Serial Number 09/642,820, filed August
22, 2000,
incorporated herein by reference.
DETAILED DESCRIPTION OF INVENTION
The present invention provides several benefits for active agent delivery.
First,
the invention can stabilize adenosine and prevent its digestion in the
stomach. In
addition, the pharmacologic effect can be prolonged by delayed release of
adenosine.
Furthermore, active agents can be combined to produce synergistic effects.
Also,
absorption of the active agent in the intestinal tract can be enhanced. The
invention also
allows targeted delivery of active agents to specifics sites of action.
Adenosine is both commercially available and readily manufactured using
published synthetic schemes by those of ordinary skill in the art.
The composition of the invention comprises adenosine covalently attached to a
polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a
homopolymer of one
of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or
more
naturally occurnng amino acids, (iv) a homopolymer of a synthetic amino acid,
(v) a
heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of
one or
more naturally occurring amino acids and one or more synthetic amino acids.
Proteins, oligopeptides and polypeptides are polymers of amino acids that have
primary, secondary and tertiary structures. The secondary structure of the
protein is the
local conformation of the polypeptide chain and consists of helices, pleated
sheets and
turns. The protein's amino acid sequence and the structural constraints on the
conformations of the chain determine the spatial arrangement of the molecule.
The
folding of the secondary structure and the spatial arrangement of the side
chains
constitute the tertiary structure.
Proteins fold because of the dynamics associated between neighboring atoms on
the protein and solvent molecules. The thermodynamics of protein folding and
unfolding
are defined by the free energy of a particular condition of the protein that
relies on a
particular model. The process of protein folding involves, amongst other
things, amino
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acid residues packing into a hydrophobic core. The amino acid side chains
inside the
protein core occupy the same volume as they do in amino acid crystals. The
folded
protein interior is therefore more like a crystalline solid than an oil drop
and so the best
model for determining forces contributing to protein stability is the solid
reference state.
The major forces contributing to the thermodynamics of protein folding are Van
der Waals interactions, hydrogen bonds, electrostatic interactions,
configurational
entropy and the hydrophobic effect. Considering protein stability, the
hydrophobic effect
refers to the energetic consequences of removing apolar groups from the
protein interior
and exposing them to water. Comparing the energy of amino acid hydrolysis with
to protein unfolding in the solid reference state, the hydrophobic effect is
the dominant
force. Hydrogen bonds are established during the protein fold process and
intramolecular
bonds are formed at the expense of hydrogen bonds with water. Water molecules
are
"pushed out" of the packed, hydrophobic protein core. All of these forces
combine and
contribute to the overall stability of the folded protein where the degree to
which ideal
packing occurs determines the degree of relative stability of the protein. The
result of
maximum packing is to produce a center of residues or hydrophobic core that
has
maximum shielding from solvent.
Since it is likely that lipophilic drugs would reside in the hydrophobic core
of a
peptide, it would require energy to unfold the peptide before the drug can be
released.
2o The unfolding process requires overcoming the hydrophobic effect by
hydrating the
amino acids or achieving the melting temperature of the protein. The heat of
hydration is
a destabilization of a protein. Typically, the folded state of a protein is
favored by only
5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at
neutral pH and
at room temperature requires chemical reagents. In fact, partial unfolding of
a protein is
often observed prior to the onset of irreversible chemical or conformation
processes.
Moreover, protein conformation generally controls the rate and extent of
deleterious
chemical reactions.
Conformational protection of active agents by proteins depends on the
stability of
the protein's folded state and the thermodynamics associated with the agent's
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decomposition. Conditions necessary for the agent's decomposition should be
different
than for protein unfolding.
Selection of the amino acids will depend on the physical properties desired.
For
instance, if increase in bulk or lipophilicity is desired, then the Garner
polypeptide will be
enriched in the amino acids in the table provided below. Polar amino acids, on
the other
hand, can be selected to increase the hydrophilicity of the polypeptide.
Ionizing amino acids can be selected for pH controlled peptide unfolding.
Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the
stomach, but will
ionize upon entry into the intestine. Conversely, basic amino acids, such as
histidine,
lysine and arginine, ionize in the stomach and are neutral in an alkaline
environment.
Other factors such as n-~ interactions between aromatic residues, kinking of
the
peptide chain by addition of proline, disulfide crosslinking and hydrogen
bonding can all
be used to select the optimum amino acid sequence for a given application.
Ordering of
the linear sequence can influence how these interactions can be maximized and
is
~ 5 important in directing the secondary and tertiary structures of the
polypeptide.
Furthermore, amino acids with reactive side chains (e.g., glutamic acid,
lysine,
aspartic acid, serine, threonine and cysteine) can be incorporated for
attaching multiple
active agents or adjuvants to the same Garner peptide. This is particularly
useful if a
synergistic effect between two or more active agents is desired.
2o As stated above, variable molecular weights of the Garner compound can have
profound effects on the active agent release kinetics. As a result, low
molecular weight
active agent delivery systems are preferred. An advantage of this invention is
that chain
length and molecular weight of the polypeptide can be optimized depending on
the level
of conformational protection desired. This property can be optimized in
concert with the
25 kinetics of the first order release mechanism. Thus, another advantage of
this invention is
that prolonged release time can be imparted by increasing the molecular weight
of the
Garner polypeptide. Another, significant advantage of the invention is that
the kinetics of
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active agent release is primarily controlled by the enzymatic hydrolysis of
the key bond
between the carrier peptide and the active agent.
Dextran is the only polysaccharide known that has been explored as a
macromolecular carrier for the covalent binding of drug for colon specific
drug delivery.
Generally, it was only possible to load up to 1/10 of the total drug-dextran
conjugate
weight with drug. As stated earlier, polysaccharides are digested mainly in
the colon and
drug absorption is mainly limited to the colon. As compared to dextran, this
invention
has two major advantages. First, peptides are hydrolyzed by any one of several
aminopeptidases found in the intestinal lumen or associated with the brush-
border
membrane and so active agent release and subsequent absorption can occur in
the
jejunum or the ileum. Second, the molecular weight of the Garner molecule can
be
controlled and, thus, active agent loading can also be controlled.
As a practical example, the following table lists the molecular weights of
lipophilic amino acids (less one water molecule) and selected analgesics and
vitamins.
t 5 TABLE
Amino acid MW Active anent MW
Glycine 57 Acetaminophen 151
Alanine 71 Vitamin B6 (Pyroxidine) 169
Valine 99 Vitamin C (Ascorbic acid) 176
Leucine 113 Aspirin 180
Isoleucine 113 Ibuprofen 206
Phenylalanine147 Retinoic acid 300
Tyrosine 163 Vitamin BZ (Riboflavin) 376
Vitamin D2 397
Vitamin E (Tocopherol) 431
Lipophilic amino acids are preferred because conformational protection through
the
stomach is important for the selected active agents, which were selected based
on ease of
covalent attachment to an oligopeptide. Eighteen was subtracted from the amino
acid's
molecular weight so that their condensation into a polypeptide is considered.
For
example, a decamer of glycine (MW=588) linked to aspirin would have a total
molecular
weight of 750 and aspirin would represent 24% of the total weight of the
active agent
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delivery composition or over two times the maximum drug loading for dextran.
This is
only for an N- or C- terminus application, for those active agents attached to
pendant
groups of decaglutamic acid, for instance, a drug with a molecular weight of
180 could
conceivably have a loading of 58%, although this may not be entirely
practical.
The alcohol, amine or carboxylic acid group of an active agent may be
covalently
attached to the N-terminus, the C-terminus or the side chain of the
oligopeptide or
polypeptide. The location of attachment depends somewhat on the functional
group
selection. For instance, if the active drug is a carboxylic acid (e.g.,
aspirin) then the N-
terminus of the oligopeptide is the preferred point of attachment. If the
active agent is an
to amine (e.g., ampicillin), then the C-terminus is the preferred point of
attachment in order
to achieve a stable peptide linked active agent. In both, the C- and N-
terminus examples,
the peptide is, in essence, extended by one monomeric unit forming a new
peptide bond.
If the active agent is an alcohol, then either the C-terminus or the N-
terminus is the
preferred point of attachment in order to achieve a stable composition. As in
the example
above where the alcohol, norethindrone, was covalently attached to
poly(hydroxypropylglutamine), an alcohol can be convened into an
alkylchloroformate
with phosgene. This invention, then, pertains to the reaction of this key
intermediate with
the N-terminus of the peptide Garner. The active ingredient can be released
from the
peptide carrier by intestinal peptidases.
2o The alcohol can be selectively bound to the gamma carboxylate of glutamic
acid
and then this conjugate covalently attached to the C-terminus of the peptide
carrier.
Because the glutamic acid-drug conjugate can be considered a dimer, this
product adds
two monomeric units to the C-terminus of the peptide carrier where the
glutamic acid
moiety serves as a spacer between the peptide and the drug as shown in Fig. 4.
Intestinal
enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drug
moiety
from the peptide carrier. The newly formed free amine of the glutamic acid
residue will
then undergo an intramolecular transamination reaction, thereby, releasing the
active
agent with coincident formation of pyroglutamic acid as shown in Fig. 5.
Alternatively,
the glutamic acid-drug dimer can be converted into the gamma ester of glutamic
acid N-
carboxyanhydride. This intermediate can then be polymerized, as described
above, using
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any suitable initiator as shown in Fig. 4. The product of this polymerization
is
polyglutamic acid with active ingredients attached to multiple pendant groups.
Hence,
maximum drug loading of the corner peptide can be achieved. In addition, other
amino
acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to
impart
specific properties to the drug delivery system.
The invention also provides a method of imparting the same mechanism of action
for other polypeptides containing functional side chains. Examples include,
but are not
limited to, polylysine, polyasparagine, polyarginine, polyserine,
polycysteine,
polytyrosine, polythreonine and polyglutamine. The mechanism can translate to
these
polypeptides through a spacer or linker on the pendant group, which is
terminated,
preferably, by the glutamic acid-drug dimer. This carrier peptide-drug
conjugate is
distinguished from the prior art by virtue of the fact that the primary
release of the drug
moiety relies on peptidases and not on esterases. Alternatively, the active
agent can be
attached directly to the pendant group where some other indigenous enzymes in
the
alimentary tract can affect release.
The active agent can be covalently attached to the N-terminus, the C-terminus
or
the side chain of the polypeptide using known techniques. Examples of linking
organic
compounds to the N-terminus type of a peptide include, but are not limited to,
the
attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides
and 1,3-
dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example,
there are
known techniques for forming peptide linked biotin and peptide linked
acridine.
In the present invention, adenosine is covalently attached to the polypeptide
via
the ribose hydroxyl group.
The polypeptide corner can be prepared using conventional techniques. A
preferred technique is copolymerization of mixtures of amino acid N-
carboxyanhydrides.
Alternatively, if a specific sequence is desired, a solid state automated
peptide synthesizer
can be used.
12
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The addition of stabilizers to the composition has the potential of
stabilizing the
polypeptide further. Stabilizers such as sugar, amino acids, polyethylene
glycol (PEG)
and salts have been shown to prevent protein unfolding. In another embodiment
of the
invention, a pre-first order release of the active agent is imparted by
microencapsulating
the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid
complex,
PEG or salts.
There is evidence that hydrophilic compounds are absorbed through the
intestinal
epithelia efficiently via specialized transporters. The entire membrane
transport system is
intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one
can expect
to that excitation of the membrane transport system will involve some sort of
specialized
adjuvant resulting in localized delivery of active agents. There are seven
known
intestinal transport systems classified according to the physical properties
of the
transported substrate. They include the amino acid, oligopeptide, glucose,
monocarboxic
acid, phosphate, bile acid and the P-glycoprotein transport systems and each
has its own
15 associated mechanism of transport. The mechanisms can depend on hydrogen
ions,
sodium ions, binding sites or other cofactors. The invention also allows
targeting the
mechanisms for intestinal epithelial transport systems to facilitate
absorption of active
agents.
In another embodiment of the invention, the composition includes one or more
2o adjuvants to enhance the bioavailability of the active agent. Addition of
an adjuvant is
particularly preferred when using an otherwise poorly absorbed active agent.
Suitable
adjuvants, for example, include: papain, which is a potent enzyme for
releasing the
catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which
activate
enzymes in the BBM; and bile acids, which have been attached to peptides to
enhance
25 absorption of the peptides.
Preferably, the resultant peptide-adenosine conjugate is formulated into a
tablet
using suitable excipients and can either be wet granulated or dry compressed.
Compositions of the invention are, in essence, the formation of amides from
acids
and amines and can be prepared by the following examples.
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Acid/N-terminus conjugation
An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to
0°C. The solution can then be treated with diisopropylcarbodiimide and
hydroxybenzotriazole followed by the amine peptide carrier. The reaction can
then be
stirred for several hours at room temperature, the urea by-product filtered
off, the product
precipitated out in ether and purified using gel permeation chromatography
(GPC) or
dialysis.
AminelC-terminus conjugation
The peptide carrier can be dissolved in DMF under nitrogen and cooled to
0°C.
The solution can then be treated with diisopropylcarbodiimide and
hydroxybenzotriazole
followed by the amine bioactive agent. The reaction can then be stirred for
several hours
at room temperature, the urea by-product filtered off, and the product
precipitated out in
ether and purified using GPC or dialysis.
AlcohoUN-Terminus Conjugation
In the following example the combination of the alcohol with triphosgene
produces a chloroformate, which when reacted with the N-terminus of the
peptide
produces a carbamate. Pursuant to this, an alcohol bioactive agent can be
treated with
triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier
is then
added slowly and the solution stirred at room temperature for several hours.
The product
is then precipitated out in ether. The crude product is suitably deprotected
and purified
using GPC.
Other solvents, activating agents, cocatalysts and bases can be used. Examples
of
other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or
chlorinated
solvents such as chloroform. Examples of other activating agents include
dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst
is N-
hydroxysuccinimide. Examples of bases include pyrrolidinopyridine,
dimethylaminopyridine, triethylamine or tributylamine.
14
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Preparation of Alkyl Glutamate
There have been over 30 different y alkyl glutamates prepared any one of which
may be suitable for the drug alcohol of choice. For example, a suspension of
glutamic
acid, the alcohol and concentrated hydrochloric acid can be prepared and
heated for
several hours. The y alkyl glutamate product can be precipitated out in
acetone, filtered,
dried and recrystallized from hot water.
Alkyl GlutamatelC-Terminus Conjugation
The peptide Garner can be dissolved in DMF under nitrogen and cooled to
0°C.
The solution can then be treated with diisopropylcarbodiimide and
hydroxybenzotriazole
1o followed by the y alkyl glutamate bioactive agent. The reaction can then be
stirred for
several hours at room temperature, the urea by-product filtered off, and the
product
precipitated out in ether and purified using GPC or dialysis.
Preparation of Alkyl Glutamate-NCA
y Alkyl glutamate can be suspended in dry THF where triphosgene is added and
15 the mixture refluxed under a nitrogen atmosphere until the mixture becomes
homogenous. The solution can be poured into heptane to precipitate the NCA
product,
which is filtered, dried and recrystallized from a suitable solvent.
Preparation of Poly[Y Alkyl Glutamate]
y Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of
2o a primary amine can be added to the solution until it becomes viscous
(typically
overnight). The product can be isolated from the solution by pouring it into
water and
filtering. The product can be purified using GPC or dialysis.
Although illustrated and described above with reference to specific
embodiments,
the invention is nevertheless not intended to be limited to the details shown.
Rather,
25 various modifications may be made in the details within the scope and range
of
equivalents of the claims and without departing from the spirit of the
invention.
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CLAIMS
What is claimed is:
1. A pharmaceutical composition comprising:
a polypeptide; and
adenosine covalently attached to said polypeptide.
2. The composition of claim 1 wherein said polypeptide is an oligopeptide.
3. The composition of claim 1 wherein said polypeptide is a homopolymer of a
naturally occurring amino acid.
4. The composition of claim 1 wherein said polypeptide is a heteropolymer of
two or more naturally occurring amino acids.
5. The composition of claim 1 wherein said polypeptide is a homopolymer of a
synthetic amino acid.
6. The composition of claim 1 wherein said polypeptide is a heteropolymer of
two or more synthetic amino acids.
7. The composition of claim 1 wherein said polypeptide is a heteropolymer of
one or more naturally occurring amino acids and one or more synthetic amino
acids.
8. The composition of claim 1 wherein adenosine is covalently attached to a
side
chain, the N-terminus or the C-terminus of said polypeptide.
9. The composition of claim 1 further comprising a microencapsulating agent.
10. The composition of claim 9 wherein said microencapsulating agent is
selected from the group consisting of polyethylene glycol (PEG), an amino
acid, a sugar
and a salt.
11. The composition of claim 1 further comprising an adjuvant.
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12. The composition of claim 11 wherein said adjuvant activates an intestinal
transporter.
13. The composition of claim 1 further comprising a pharmaceutically
acceptable
excipient.
14. The composition of claim 1 wherein said composition is in the form of an
ingestable tablet.
15. The composition of claim 1 wherein said composition is in the form of an
intravenous preparation.
16. The composition of claim 1 wherein said composition is in the form of an
oral suspension.
17. The composition of claim 1 wherein adenosine is conformationally protected
by folding of said polypeptide about said active agent.
18. The composition of claim 1 wherein said polypeptide is capable of
releasing
adenosine from said composition in a pH-dependent manner.
19. A method for protecting adenosine from degradation comprising covalently
attaching said active agent to a polypeptide.
20. A method for controlling release of adenosine from a composition wherein
said composition comprises a polypeptide, said method comprising covalently
attaching
adenosine to said polypeptide.
21. A method for delivering adenosine to a patient comprising administering to
said patient a composition comprising:
a polypeptide; and
adenosine covalently attached to said polypeptide.
22. The method of claim 21 wherein adenosine is released from said composition
by an enzyme-catalyzed release.
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23. The method of claim 21 wherein adenosine is released from said composition
by a pH-dependent unfolding of said polypeptide.
24. The method of claim 21 wherein said active agent is released from said
composition in a sustained release.
25. The method of claim 21 wherein said composition further comprises an
adjuvant covalently attached to said polypeptide and wherein release of said
adjuvant
from said composition is controlled by said polypeptide.
~8
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Abstract
A composition comprising a polypeptide and adenosine covalently attached to
the
polypeptide. Also provided is a method for delivery of adenosine to a patient
comprising
administering to the patient a composition comprising a polypeptide and
adenosine
covalently attached to the polypeptide. Also provided is a method for
protecting
adenosine from degradation comprising covalently attaching it to a
polypeptide. Also
provided is a method for controlling release of adenosine from a composition
comprising
covalently attaching it to the polypeptide.
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A NOVEL PHARMACEUTICAL COMPOUND CONTAINING
ADRENOCORTICOTROPIC HORMONE AND METHODS OF MAKING AND
USING SAME
FIELD OF THE INVENTION
The present invention relates to a novel pharmaceutical compound that
comprises
a polypeptide that is preferably covalently attached to adrenocorticotropic
hormone, as
well as methods for protecting and administering adrenocorticotropic hormone.
This
novel compound, referred to as a CARRIERWAVETM Molecular Analogue (CMA), has
1o the benefit of taking a known effective pharmaceutical agent that is both
well studied and
occupies a known segment of the pharmaceutical market, and combining it with a
carrier
compound that enhances the usefulness of the pharmaceutical agent without
compromising its pharmaceutical effectiveness.
BACKGROUND OF THE INVENTION
15 Adrenocorticotropic hormone is a known pharmaceutical agent that is useful
for
the diagnosis of Addison's disease and other conditions in which the
functionality of the
adrenal cortex is to be determined. It is both commercially available and
readily
manufactured using published synthetic schemes by those of ordinary skill in
the art.
The novel pharmaceutical compound of the present invention is useful in
20 accomplishing one or more of the following goals: enhancement of the
chemical stability
of the original compound; alteration of the release profile of an orally
administered
product; enhanced digestion or absorption; targeted delivery to particular
tissue/cell type;
and provision for an oral dosage form when none exists. The novel
pharmaceutical
compound may contain one or more of the following: another active
pharmaceutical
25 agent, an adjuvant, or an inhibitor.
Active agent delivery systems are often critical for the effective delivery of
a
biologically active agent (active agent) to the appropriate target. The
importance of these
systems becomes magnified when patient compliance and active agent stability
are taken
under consideration. For instance, one would expect patient compliance to
increase
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markedly if an active agent is administered orally in lieu of an injection or
another
invasive technique. Increasing the stability of the active agent, such as
prolonging shelf
life or survival in the stomach, will assure dosage reproducibility and
perhaps even
reduce the number of dosages required which could improve patient compliance.
Absorption of an orally administered active agent is often blocked by the
harshly
acidic stomach milieu, powerful digestive enzymes in the GI tract,
permeability of
cellular membranes and transport across lipid bilayers. Incorporating
adjuvants such as
resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance
permeability of
cellular membranes. Microencapsulating active agents using protenoid
microspheres,
l0 liposomes or polysaccharides have been effective in abating enzyme
degradation of the
active agent. Enzyme inhibiting adjuvants have also been used to prevent
enzyme
degradation. Enteric coatings have been used as a protector of pharmaceuticals
in the
stomach.
Active agent delivery systems also provide the ability to control the release
of the
active agent. For example, formulating diazepam with a copolymer of glutamic
acid and
aspartic acid enables a sustained release of the active agent. As another
example,
copolymers of lactic acid and glutaric acid are used to provide timed release
of human
growth hormone. A wide range of pharmaceuticals purportedly provide sustained
release
through microencapsulation of the active agent in amides of dicarboxylic
acids, modified
2o amino acids or thermally condensed amino acids. Slow release rendering
additives can
also be intermixed with a large array of active agents in tablet formulations.
Each of these technologies imparts enhanced stability and time-release
properties
to active agent substances. Unfortunately, these technologies suffer from
several
shortcomings. Incorporation of the active agent is often dependent on
diffusion into the
microencapsulating matrix, which may not be quantitative and may complicate
dosage
reproducibility. In addition, encapsulated drugs rely on diffusion out of the
matrix, which
is highly dependant on the water solubility of the active agent. Conversely,
water-soluble
microspheres swell by an infinite degree and, unfortunately, may release the
active agent
in bursts with little active agent available for sustained release.
Furthermore, in some
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technologies, control of the degradation process required for active agent
release is
unreliable. For example, an enterically coated active agent depends on pH to
release the
active agent and, as such, is difficult to control the rate of release.
In the past, use has been made of amino acid side chains of polypeptides as
pendant groups to which active agents can be attached. These technologies
typically
require the use of spacer groups between the amino acid pendant group and the
active
agent. The peptide-drug conjugates of this class of drug delivery system rely
on enzymes
in the bloodstream for the release of the drug and, as such, are not used for
oral
administration. Examples of timed and targeted release of injectable or
subcutaneous
1o pharmaceuticals include: linking of norethindrone, via a hydroxypropyl
spacer, to the
gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a
peptide
spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been
covalently
attached directly to the beta carboxylate of polyaspartic acid without a
spacer group.
This prodrug formulation was designed as a colon-specific drug delivery system
where
the drug is released by bacterial hydrolytic enzymes residing in the large
intestines. The
released dexamethasone active agent, in turn, was targeted to treat large
bowel disorders
and was not intended to be absorbed into the bloodstream. Yet another
technology
combines the advantages of covalent drug attachment with liposome formation
where the
active ingredient is attached to highly ordered lipid films (known as HARs)
via a peptide
linker. Thus, there has been no drug delivery system, heretofore reported,
that
incorporates the concept of attaching an active ingredient to a polygeptide
pendant group
with its targeted delivery into the bloodstream via oral administration.
It is also important to control the molecular weight, molecular size and
particle
size of the active agent delivery system. Variable molecular weights have
unpredictable
diffusion rates and pharmacokinetics. High molecular weight carriers are
digested slowly
or late, as in the case of naproxen-linked dextran, which is digested almost
exclusively in
the colon by bacterial enzymes. High molecular weight microspheres usually
have high
moisture content which may present a problem with water labile active
ingredients.
Particle size not only becomes a problem with injectable drugs, as in the HAR
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application, but absorption through the brush-border membrane of the
intestines is limited
to less than 5 microns.
SUMMARY OF THE INVENTION
The present invention provides covalent attachment of the active agent
(adrenocorticotropic hormone) to a polymer of peptides or amino acids. The
invention is
distinguished from the above-mentioned technologies by virtue of covalently
attaching
adrenocorticotropic hormone to the N-terminus, the C-terminus or directly to
the amino
acid side chain of an oligopeptide or polypeptide, also referred to herein as
a carrier
peptide. In certain applications, the polypeptide will stabilize the active
agent, primarily
1o in the stomach, through conformational protection. In these applications,
delivery of the
active agent is controlled, in part, by the kinetics of unfolding of the
carrier peptide.
Upon entry into the upper intestinal tract, indigenous enzymes release the
active
ingredient for absorption by the body by selectively hydrolyzing the peptide
bonds of the
carrier peptide. This enzymatic action introduces a second order sustained
release
mechanism.
Alternatively, the present invention provides a pharmaceutical composition
comprising adrenocorticotropic hormone microencapsulated by a polypeptide.
The invention provides a composition comprising a polypeptide and
adrenocorticotropic hormone covalently attached to the polypeptide.
Preferably, the
2o polypeptide is (i) an oligopeptide, (ii) a homopolymer of one of the twenty
naturally
occurring amino acids, (iii) a heteropolymer of two or more naturally occurnng
amino
acids, (iv) a homopolymer of a synthetic amino acid, (v) a heteropolymer of
two or more
synthetic amino acids or (vi) a heteropolymer of one or more naturally
occurring amino
acids and one or more synthetic amino acids.
Adrenocorticotropic hormone preferably is covalently attached to a side chain,
the N-terminus or the C-terminus of the polypeptide. In a preferred
embodiment, the
active agent is a carboxylic acid and is covalently attached to the N-terminus
of the
polypeptide. In another preferred embodiment, the active agent is an amine and
is
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covalently attached to the C-terminus of the polypeptide. In another preferred
embodiment, the active agent is an alcohol and is covalently attached to the C-
terminus
of the polypeptide. In yet another preferred embodiment, the active agent is
an alcohol
and is covalently attached to the N-terminus of the polypeptide.
The composition of the invention can also include one or more of a
microencapsulating agent, an adjuvant and a pharmaceutically acceptable
excipient. The
microencapsulating agent can be selected from polyethylene glycol (PEG), an
amino
acid, a sugar and a salt. When an adjuvant is included in the composition, the
adjuvant
preferably activates an intestinal transporter.
Preferably, the composition of the invention is in the form of an ingestable
tablet,
an intravenous preparation or an oral suspension. The active agent can be
conformationally protected by folding of the polypeptide about the active
agent. In
another embodiment, the polypeptide is capable of releasing the active agent
from the
composition in a pH-dependent manner.
The invention also provides a method for protecting adrenocorticotropic
hormone
from degradation comprising covalently attaching it to a polypeptide.
The invention also provides a method for delivering adrenocorticotropic
hormone
to a patient, the patient being a human or a non-human animal, comprising
administering
to the patient a composition comprising a polypeptide and an active agent
covalently
attached to the polypeptide. In a preferred embodiment, adrenocorticotropic
hormone is
released from the composition by an enzyme-catalyzed release. In another
preferred
embodiment, adrenocorticotropic hormone is released in a time-dependent manner
based
on the pharmacokinetics of the enzyme-catalyzed release. In another preferred
embodiment, the composition further comprises a microencapsulating agent and
adrenocorticotropic hormone is released from the composition by dissolution of
the
microencapsulating agent. In another preferred embodiment, adrenocorticotropic
hormone is released from the composition by a pH-dependent unfolding of the
polypeptide. In another preferred embodiment, adrenocorticotropic hormone is
released
from the composition in a sustained release. In yet another preferred
embodiment, the
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composition further comprises an adjuvant covalently attached to the
polypeptide and
release of the adjuvant from the composition is controlled by the polypeptide.
The
adjuvant can be microencapsulated into a carrier peptide-drug conjugate for
biphasic
release of active ingredients.
The invention also provides a method for preparing a composition comprising a
polypeptide and an active agent covalently attached to the polypeptide. The
method
comprises the steps of:
(a) attaching adrenocorcicotropic hormone to a side chain of an amino acid to
form an
active agent/amino acid complex;
(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA)
from the active agentJamino acid complex; and
(c) polymerizing the active agendamino acid complex N-carboxyanhydride
(NCA).
In a preferred embodiment, steps (a) and (b) are repeated prior to step (c)
with a
second active agent. When steps (a) and (b) are repeated prior to step (c)
with a second
agent, adrenocorticotropic hormone and a second active agent can be
copolymerized in
step (c). In another preferred embodiment, the amino acid is glutamic acid and
the active
agent is released from the glutamic acid as a dimer upon a hydrolysis of the
polypeptide
and wherein the active agent is released from the glutamic acid by coincident
intramolecular transamination. In another preferred embodiment, the glutamic
acid is
replaced by an amino acid selected from the group consisting of aspartic acid,
arginine,
asparagine, cysteine, lysine, threonine, and serine, and wherein the active
agent is
attached to the side chain of the amino acid to form an amide, a thioester, an
ester, an
ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another
preferred
embodiment, the glutamic acid is replaced by a synthetic amino acid with a
pendant
group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an
acid
functionality.
It is to be understood that both the foregoing general description and the
following detailed description are exemplary, but are not restrictive, of the
invention.
The general applications of this invention to other active pharmaceutical
agents is
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described in U.S. Patent Application Serial Number 09/642,820, filed August
22, 2000,
incorporated herein by reference.
DETAILED DESCRIPTION OF INVENTION
The present invention provides several benefits for active agent delivery.
First,
the invention can stabilize adrenocorticotropic hormone and prevent its
digestion in the
stomach. In addition, the pharmacologic effect can be prolonged by delayed
release of
adrenocorticotropic hormone. Furthermore, active agents can be combined to
produce
synergistic effects. Also, absorption of the active agent in the intestinal
tract can be
enhanced. The invention also allows targeted delivery of active agents to
specifics sites
of action.
The composition of the invention comprises adrenocorticotropic hormone
covalently attached to a polypeptide. Preferably, the polypeptide is (i) an
oligopeptide,
(ii) a homopolymer of one of the twenty naturally occurnng amino acids, (iii)
a
heteropolymer of two or more naturally occurring amino acids, (iv) a
homopolymer of a
IS synthetic amino acid, (v) a heteropolymer of two or more synthetic amino
acids or (vi) a
heteropolymer of one or more naturally occurring amino acids and one or more
synthetic
amino acids.
Proteins, oligopeptides and polypeptides are polymers of amino acids that have
primary, secondary and tertiary structures. The secondary structure of the
protein is the
local conformation of the polypeptide chain and consists of helices, pleated
sheets and
turns. The protein's amino acid sequence and the structural constraints on the
conformations of the chain determine the spatial arrangement of the molecule.
The
folding of the secondary structure and the spatial arrangement of the side
chains
constitute the tertiary structure.
Proteins fold because of the dynamics associated between neighboring atoms on
the protein and solvent molecules. The thermodynamics of protein folding and
unfolding
are defined by the free energy of a particular condition of the protein that
relies on a
particular model. The process of protein folding involves, amongst other
things, amino
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acid residues packing into a hydrophobic core. The amino acid side chains
inside the
protein core occupy the same volume as they do in amino acid crystals. The
folded
protein interior is therefore more like a crystalline solid than an oil drop
and so the best
model for determining forces contributing to protein stability is the solid
reference state.
The major forces contributing to the thermodynamics of protein folding are Van
der Waals interactions, hydrogen bonds, electrostatic interactions,
configurational
entropy and the hydrophobic effect. Considering protein stability, the
hydrophobic effect
refers to the energetic consequences of removing apolar groups from the
protein interior
and exposing them to water. Comparing the energy of amino acid hydrolysis with
protein unfolding in the solid reference state, the hydrophobic effect is the
dominant
force. Hydrogen bonds are established during the protein fold process and
intramolecular
bonds are formed at the expense of hydrogen bonds with water. Water molecules
are
"pushed out" of the packed, hydrophobic protein core. All of these forces
combine and
contribute to the overall stability of the folded protein where the degree to
which ideal
packing occurs determines the degree of relative stability of the protein. The
result of
maximum packing is to produce a center of residues or hydrophobic core that
has
maximum shielding from solvent.
Since it is likely that lipophilic drugs would reside in the hydrophobic core
of a
peptide, it would require energy to unfold the peptide before the drug can be
released.
2o The unfolding process requires overcoming the hydrophobic effect by
hydrating the
amino acids or achieving the melting temperature of the protein. The heat of
hydration is
a destabilization of a protein. Typically, the folded state of a protein is
favored by only
5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at
neutral pH and
at room temperature requires chemical reagents. In fact, partial unfolding of
a grotein is
often observed prior to the onset of irreversible chemical or conformation
processes.
Moreover, protein conformation generally controls the rate and extent of
deleterious
chemical reactions.
Conformational protection of active agents by proteins depends on the
stability of
the protein's folded state and the thermodynamics associated with the agent's
8
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decomposition. Conditions necessary for the agent's decomposition should be
different
than for protein unfolding.
Selection of the amino acids will depend on the physical properties desired.
For
instance, if increase in bulk or lipophilicity is desired, then the carrier
polypeptide will be
enriched in the amino acids in the table provided below. Polar amino acids, on
the other
hand, can be selected to increase the hydrophilicity of the polypeptide.
Ionizing amino acids can be selected for pH controlled peptide unfolding.
Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the
stomach, but will
ionize upon entry into the intestine. Conversely, basic amino acids, such as
histidine,
lysine and arginine, ionize in the stomach and are neutral in an alkaline
environment.
Other factors such as ~-n interactions between aromatic residues, kinking of
the
peptide chain by addition of proline, disulfide crosslinking and hydrogen
bonding can all
be used to select the optimum amino acid sequence for a given application.
Ordering of
the linear sequence can influence how these interactions can be maximized and
is
important in directing the secondary and tertiary structures of the
polypeptide.
Furthermore, amino acids with reactive side chains (e.g., glutamic acid,
lysine,
aspartic acid, serine, threonine and cysteine) can be incorporated for
attaching multiple
active agents or adjuvants to the same Garner peptide. This is particularly
useful if a
synergistic effect between two or more active agents is desired.
As stated above, variable molecular weights of the carrier compound can have
profound effects on the active agent release kinetics. As a result, low
molecular weight
active agent delivery systems are preferred. An advantage of this invention is
that chain
length and molecular weight of the polypeptide can be optimized depending on
the level
of conformational protection desired. This property can be optimized in
concert with the
kinetics of the first order release mechanism. Thus, another advantage of this
invention is
that prolonged release time can be imparted by increasing the molecular weight
of the
carrier polypeptide. Another, significant advantage of the invention is that
the kinetics of
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active agent release is primarily controlled by the enzymatic hydrolysis of
the key bond
between the Garner peptide and the active agent.
Dextran is the only polysaccharide known that has been explored as a
macromolecular carrier for the covalent binding of drug for colon specific
drug delivery.
Generally, it was only possible to load up to 1/10 of the total drug-dextran
conjugate
weight with drug. As stated earlier, polysaccharides are digested mainly in
the colon and
drug absorption is mainly limited to the colon. As compared to dextran, this
invention
has two major advantages. First, peptides are hydrolyzed by any one of several
aminopeptidases found in the intestinal lumen or associated with the brush-
border
membrane and so active agent release and subsequent absorption can occur in
the
jejunum or the ileum. Second, the molecular weight of the carrier molecule can
be
controlled and, thus, active agent loading can also be controlled.
As a practical example, the following table lists the molecular weights of
lipophilic amino acids (less one water molecule) and selected analgesics and
vitamins.
TABLE
Amino acid MW Active a,.gent MW
Glycine 57 Acetaminophen 151
Alanine 71 Vitamin B6 (Pyroxidine) 169
Valine 99 Vitamin C (Ascorbic acid) 176
Leucine 113 Aspirin 180
Isoleucine 113 Ibuprofen 206
Phenylalanine147 Retinoic acid 300
Tyrosine 163 Vitamin BZ (Riboflavin) 3?6
Vitamin DZ 397
Vitamin E (Tocopherol) 431
Lipophilic amino acids are preferred because conformational protection through
the
stomach is important for the selected active agents, which were selected based
on ease of
covalent attachment to an oligopeptide. Eighteen was subtracted from the amino
acid's
molecular weight so that their condensation into a polypeptide is considered.
For
example, a decamer of glycine (MW=588) linked to aspirin would have a total
molecular
weight of 750 and aspirin would represent 24% of the total weight of the
active agent
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delivery composition or over two times the maximum drug loading for dextran.
This is
only for an N- or C- terminus application, for those active agents attached to
pendant
groups of decaglutamic acid, for instance, a drug with a molecular weight of
180 could
conceivably have a loading of 58%, although this may not be entirely
practical.
The alcohol, amine or carboxylic acid group of an active agent may be
covalently
attached to the N-terminus, the C-terminus or the side chain of the
oligopeptide or
polypeptide. The location of attachment depends somewhat on the functional
group
selection. For instance, if the active drug is a carboxylic acid (e.g.,
aspirin) then the N-
terminus of the oligopeptide is the preferred point of attachment. If the
active agent is an
to amine (e.g., ampicillin), then the C-terminus is the preferred point of
attachment in order
to achieve a stable peptide linked active agent. In both, the C- and N-
terminus examples,
the peptide is, in essence, extended by one monomeric unit forming a new
peptide bond.
If the active agent is an alcohol, then either the C-terminus or the N-
terminus is the
preferred point of attachment in order to achieve a stable composition. As in
the example
above where the alcohol, norethindrone, was covalently attached to
poly(hydroxypropylglutamine), an alcohol can be converted into an
alkylchloroformate
with phosgene. This invention, then, pertains to the reaction of this key
intermediate with
the N-terminus of the peptide carrier. The active ingredient can be released
from the
peptide carrier by intestinal peptidases.
2o The alcohol can be selectively bound to the gamma carboxylate of glutamic
acid
and then this conjugate covalently attached to the C-terminus of the peptide
carrier.
Because the glutamic acid-drug conjugate can be considered a dimer, this
product adds
two monomeric units to the C-terminus of the peptide carrier where the
glutamic acid
moiety serves as a spacer between the peptide and the drug as shown in Fig. 4.
Intestinal
enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drug
moiety
from the peptide carrier. The newly formed free amine of the glutamic acid
residue will
then undergo an intramolecular transamination reaction, thereby, releasing the
active
agent with coincident formation of pyroglutamic acid as shown in Fig. 5.
Alternatively,
the glutamic acid-drug dimer can be converted into the gamma ester of glutamic
acid N-
3o carboxyanhydride. This intermediate can then be polymerized, as described
above, using
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any suitable initiator as shown in Fig. 4. The product of this polymerization
is
polyglutamic acid with active ingredients attached to multiple pendant groups.
Hence,
maximum drug loading of the Garner peptide can be achieved. In addition, other
amino
acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to
impart
specific properties to the drug delivery system.
The invention also provides a method of imparting the same mechanism of action
for other polypeptides containing functional side chains. Examples include,
but are not
limited to, polylysine, polyasparagine, polyarginine, polyserine,
polycysteine,
polytyrosine, polythreonine and polyglutamine. The mechanism can translate to
these
l0 polypeptides through a spacer or linker on the pendant group, which is
terminated,
preferably, by the glutamic acid-drug dimer. This carrier peptide-drug
conjugate is
distinguished from the prior art by virtue of the fact that the primary
release of the drug
moiety relies on peptidases and not on esterases. Alternatively, the active
agent can be
attached directly to the pendant group where some other indigenous enzymes in
the
15 alimentary tract can affect release.
The active agent can be covalently attached to the N-terminus, the C-terminus
or
the side chain of the polypeptide using known techniques. Examples of linking
organic
compounds to the N-terminus type of a peptide include, but are not limited to,
the
attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides
and 1,3-
20 dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another
example, there are
known techniques for forming peptide linked biotin and peptide linked
acridine.
In the present invention, adrenocorticotropic hormone is covalently attached
to
the polypeptide via an amide bond.
The polypeptide carrier can be prepared using conventional techniques. A
25 preferred technique is copolymerization of mixtures of amino acid N-
carboxyanhydrides.
Alternatively, if a specific sequence is desired, a solid state automated
peptide synthesizer
can be used.
12
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The addition of stabilizers to the composition has the potential of
stabilizing the
polypeptide further. Stabilizers such as sugar, amino acids, polyethylene
glycol (PEG)
and salts have been shown to prevent protein unfolding. In another embodiment
of the
invention, a pre-first order release of the active agent is imparted by
microencapsulating
the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid
complex,
PEG or salts.
There is evidence that hydrophilic compounds are absorbed through the
intestinal
epithelia efficiently via specialized transporters. The entire membrane
transport system is
intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one
can expect
1o that excitation of the membrane transport system will involve some sort of
specialized
adjuvant resulting in localized delivery of active agents. There are seven
known
intestinal transport systems classified according to the physical properties
of the
transported substrate. They include the amino acid, oligopeptide, glucose,
monocarboxic
acid, phosphate, bile acid and the P-glycoprotein transport systems and each
has its own
15 associated mechanism of transport. The mechanisms can depend on hydrogen
ions,
sodium ions, binding sites or other cofactors. The invention also allows
targeting the
mechanisms for intestinal epithelial transport systems to facilitate
absorption of active
agents.
In another embodiment of the invention, the composition includes one or more
2o adjuvants to enhance the bioavailability of the active agent. Addition of
an adjuvant is
particularly preferred when using an otherwise poorly absorbed active agent.
Suitable
adjuvants, for example, include: papain, which is a potent enzyme for
releasing the
catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which
activate
enzymes in the BBM; and bile acids, which have been attached to peptides to
enhance
25 absorption of the peptides.
Preferably, the resultant peptide-adrenocorticotropic hormone conjugate is
formulated into a tablet using suitable excipients and can either be wet
granulated or dry
compressed.
13
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Compositions of the invention are, in essence, the formation of amides from
acids
and amines and can be prepared by the following examples.
Acid/N-terminus conjugation
An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to
0°C. The solution can then be treated with diisopropylcarbodiimide and
hydroxybenzotriazole followed by the amine peptide Garner. The reaction can
then be
stirred for several hours at room temperature, the urea by-product filtered
off, the product
precipitated out in ether and purified using gel permeation chromatography
(GPC) or
dialysis.
to AmineJC-terminus conjugation
The peptide carrier can be dissolved in DMF under nitrogen and cooled to
0°C.
The solution can then be treated with diisopropylcarbodiimide and
hydroxybenzotriazole
followed by the amine bioactive agent. The reaction can then be stirred for
several hours
at room temperature, the urea by-product filtered off, and the product
precipitated out in
15 ether and purified using GPC or dialysis.
AlcohollN-Terminus Conjugation
In the following example the combination of the alcohol with triphosgene
produces a chloroformate, which when reacted with the N-terminus of the
peptide
produces a carbamate. Pursuant to this, an alcohol bioactive agent can be
treated with
2o triphosgene in dry DMF under nitrogen. The suitably protected peptide
Garner is then
added slowly and the solution stirred at room temperature for several hours.
The product
is then precipitated out in ether. The crude product is suitably deprotected
and purified
using GPC.
Other solvents, activating agents, cocatalysts and bases can be used. Examples
of
25 other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or
chlorinated
solvents such as chloroform. Examples of other activating agents include
dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst
is N-
14
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hydroxysuccinimide. Examples of bases include pyrrolidinopyridine,
dimethylaminopyridine, triethylamine or tributylamine.
Preparation of Alkyl Glutamate
There have been over 30 different y alkyl glutamates prepared any one of which
may be suitable for the drug alcohol of choice. For example, a suspension of
glutamic
acid, the alcohol and concentrated hydrochloric acid can be prepared and
heated for
several hours. The y alkyl glutamate product can be precipitated out in
acetone, filtered,
dried and recrystallized from hot water.
y~Alkyl Glutamate/C-Terminus Coqjugation
1o The peptide Garner can be dissolved in DMF under nitrogen and cooled to
0°C.
The solution can then be treated with diisopropylcarbodiimide and
hydroxybenzotriazole
followed by the y alkyl glutamate bioactive agent. The reaction can then be
stirred for
several hours at room temperature, the urea by-product filtered off, and the
product
precipitated out in ether and purified using GPC or dialysis.
is Preparation of y~Alkyl Glutamate-NCA
y Alkyl glutamate can be suspended in dry THF where triphosgene is added and
the mixture refluxed under a nitrogen atmosphere until the mixture becomes
homogenous. The solution can be poured into heptane to precipitate the NCA
product,
which is filtered, dried and recrystallized from a suitable solvent.
2o Preparation of Poly[fir Alkyl Glutamate]
y Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of
a primary amine can be added to the solution until it becomes viscous
(typically
overnight). The product can be isolated from the solution by pouring it into
water and
filtering. The product can be purified using GPC or dialysis.
2s Although illustrated and described above with reference to specific
embodiments,
the invention is nevertheless not intended to be limited to the details shown.
Rather,
IS
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various modifications may be made in the details within the scope and range of
equivalents of the claims and without departing from the spirit of the
invention.
16
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CLAIMS
What is claimed is:
1. A pharmaceutical composition comprising:
a polypeptide; and
adrenocorticotropic hormone covalently attached to said polypeptide.
2. The composition of claim 1 wherein said polypeptide is an oligopeptide.
3. The composition of claim 1 wherein said polypeptide is a homopolymer of a
naturally occurnng amino acid.
4. The composition of claim 1 wherein said polypeptide is a heteropolymer of
1o two or more naturally occurring amino acids.
5. The composition of claim 1 wherein said polypeptide is a homopolymer of a
synthetic amino acid.
6. The composition of claim 1 wherein said polypeptide is a heteropolymer of
two or more synthetic amino acids.
7. The composition of claim 1 wherein said polypeptide is a heteropolymer of
one or more naturally occurring amino acids and one or more synthetic amino
acids.
8. The composition of claim 1 wherein adrenocorticotropic hormone is
covalently attached to a side chain, the N-terminus or the C-terminus of said
polypeptide.
9. The composition of claim 1 further comprising a microencapsulating agent.
10. The composition of claim 9 wherein said microencapsulating agent is
selected from the group consisting of polyethylene glycol (PEG), an amino
acid, a sugar
and a salt.
11. The composition of claim 1 further comprising an adjuvant.
17
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12. The composition of claim 11 wherein said adjuvant activates an intestinal
transporter.
13. The composition of claim 1 further comprising a pharmaceutically
acceptable
excipient.
14. The composition of claim 1 wherein said composition is in the form of an
ingestable tablet.
15. The composition of claim 1 wherein said composition is in the form of an
intravenous preparation.
16. The composition of claim 1 wherein said composition is in the form of an
to oral suspension.
17. The composition of claim 1 wherein adrenocorticotropic hormone is
conformationally protected by folding of said polypeptide about said active
agent.
18. The composition of claim 1 wherein said polypeptide is capable of
releasing
adrenocorticotropic hormone from said composition in a pH-dependent manner.
19. A method for protecting adrenocorticotropic hormone from degradation
comprising covalently attaching said active agent to a polypeptide.
20. A method for controlling release of adrenocorticotropic hormone from a
composition wherein said composition comprises a polypeptide, said method
comprising
covalently attaching adrenocorticotropic hormone to said polypeptide.
21. A method for delivering adrenocorticotropic hormone to a patient
comprising administering to said patient a composition comprising:
a polypeptide; and
adrenocorticotropic hormone covalently attached to said polypeptide.
22. The method of claim 21 wherein adrenocorticotropic hormone is released
from said composition by an enzyme-catalyzed release.
~8
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23. The method of claim 21 wherein adrenocorticotropic hormone is released
from said composition by a pH-dependent unfolding of said polypeptide.
24. The method of claim 21 wherein said active agent is released from said
composition in a sustained release.
25. The method of claim 21 wherein said composition further comprises an
adjuvant covalently attached to said polypeptide and wherein release of said
adjuvant
from said composition is controlled by said polypeptide.
Abstract
to A composition comprising a polypeptide and adrenocorticotropic hormone
covalently attached to the polypeptide. Also provided is a method for delivery
of
adrenocorticotropic hormone to a patient comprising administering to the
patient a
composition comprising a polypeptide and adrenocorticotropic hormone
covalently
attached to the polypeptide. Also provided is a method for protecting
adrenocorticotropic
hormone from degradation comprising covalently attaching it to a polypeptide.
Also
provided is a method for controlling release of adrenocorticotropic hormone
from a
composition comprising covalently attaching it to the polypeptide.
19
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A NOVEL PHARMACEUTICAL COMPOUND CONTAINING ALBUTEROL
AND METHODS OF MAKING AND USING SAME
FIELD OF THE INVENTION
The present invention relates to a novel pharmaceutical compound that
comprises
a polypeptide that is preferably covalently attached to albuterol, as well as
methods for
protecting and administering albuterol. This novel compound, referred to as a
CARR1ERWAVETM Molecular Analogue (CMA), has the benefit of taking a known
effective pharmaceutical agent that is both well studied and occupies a known
segment of
t0 the pharmaceutical market, and combining it with a carrier compound that
enhances the
usefulness of the pharmaceutical agent without compromising its pharmaceutical
effectiveness.
BACKGROUND OF THE INVENTION
Albuterol is a known pharmaceutical agent that is used for the symptomatic
15 management of bronchospasm in patients with reversible, obstructive airway
disease.
It is both commercially available and readily manufactured using published
synthetic
schemes by those of ordinary skill in the art. Its structure is:
~H
H
H~ l N CHI
f _CH3
CHI
ND
The novel pharmaceutical compound of the present invention is useful in
2o accomplishing one or more of the following goals: enhancement of the
chemical stability
of the original compound; alteration of the release profile of an orally
administered
product; enhanced digestion or absorption; targeted delivery to particular
tissue/cell type;
and provision for an oral dosage form when none exists. The novel
pharmaceutical
compound may contain one or more of the following: another active
pharmaceutical
25 agent, an adjuvant, or an inhibitor.
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Active agent delivery systems are often critical for the effective delivery of
a
biologically active agent (active agent) to the appropriate target. The
importance of these
systems becomes magnified when patient compliance and active agent stability
are taken
under consideration. For instance, one would expect patient compliance to
increase
markedly if an active agent is administered orally in lieu of an injection or
another
invasive technique. Increasing the stability of the active agent, such as
prolonging shelf
life or survival in the stomach, will assure dosage reproducibility and
perhaps even
reduce the number of dosages required which could improve patient compliance.
Absorption of an orally administered active agent is often blocked by the
harshly
to acidic stomach milieu, powerful digestive enzymes in the GI tract,
permeability of
cellular membranes and transport across lipid bilayers. Incorporating
adjuvants such as
resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance
permeability of
cellular membranes. Microencapsulating active agents using protenoid
microspheres,
liposomes or polysaccharides have been effective in abating enzyme degradation
of the
15 active agent. Enzyme inhibiting adjuvants have also been used to prevent
enzyme
degradation. Enteric coatings have been used as a protector of pharmaceuticals
in the
stomach.
Active agent delivery systems also provide the ability to control the release
of the
active agent. For example, formulating diazepam with a copolymer of glutamic
acid and
2o aspartic acid enables a sustained release of the active agent. As another
example,
copolymers of lactic acid and glutaric acid are used to provide timed release
of human
growth hormone. A wide range of pharmaceuticals purportedly provide sustained
release
through microencapsulation of the active agent in amides of dicarboxylic
acids, modified
amino acids or thermally condensed amino acids. Slow release rendering
additives can
25 also be intermixed with a large array of active agents in tablet
formulations.
Each of these technologies imparts enhanced stability and time-release
properties
to active agent substances. Unfortunately, these technologies suffer from
several
shortcomings. Incorporation of the active agent is often dependent on
diffusion into the
microencapsulating matrix, which may not be quantitative and may complicate
dosage
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reproducibility. In addition, encapsulated drugs rely on diffusion out of the
matrix, which
is highly dependant on the water solubility of the active agent. Conversely,
water-soluble
microspheres swell by an infinite degree and, unfortunately, may release the
active agent
in bursts with little active agent available for sustained release.
Furthermore, in some
technologies, control of the degradation process required for active agent
release is
unreliable. For example, an enterically coated active agent depends on pH to
release the
active agent and, as such, is difficult to control the rate of release.
In the past, use has been made of amino acid side chains of polypeptides as
pendant groups to which active agents can be attached. These technologies
typically
l0 require the use of spacer groups between the amino acid pendant group and
the active
agent. The peptide-drug conjugates of this class of drug delivery system rely
on enzymes
in the bloodstream for the release of the drug and, as such, are not used for
oral
administration. Examples of timed and targeted release of injectable or
subcutaneous
pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer,
to the
15 gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard,
via a peptide
spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been
covalently
attached directly to the beta carboxylate of polyaspartic acid without a
spacer group.
This prodrug formulation was designed as a colon-specific drug delivery system
where
the drug is released by bacterial hydrolytic enzymes residing in the large
intestines. The
20 released dexamethasone active agent, in turn, was targeted to treat large
bowel disorders
and was not intended to be absorbed into the bloodstream. Yet another
technology
combines the advantages of covalent drug attachment with liposome formation
where the
active ingredient is attached to highly ordered lipid films (known as HARs)
via a peptide
linker. Thus, there has been no drug delivery system, heretofore reported,
that
25 incorporates the concept of attaching an active ingredient to a polypeptide
pendant group
with its targeted delivery into the bloodstream via oral administration.
It is also important to control the molecular weight, molecular size and
particle
size of the active agent delivery system. Variable molecular weights have
unpredictable
diffusion rates and pharmacokinetics. High molecular weight Garners are
digested slowly
30 or late, as in the case of naproxen-linked dextran, which is digested
almost exclusively in
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the colon by bacterial enzymes. High molecular weight microspheres usually
have high
moisture content which may present a problem with water labile active
ingredients.
Particle size not only becomes a problem with injectable drugs, as in the HAR
application, but absorption through the brush-border membrane of the
intestines is limited
to less than 5 microns.
SUMMARY OF THE INVENTION
The present invention provides covalent attachment of the active agent
(albuterol)
to a polymer of peptides or amino acids. The invention is distinguished from
the above-
mentioned technologies by virtue of covalently attaching albuterol to the N-
terminus, the
to C-terminus or directly to the amino acid side chain of an oligopeptide or
polypeptide,
also referred to herein as a Garner peptide. In certain applications, the
polypeptide will
stabilize the active agent, primarily in the stomach, through conformational
protection.
In these applications, delivery of the active agent is controlled, in part, by
the kinetics of
unfolding of the carrier peptide. Upon entry into the upper intestinal tract,
indigenous
15 enzymes release the active ingredient for absorption by the body by
selectively
hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action
introduces a
second order sustained release mechanism.
Alternatively, the present invention provides a pharmaceutical composition
comprising albuterol microencapsulated by a polypeptide.
2o The invention provides a composition comprising a polypeptide and albuterol
covalently attached to the polypeptide. Preferably, the polypeptide is (i) an
oligopeptide,
(ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii)
a
heteropolymer of two or more naturally occurring amino acids, (iv) a
homopolymer of a
synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids
or (vi) a
25 heteropolymer of one or more naturally occurring amino acids and one or
more synthetic
amino acids.
Albuterol preferably is covalently attached to a side chain, the N-terminus or
the
C-terminus of the polypeptide. In a preferred embodiment, the active agent is
a
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carboxylic acid and is covalently attached to the N-terminus of the
polypeptide. In
another preferred embodiment, the active agent is an amine and is covalently
attached to
the C-terminus of the polypeptide. In another preferred embodiment, the active
agent is
an alcohol and is covalently attached to the C-terminus of the polypeptide. In
yet another
preferred embodiment, the active agent is an alcohol and is covalently
attached to the N-
terminus of the polypeptide.
The composition of the invention can also include one or more of a
microencapsulating agent, an adjuvant and a pharmaceutically acceptable
excipient. The
microencapsulating agent can be selected from polyethylene glycol (PEG), an
amino
acid, a sugar and a salt. When an adjuvant is included in the composition, the
adjuvant
preferably activates an intestinal transporter.
Preferably, the composition of the invention is in the form of an ingestable
tablet,
an intravenous preparation or an oral suspension. The active agent can be
conformationally protected by folding of the polypeptide about the active
agent. In
another embodiment, the polypeptide is capable of releasing the active agent
from the
composition in a pH-dependent manner.
The invention also provides a method for protecting albuterol from degradation
comprising covalently attaching it to a polypeptide.
The invention also provides a method for delivering albuterol to a patient,
the
2o patient being a human or a non-human animal, comprising administering to
the patient a
composition comprising a polypeptide and an active agent covalently attached
to the
polypeptide. In a preferred embodiment, albuterol is released from the
composition by an
enzyme-catalyzed release. In another preferred embodiment, albuterol is
released in a
time-dependent manner based on the phannacokinetics of the enzyme-catalyzed
release.
In another preferred embodiment, the composition further comprises a
microencapsulating agent and albuterol is released from the composition by
dissolution
of the microencapsulating agent. In another preferred embodiment, albuterol is
released
from the composition by a pH-dependent unfolding of the polypeptide. In
another
preferred embodiment, albuterol is released from the composition in a
sustained release.
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In yet another preferred embodiment, the composition further comprises an
adjuvant
covalently attached to the polypeptide and release of the adjuvant from the
composition is
controlled by the polypeptide. The adjuvant can be microencapsulated into a
earner
peptide-drug conjugate for biphasic release of active ingredients.
The invention also provides a method for preparing a composition comprising a
polypeptide and an active agent covalently attached to the polypeptide. The
method
comprises the steps of:
(a) attaching atbuterol to a side chain of an amino acid to form an active
agent/amino acid complex;
(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA)
from the active agent/amino acid complex; and
(c) polymerizing the active agent/amino acid complex N-carboxyanhydride
(NCA).
In a preferred embodiment, steps (a) and (b) are repeated prior to step (c)
with a
second active agent. When steps (a) and (b) are repeated prior to step (c)
with a second
agent, albuterol and a second active agent can be copolymerized in step (c).
In another
preferred embodiment, the amino acid is glutamic acid and the active agent is
released
from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and
wherein the
active agent is released from the glutamic acid by coincident intramolecular
2o transamination. In another preferred embodiment, the glutamic acid
is.replaced by an
amino acid selected from the group consisting of aspartic acid, arginine,
asparagine,
cysteine, lysine, threonine, and serine, and wherein the active agent is
attached to the side
chain of the amino acid to form an amide, a thioester, an ester, an ether, a
urethane, a
carbonate, an anhydride or a carbamate. In yet another preferred embodiment,
the
glutamic acid is replaced by a synthetic amino acid with a pendant group
comprising an
amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.
It is to be understood that both the foregoing general description and the
following detailed description are exemplary, but are not restrictive, of the
invention.
The general applications of this invention to other active pharmaceutical
agents is
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described in U.S. Patent Application Serial Number 09/642,820, filed August
22, 2000,
incorporated herein by reference.
DETAILED DESCRIPTION OF INVENTION
The present invention provides several benefits for active agent delivery.
First,
the invention can stabilize albuterol and prevent its digestion in the
stomach. In addition,
the pharmacologic effect can be prolonged by delayed release of albuterol.
Furthermore,
active agents can be combined to produce synergistic effects. Also, absorption
of the
active agent in the intestinal tract can be enhanced. The invention also
allows targeted
delivery of active agents to specifics sites of action.
The composition of the invention comprises albuterol covalently attached to a
polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a
homopolymer of one
of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or
more
naturally occurnng amino acids, (iv) a homopolymer of a synthetic amino acid,
(v) a
heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer of
one or
more naturally occurnng amino acids and one or more synthetic amino acids.
Proteins, oligopeptides and polypeptides are polymers of amino acids that have
primary, secondary and tertiary structures. The secondary structure of the
protein is the
local conformation of the polypeptide chain and consists of helices, pleated
sheets and
turns. The protein's amino acid sequence and the structural constraints on the
conformations of the chain determine the spatial arrangement of the molecule.
The
folding of the secondary structure and the spatial arrangement of the side
chains
constitute the tertiary structure.
Proteins fold because of the dynamics associated between neighboring atoms on
the protein and solvent molecules. The thermodynamics of protein folding and
unfolding
are defined by the free energy of a particular condition of the protein that
relies on a
particular model. The process of protein folding involves, amongst other
things, amino
acid residues packing into a hydrophobic core. The amino acid side chains
inside the
protein core occupy the same volume as they do in amino acid crystals. The
folded
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protein interior is therefore more like a crystalline solid than an oil drop
and so the best
model for determining forces contributing to protein stability is the solid
reference state.
The major forces contributing to the thermodynamics of protein folding are Van
der Waals interactions, hydrogen bonds, electrostatic interactions,
configurational
entropy and the hydrophobic effect. Considering protein stability, the
hydrophobic effect
refers to the energetic consequences of removing apolar groups from the
protein interior
and exposing them to water. Comparing the energy of amino acid hydrolysis with
protein unfolding in the solid reference state, the hydrophobic effect is the
dominant
force. Hydrogen bonds are established during the protein fold process and
intramolecular
1o bonds are formed at the expense of hydrogen bonds with water. Water
molecules are
"pushed out" of the packed, hydrophobic protein core. All of these forces
combine and
contribute to the overall stability of the folded protein where the degree to
which ideal
packing occurs determines the degree of relative stability of the protein. The
result of
maximum packing is to produce a center of residues or hydrophobic core that
has
maximum shielding from solvent.
Since it is likely that lipophilic drugs would reside in the hydrophobic core
of a
peptide, it would require energy to unfold the peptide before the drug can be
released.
The unfolding process requires overcoming the hydrophobic effect by hydrating
the
amino acids or achieving the melting temperature of the protein. The heat of
hydration is
a destabilization of a protein. Typically, the folded state of a protein is
favored by only
5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at
neutral pH and
at room temperature requires chemical reagents. In fact, partial unfolding of
a protein is
often observed prior to the onset of irreversible chemical or conformation
processes.
Moreover, protein conformation generally controls the rate and extent of
deleterious
chemical reactions.
Conformational protection of active agents by proteins depends on the
stability of
the protein's folded state and the thermodynamics associated with the agent's
decomposition. Conditions necessary for the agent's decomposition should be
different
than for protein unfolding.
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Selection of the amino acids will depend on the physical properties desired.
For
instance, if increase in bulk or lipophilicity is desired, then the carrier
polypeptide will be
enriched in the amino acids in the table provided below. Polar amino acids, on
the other
hand, can be selected to increase the hydrophilicity of the polypeptide.
Ionizing amino acids can be selected for pH controlled peptide unfolding.
Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the
stomach, but will
ionize upon entry into the intestine. Conversely, basic amino acids, such as
histidine,
lysine and arginine, ionize in the stomach and are neutral in an alkaline
environment.
Other factors such as ~-n interactions between aromatic residues, kinking of
the
peptide chain by addition of proline, disulfide crosslinking and hydrogen
bonding can all
be used to select the optimum amino acid sequence for a given application.
Ordering of
the linear sequence can influence how these interactions can be maximized and
is
important in directing the secondary and tertiary structures of the
polypeptide.
Furthermore, amino acids with reactive side chains (e.g., glutamic acid,
lysine,
aspartic acid, serine, threonine and cysteine) can be incorporated for
attaching multiple
active agents or adjuvants to the same Garner peptide. This is particularly
useful if a
synergistic effect between two or more active agents is desired.
As stated above, variable molecular weights of the carrier compound can have
profound effects on the active agent release kinetics. As a result, low
molecular weight
active agent delivery systems are preferred. An advantage of this invention is
that chain
length and molecular weight of the polypeptide can be optimized depending on
the level
of conformational protection desired. This property can be optimized in
concert with the
kinetics of the first order release mechanism. Thus, another advantage of this
invention is
that prolonged release time can be imparted by increasing the molecular weight
of the
carrier polypeptide. Another, significant advantage of the invention is that
the kinetics of
active agent release is primarily controlled by the enzymatic hydrolysis of
the key bond
between the Garner peptide and the active agent.
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Dextran is the only polysaccharide known that has been explored as a
macromolecular Garner for the covalent binding of drug for colon specific drug
delivery.
Generally, it was only possible to load up to 1/10 of the total drug-dextran
conjugate
weight with drug. As stated earlier, polysaccharides are digested mainly in
the colon and
drug absorption is mainly limited to the colon. As compared to dextran, this
invention
has two major advantages. First, peptides are hydrolyzed by any one of several
aminopeptidases found in the intestinal lumen or associated with the brush-
border
membrane and so active agent release and subsequent absorption can occur in
the
jejunum or the ileum. Second, the molecular weight of the Garner molecule can
be
controlled and, thus, active agent loading can also be controlled.
As a practical example, the following table lists the molecular weights of
lipophilic amino acids (less one water molecule) and selected analgesics and
vitamins.
TABLE
Amino acid MW Active a, ent MW
Glycine 57 Acetaminophen 151
Alanine 71 Vitamin B6 (Pyroxidine) 169
Valine 99 Vitamin C (Ascorbic acid) 176
Leucine 113 Aspirin 180
Isoleucine 113 Ibuprofen 206
Phenylalanine147 Retinoic acid 300
Tyrosine 163 Vitamin BZ (Riboflavin) 376
Vitamin D2 397
Vitamin E (Tocopherol) 431
Lipophilic amino acids are preferred because conformational protection through
the
~5 stomach is important for the selected active agents, which were selected
based on ease of
covalent attachment to an oligopeptide. Eighteen was subtracted from the amino
acid's
molecular weight so that their condensation into a polypeptide is considered.
For
example, a decamer of glycine (MW=588) linked to aspirin would have a total
molecular
weight of 750 and aspirin would represent 24% of the total weight of the
active agent
delivery composition or over two times the maximum drug loading for dextran.
This is
only for an N- or C- terminus application, for those active agents attached to
pendant
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groups of decaglutamic acid, for instance, a drug with a molecular weight of
180 could
conceivably have a loading of 58%, although this may not be entirely
practical.
The alcohol, amine or carboxylic acid group of an active agent may be
covalently
attached to the N-terminus, the C-terminus or the side chain of the
oligopeptide or
polypeptide. The location of attachment depends somewhat on the functional
group
selection. For instance, if the active drug is a carboxylic acid (e.g.,
aspirin) then the N-
terminus of the oligopeptide is the preferred point of attachment. If the
active agent is an
amine (e.g., ampicillin), then the C-terminus is the preferred point of
attachment in order
to achieve a stable peptide linked active agent. In both, the C- and N-
terminus examples,
1o the peptide is, in essence, extended by one monomeric unit forming a new
peptide bond.
If the active agent is an alcohol, then either the C-terminus or the N-
terminus is the
preferred point of attachment in order to achieve a stable composition. As in
the example
above where the alcohol, norethindrone, was covalently attached to
poly(hydroxypropylglutamine), an alcohol can be converted into an
alkylchloroformate
with phosgene. This invention, then, pertains to the reaction of this key
intermediate with
the N-terminus of the peptide Garner. The active ingredient can be released
from the
peptide Garner by intestinal peptidases.
The alcohol can be selectively bound to the gamma carboxylate of glutamic acid
and then this conjugate covalently attached to the C-terminus of the peptide
carrier.
2o Because the glutamic acid-drug conjugate can be considered a dimer, this
product adds
two monomeric units to the C-terminus of the peptide Garner where the glutamic
acid
moiety serves as a spacer between the peptide and the drug as shown in Fig. 4.
Intestinal
enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drug
moiety
from the peptide Garner. The newly formed free amine of the glutamic acid
residue will
then undergo an intramolecular transamination reaction, thereby, releasing the
active
agent with coincident formation of pyroglutamic acid as shown in Fig. 5.
Alternatively,
the glutamic acid-drug dimer can be converted into the gamma ester of glutamic
acid N-
carboxyanhydride. This intermediate can then be polymerized, as described
above, using
any suitable initiator as shown in Fig. 4. The product of this polymerization
is
3o polyglutamic acid with active ingredients attached to multiple pendant
groups. Hence,
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maximum drug loading of the carrier peptide can be achieved. In addition,
other amino
acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to
impart
specific properties to the drug delivery system.
The invention also provides a method of imparting the same mechanism of action
for other polypeptides containing functional side chains. Examples include,
but are not
limited to, polylysine, polyasparagine, polyarginine, polyserine,
polycysteine,
polytyrosine, polythreonine and polyglutamine. The mechanism can translate to
these
polypeptides through a spacer or linker on the pendant group, which is
terminated,
preferably, by the glutamic acid-drug dimer. This carrier peptide-drug
conjugate is
distinguished from the prior art by virtue of the fact that the primary
release of the drug
moiety relies on peptidases and not on esterases. Alternatively, the active
agent can be
attached directly to the pendant group where some other indigenous enzymes in
the
alimentary tract can affect release.
The active agent can be covalently attached to the N-terminus, the C-terminus
or
the side chain of the polypeptide using known techniques. Examples of linking
organic
compounds to the N-terminus type of a peptide include, but are not limited to,
the
attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides
and 1,3-
dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example,
there are
known techniques for forming peptide linked biotin and peptide linked
acridine.
2o In the present invention, albuterol is covalently attached to the
polypeptide via
one of the hydroxyl groups.
The polypeptide Garner can be prepared using conventional techniques. A
preferred technique is copolymerization of mixtures of amino acid N-
carboxyanhydrides.
Alternatively, if a specific sequence is desired, a solid state automated
peptide synthesizer
can be used.
The addition of stabilizers to the composition has the potential of
stabilizing the
polypeptide further. Stabilizers such as sugar, amino acids, polyethylene
glycol (PEG)
and salts have been shown to prevent protein unfolding. In another embodiment
of the
12
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invention, a pre-first order release of the active agent is imparted by
microencapsulating
the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid
complex,
PEG or salts.
There is evidence that hydrophilic compounds are absorbed through the
intestinal
epithelia efficiently via specialized transporters. The entire membrane
transport system is
intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one
can expect
that excitation of the membrane transport system will involve some sort of
specialized
adjuvant resulting in localized delivery of active agents. There are seven
known
intestinal transport systems classified according to the physical properties
of the
transported substrate. They include the amino acid, oligopeptide, glucose,
monocarboxic
acid, phosphate, bile acid and the P-glycoprotein transport systems and each
has its own
associated mechanism of transport. The mechanisms can depend on hydrogen ions,
sodium ions, binding sites or other cofactors. The invention also allows
targeting the
mechanisms for intestinal epithelial transport systems to facilitate
absorption of active
agents.
In another embodiment of the invention, the composition includes one or more
adjuvants to enhance the bioavailability of the active agent. Addition of an
adjuvant is
particularly preferred when using an otherwise poorly absorbed active agent.
Suitable
adjuvants, for example, include: papain, which is a potent enzyme for
releasing the
catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which
activate
enzymes in the BBM; and bile acids, which have been attached to peptides to
enhance
absorption of the peptides.
Preferably, the resultant peptide-albuterol conjugate is formulated into a
tablet
using suitable excipients and can either be wet granulated or dry compressed.
Compositions of the invention are, in essence, the formation of amides from
acids
and amines and can be prepared by the following examples.
l3
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Acid/N-terminus conjugation
An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to
0°C. The solution can then be treated with diisopropylcarbodiimide and
hydroxybenzotriazole followed by the amine peptide carrier. The reaction can
then be
stirred for several hours at room temperature, the urea by-product filtered
off, the product
precipitated out in ether and purified using gel permeation chromatography
(GPC) or
dialysis.
AminelC-terminus conjugation
The peptide carrier can be dissolved in DMF under nitrogen and cooled to
0°C.
to The solution can then be treated with diisopropylcarbodiimide and
hydroxybenzotriazole
followed by the amine bioactive agent. The reaction can then be stirred for
several hours
at room temperature, the urea by-product filtered off, and the product
precipitated out in
ether and purified using GPC or dialysis.
Alcohol/N-Terminus Conjugation
15 In the following example the combination of the alcohol with triphosgene
produces a chloroformate, which when reacted with the N-terminus of the
peptide
produces a carbamate. Pursuant to this, an alcohol bioactive agent can be
treated with
triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier
is then
added slowly and the solution stirred at room temperature for several hours.
The product
2o is then precipitated out in ether. The crude product is suitably
deprotected and purified
using GPC.
Other solvents, activating agents, cocatalysts and bases can be used. Examples
of
other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or
chlorinated
solvents such as chloroform. Examples of other activating agents include
25 dicyclohexylcarbodiimide or thionyl chloride. An example of another
cocatalyst is N-
hydroxysuccinimide. Examples of bases include pyrrolidinopyridine,
dimethylaminopyridine, triethylamine or tributylamine.
14
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Preparation of ~-Alkyl Glutamate
There have been over 30 different y alkyl glutamates prepared any one of which
may be suitable for the drug alcohol of choice. For example, a suspension of
glutamic
acid, the alcohol and concentrated hydrochloric acid can be prepared and
heated for
several hours. The y alkyl glutamate product can be precipitated out in
acetone, filtered,
dried and recrystallized from hot water.
~y~-Alkyl GlutamatelC-Terminus Conjugation
The peptide carrier can be dissolved in DNiF under nitrogen and cooled to
0°C.
The solution can then be treated with diisopropylcarbodiimide and
hydroxybenzotriazole
followed by the y alkyl glutamate bioactive agent. The reaction can then be
stirred for
several hours at room temperature, the urea by-product filtered off, and the
product
precipitated out in ether and purified using GPC or dialysis.
Preparation of y~Alkyl Glutamate-NCA
y Alkyl glutamate can be suspended in dry THF where triphosgene is added and
the mixture refluxed under a nitrogen atmosphere until the mixture becomes
homogenous. The solution can be poured into heptane to precipitate the NCA
product,
which is filtered, dried and recrystallized from a suitable solvent.
Preparation of Poly[7 Alkyl Glutamate]
y Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of
a primary amine can be added to the solution until it becomes viscous
(typically
overnight). The product can be isolated from the solution by pouring it into
water and
filtering. The product can be purified using GPC or dialysis.
Although illustrated and described above with reference to specific
embodiments,
the invention is nevertheless not intended to be limited to the details shown.
Rather,
various modifications may be made in the details within the scope and range of
equivalents of the claims and without departing from the spirit of the
invention.
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CLAIMS
What is claimed is:
1. A pharmaceutical composition comprising:
a polypeptide; and
albuterol covalently attached to said polypeptide.
2. The composition of claim 1 wherein said polypeptide is an oligopeptide.
3. The composition of claim 1 wherein said polypeptide is a homopolymer of a
naturally occurring amino acid.
4. The composition of claim 1 wherein said polypeptide is a heteropolymer of
to two or more naturally occurring amino acids.
5. The composition of claim 1 wherein said polypeptide is a homopolymer of a
synthetic amino acid.
6. The composition of claim 1 wherein said polypeptide is a heteropolymer of
two or more synthetic amino acids.
7. The composition of claim 1 wherein said polypeptide is a heteropolymer of
one or more naturally occurnng amino acids and one or more synthetic amino
acids.
8. The composition of claim 1 wherein albuterol is covalently attached to a
side
chain, the N-terminus or the C-terminus of said polypeptide.
9. The composition of claim 1 further comprising a microencapsulating agent.
10. The composition of claim 9 wherein said microencapsulating agent is
selected from the group consisting of polyethylene glycol (PEG), an amino
acid, a sugar
and a salt.
11. The composition of claim 1 further comprising an adjuvant.
16
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12. The composition of claim 11 wherein said adjuvant activates an intestinal
transporter.
13. The composition of claim 1 further comprising a pharmaceutically
acceptable
excipient.
14. The composition of claim 1 wherein said composition is in the form of an
ingestable tablet.
15. The composition of claim 1 wherein said composition is in the form of an
intravenous preparation.
16. The composition of claim 1 wherein said composition is in the form of an
oral suspension.
17. The composition of claim 1 wherein albuterol is conformationally protected
by folding of said polypeptide about said active agent.
18. The composition of claim 1 wherein said polypeptide is capable of
releasing
albuterol from said composition in a pH-dependent manner.
19. A method for protecting albuterol from degradation comprising covalently
attaching said active agent to a polypeptide.
20. A method for controlling release of albuterol from a composition wherein
said composition comprises a polypeptide, said method comprising covalently
attaching
albuterol to said polypeptide.
21. A method for delivering albuterol to a patient comprising administering to
said patient a composition comprising:
a polypeptide; and
albuterol covalently attached to said polypeptide.
22. The method of claim 21 wherein albuterol is released from said composition
by an enzyme-catalyzed release.
17
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23. The method of claim 21 wherein albuterol is released from said composition
by a pH-dependent unfolding of said polypeptide.
24. The method of claim 21 wherein said active agent is released from said
composition in a sustained release.
25. The method of claim 21 wherein said composition further comprises an
adjuvant covalently attached to said polypeptide and wherein release of said
adjuvant
from said composition is controlled by said polypeptide.
18
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Abstract
A composition comprising a polypeptide and albuterol covalently attached to
the
polypeptide. Also provided is a method for delivery of albuterol to a patient
comprising
administering to the patient a composition comprising a polypeptide and
albuterol
covalently attached to the polypeptide. Also provided is a method for
protecting
albuterol from degradation comprising covalently attaching it to a
polypeptide. Also
provided is a method for controlling release of albuterol from a composition
comprising
covalently attaching it to the polypeptide.
19
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A NOVEL PHARMACEUTICAL COMPOUND CONTAINING ALENDRONATE
AND METHODS OF MAKING AND USING SAME
FIELD OF THE INVENTION
The present invention relates to a novel pharmaceutical compound that
comprises
a polypeptide that is preferably covalently attached to alendronate, as well
as methods for
protecting and administering alendronate. This novel compound, referred to as
a
CARRIERWAVETM Molecular Analogue (CMA), has the benefit of taking a known
effective pharmaceutical agent that is both well studied and occupies a known
segment of
the pharmaceutical market, and combining it with a carrier compound that
enhances the
usefulness of the pharmaceutical agent without compromising its pharmaceutical
effectiveness.
BACKGROUND OF THE INVENTION
Alendronate is a known pharmaceutical agent that is used for controlling
osteoporosis in men. Its chemical name is (4-amino-1-
hydroxybutylidene)bisphosphonic
acid. Its structure is:
0
0
P
~O
P~O
O~~
O
The novel pharmaceutical compound of the present invention is useful in
accomplishing one or more of the following goals: enhancement of the chemical
stability
of the original compound; alteration of the release profile of an orally
administered
product; enhanced digestion or absorption; targeted delivery to particular
tissue/cell type;
and provision for an oral dosage form when none exists. The novel
pharmaceutical
compound may contain one or more of the following: another active
pharmaceutical
agent, an adjuvant, or an inhibitor.
Active agent delivery systems are often critical for the effective delivery of
a
biologically active agent (active agent) to the appropriate target. The
importance of these
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systems becomes magnified when patient compliance and active agent stability
are taken
under consideration. For instance, one would expect patient compliance to
increase
markedly if an active agent is administered orally in lieu of an injection or
another
invasive technique. Increasing the stability of the active agent, such as
prolonging shelf
life or survival in the stomach, will assure dosage reproducibility and
perhaps even
reduce the number of dosages required which could improve patient compliance.
Absorption of an orally administered active agent is often blocked by the
harshly
acidic stomach milieu, powerful digestive enzymes in the GI tract,
permeability of
cellular membranes and transport across lipid bilayers. Incorporating
adjuvants such as
resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance
permeability of
cellular membranes. Microencapsulating active agents using protenoid
microspheres,
liposomes or polysaccharides have been effective in abating enzyme degradation
of the
active agent. Enzyme inhibiting adjuvants have also been used to prevent
enzyme
degradation. Enteric coatings have been used as a protector of pharmaceuticals
in the
stomach.
Active agent delivery systems also provide the ability to control the release
of the
active agent. For example, formulating diazepam with a copolymer of glutamic
acid and
aspartic acid enables a sustained release of the active agent. As another
example,
copolymers of lactic acid and glutaric acid are used to provide timed release
of human
growth hormone. A wide range of pharmaceuticals purportedly provide sustained
release
through microencapsulation of the active agent in amides of dicarboxylic
acids, modified
amino acids or thermally condensed amino acids. Slow release rendering
additives can
also be intermixed with a large array of active agents in tablet formulations.
Each of these technologies imparts enhanced stability and time-release
properties
to active agent substances. Unfortunately, these technologies suffer from
several
shortcomings. Incorporation of the active agent is often dependent on
diffusion into the
microencapsulating matrix, which may not be quantitative and may complicate
dosage
reproducibility. In addition, encapsulated drugs rely on diffusion out of the
matrix, which
is highly dependant on the water solubility of the active agent. Conversely,
water-soluble
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microspheres swell by an infinite degree and, unfortunately, may release the
active agent
in bursts with little active agent available for sustained release.
Furthermore, .in some
technologies, control of the degradation process required for active agent
release is
unreliable. For example, an enterically coated active agent depends on pH to
release the
active agent and, as such, is difficult to control the rate of release.
In the past, use has been made of amino acid side chains of polypeptides as
pendant groups to which active agents can be attached. These technologies
typically
require the use of spacer groups between the amino acid pendant group and the
active
agent. The peptide-drug conjugates of this class of drug delivery system rely
on enzymes
in the bloodstream for the release of the drug and, as such, are not used for
oral
administration. Examples of timed and targeted release of injectable or
subcutaneous
pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer,
to the
gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a
peptide
spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been
covalently
attached directly to the beta carboxylate of polyaspartic acid without a
spacer group.
This prodrug formulation was designed as a colon-specific drug delivery system
where
the drug is released by bacterial hydrolytic enzymes residing in the large
intestines. The
released dexamethasone active agent, in turn, was targeted to treat large
bowel disorders
and was not intended to be absorbed into the bloodstream. Yet another
technology
combines the advantages of covalent drug attachment with liposome formation
where the
active ingredient is attached to highly ordered lipid films (known as HARs)
via a peptide
linker. Thus, there has been no drug delivery system, heretofore reported,
that
incorporates the concept of attaching an active ingredient to a polypeptide
pendant group
with its targeted delivery into the bloodstream via oral administration.
It is also important to control the molecular weight, molecular size and
particle
size of the active agent delivery system. Variable molecular weights have
unpredictable
diffusion rates and pharmacokinetics. High molecular weight carriers are
digested slowly
or late, as in the case of naproxen-linked dextran, which is digested almost
exclusively in
the colon by bacterial enzymes. High molecular weight microspheres usually
have high
moisture content which may present a problem with water labile active
ingredients.
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Particle size not only becomes a problem with injectable drugs, as in the HAR
application, but absorption through the brush-border membrane of the
intestines is limited
to less than 5 microns.
SUMMARY OF THE INVENTION
The present invention provides covalent attachment of the active agent
(alendronate) to a polymer of peptides or amino acids. The invention is
distinguished
from the above-mentioned technologies by virtue of covalently attaching
alendronate to
the N-terminus, the C-terminus or directly to the amino acid side chain of an
oligopeptide
or polypeptide, also referred to herein as a carrier peptide. In certain
applications, the
1o polypeptide will stabilize the active agent, primarily in the stomach,
through
conformational protection. In these applications, delivery of the active agent
is
controlled, in part, by the kinetics of unfolding of the Garner peptide. Upon
entry into the
upper intestinal tract, indigenous enzymes release the active ingredient for
absorption by
the body by selectively hydrolyzing the peptide bonds of the Garner peptide.
This
15 enzymatic action introduces a second order sustained release mechanism.
Alternatively, the present invention provides a pharmaceutical composition
comprising alendronate microencapsulated by a polypeptide.
The invention provides a composition comprising a polypeptide and alendronate
covalently attached to the polypeptide. Preferably, the polypeptide is (i) an
oligopeptide,
20 (ii) a homopolymer of one of the twenty naturally occurring amino acids,
(iii) a
heteropolymer of two or more naturally occurring amino acids, (iv) a
homopolymer of a
synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids
or (vi) a
heteropolymer of one or more naturally occurring amino acids and one or more
synthetic
amino acids.
25 Alendronate preferably is covalently attached to a side chain, the N-
terminus or
the C-terminus of the polypeptide. In a preferred embodiment, the active agent
is a
carboxylic acid and is covalently attached to the N-terminus of the
polypeptide. In
another preferred embodiment, the active agent is an amine and is covalently
attached to
4
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the C-terminus of the polypeptide. In another preferred embodiment, the active
agent is
an alcohol and is covalently attached to the C-terminus of the polypeptide. In
yet another
preferred embodiment, the active agent is an alcohol and is covalently
attached to the N-
terminus of the polypeptide.
The composition of the invention can also include one or more of a
microencapsulating agent, an adjuvant and a pharmaceutically acceptable
excipient. The
microencapsulating agent can be selected from polyethylene glycol (PEG), an
amino
acid, a sugar and a salt. When an adjuvant is included in the composition, the
adjuvant
preferably activates an intestinal transporter.
1o Preferably, the composition of the invention is in the form of an
ingestable tablet,
an intravenous preparation or an oral suspension. The active agent can be
conformationally protected by folding of the polypeptide about the active
agent. In
another embodiment, the polypeptide is capable of releasing the active agent
from the
composition in a pH-dependent manner.
15 The invention also provides a method for protecting alendronate from
degradation
comprising covalently attaching it to a polypeptide.
The invention also provides a method for delivering alendronate to a patient,
the
patient being a human or a non-human animal, comprising administering to the
patient a
composition comprising a polypeptide and an active agent covalently attached
to the
2o polypeptide. In a preferred embodiment, alendronate is released from the
composition by
an enzyme-catalyzed release. In another preferred embodiment, alendronate is
released
in a time-dependent manner based on the pharmacokinetics of the enzyme-
catalyzed
release. In another preferred embodiment, the composition further comprises a
microencapsulating agent and alendronate is released from the composition by
25 dissolution of the microencapsulating agent. In another preferred
embodiment,
alendronate is released from the composition by a pH-dependent unfolding of
the
polypeptide. 1n another preferred embodiment, alendronate is released from the
composition in a sustained release. In yet another preferred embodiment, the
composition further comprises an adjuvant covalently attached to the
polypeptide and
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release of the adjuvant from the composition is controlled by the polypeptide.
The
adjuvant can be microencapsulated into a Garner peptide-drug conjugate for
biphasic
release of active ingredients.
The invention also provides a method for preparing a composition comprising a
polypeptide and an active agent covalently attached to the polypeptide. The
method
comprises the steps of:
(a) attaching alendronate to a side chain of an amino acid to form an active
agendamino acid complex;
(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA)
0 from the active agendamino acid complex; and
(c) polymerizing the active agent/amino acid complex N-carboxyanhydride
(NCA).
In a preferred embodiment, steps (a) and (b) are repeated prior to step (c)
with a
second active agent. When steps (a) and (b) are repeated prior to step (c)
with a second
agent, alendronate and a second active agent can be copolymerized in step (c).
In another
preferred embodiment, the amino acid is glutamic acid and the active agent is
released
from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and
wherein the
active agent is released from the glutamic acid by coincident intramolecular
transamination. In another preferred embodiment, the glutamic acid is replaced
by an
amino acid selected from the group consisting of aspartic acid, arginine,
asparagine, .
cysteine, lysine, threonine, and serine, and wherein the active agent is
attached to the side
chain of the amino acid to form an amide, a thioester, an ester, an ether, a
urethane, a
carbonate, an anhydride or a carbamate. In yet another preferred embodiment,
the
glutamic acid is replaced by a synthetic amino acid with a pendant group
comprising an
amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.
It is to be understood that both the foregoing general description and the
following detailed description are exemplary, but are not restrictive, of the
invention.
The general applications of this invention to other active pharmaceutical
agents is
described in U.S. Patent Application Serial Number 09/642,820, filed August
22, 2000,
incorporated herein by reference.
6
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DETAILED DESCRIPTION OF INVENTION
The present invention provides several benefits for active agent delivery.
First,
the invention can stabilize alendronate and prevent its digestion in the
stomach. In
addition, the pharmacologic effect can be prolonged by delayed release of
alendronate.
Furthermore, active agents can be combined to produce synergistic effects.
Also,
absorption of the active agent in the intestinal tract can be enhanced. The
invention also
allows targeted delivery of active agents to specifics sites of action.
Alendronate is the subject of U.S. Patent Numbers 4,621,077, 5,358,941,
5,681,950, 5,804,570, 5,849,726, 6,008,207, and 6,090,410, herein incorporated
by
1o reference, which describe how to make that drug.
The composition of the invention comprises alendronate covalently attached to
a
polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a
homopolymer of one
of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or
more
naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid,
(v) a
15 heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer
of one or
more naturally occurnng amino acids and one or more synthetic amino acids.
Proteins, oligopeptides and polypeptides are polymers of amino acids that have
primary, secondary and tertiary structures. The secondary structure of the
protein is the
local conformation of the polypeptide chain and consists of helices, pleated
sheets and
20 turns. The protein's amino acid sequence and the structural constraints on
the
conformations of the chain determine the spatial arrangement of the molecule.
The
folding of the secondary structure and the spatial arrangement of the side
chains
constitute the tertiary structure.
Proteins fold because of the dynamics associated between neighboring atoms on
25 the protein and solvent molecules. The thermodynamics of protein folding
and unfolding
are defined by the free energy of a particular condition of the protein that
relies on a
particular model. The process of protein folding involves, amongst other
things, amino
acid residues packing into a hydrophobic core. The amino acid side chains
inside the
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protein core occupy the same volume as they do in amino acid crystals. The
folded
protein interior is therefore more like a crystalline solid than an oil drop
and so the best
model for determining forces contributing to protein stability is the solid
reference state.
The major forces contributing to the thermodynamics of protein folding are Van
der Waals interactions, hydrogen bonds, electrostatic interactions,
configurational
entropy and the hydrophobic effect. Considering protein stability, the
hydrophobic effect
refers to the energetic consequences of removing apolar groups from the
protein interior
and exposing them to water. Comparing the energy of amino acid hydrolysis with
protein unfolding in the solid reference state, the hydrophobic effect is the
dominant
force. Hydrogen bonds are established during the protein fold process and
intramolecular
bonds are formed at the expense of hydrogen bonds with water. Water molecules
are
"pushed out" of the packed, hydrophobic protein core. All of these forces
combine and
contribute to the overall stability of the folded protein where the degree to
which ideal
packing occurs determines the degree of relative stability of the protein. The
result of
maximum packing is to produce a center of residues or hydrophobic core that
has
maximum shielding from solvent.
Since it is likely that lipophilic drugs would reside in the hydrophobic core
of a
peptide, it would require energy to unfold the peptide before the drug can be
released.
The unfolding process requires overcoming the hydrophobic effect by hydrating
the
amino acids or achieving the melting temperature of the protein. The heat of
hydration is
a destabilization of a protein. Typically, the folded state of a protein is
favored by only
5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at
neutral pH and
at room temperature requires chemical reagents. In fact, partial unfolding of
a protein is
often observed prior to the onset of irreversible chemical or conformation
processes.
Moreover, protein conformation generally controls the rate and extent of
deleterious
chemical reactions.
Conformational protection of active agents by proteins depends on the
stability of
the protein's folded state and the thermodynamics associated with the agent's
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decomposition. Conditions necessary for the agent's decomposition should be
different
than for protein unfolding.
Selection of the amino acids will depend on the physical properties desired.
For
instance, if increase in bulk or lipophilicity is desired, then the carrier
polypeptide will be
enriched in the amino acids in the table provided below. Polar amino acids, on
the other
hand, can be selected to increase the hydrophilicity of the polypeptide.
Ionizing amino acids can be selected for pH controlled peptide unfolding.
Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the
stomach, but will
ionize upon entry into the intestine. Conversely, basic amino acids, such as
hisddine,
t o lysine and arginine, ionize in the stomach and are neutral in an alkaline
environment.
Other factors such as ~-~ interactions between aromatic residues, kinking of
the
peptide chain by addition of proline, disulfide crosslinking and hydrogen
bonding can all
be used to select the optimum amino acid sequence for a given application.
Ordering of
the linear sequence can influence how these interactions can be maximized and
is
15 important in directing the secondary and tertiary structures of the
polypeptide.
Furthermore, amino acids with reactive side chains (e.g., glutamic acid,
lysine,
aspartic acid, serine, threonine and cysteine) can be incorporated for
attaching multiple
active agents or adjuvants to the same Garner peptide. This is particularly
useful if a
synergistic effect between two or more active agents is desired.
20 As stated above, variable molecular weights of the carrier compound can
have
profound effects on the active agent release kinetics. As a result, low
molecular weight
active agent delivery systems are preferred. An advantage of this invention is
that chain
length and molecular weight of the polypeptide can be optimized depending on
the level
of conformational protection desired. This property can be optimized in
concert with the
25 kinetics of the first order release mechanism. Thus, another advantage of
this invention is
that prolonged release time can be imparted by increasing the molecular weight
of the
carrier polypeptide. Another, significant advantage of the invention is that
the kinetics of
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active agent release is primarily controlled by the enzymatic hydrolysis of
the key bond
between the Garner peptide and the active agent.
Dextran is the only polysaccharide known that has been explored as a
macromolecular carrier for the covalent binding of drug for colon specific
drug delivery.
Generally, it was only possible to load up to 1/10 of the total drug-dextran
conjugate
weight with drug. As stated earlier, polysaccharides are digested mainly in
the colon and
drug absorption is mainly limited to the colon. As compared to dextran, this
invention
has two major advantages. First, peptides are hydrolyzed by any one of several
aminopeptidases found in the intestinal lumen or associated with the brush-
border
membrane and so active agent release and subsequent absorption can occur in
the
jejunum or the ileum. Second, the molecular weight of the carrier molecule can
be
controlled and, thus, active agent loading can also be controlled.
As a practical example, the following table lists the molecular weights of
lipophilic amino acids (less one water molecule) and selected analgesics and
vitamins.
TABLE
Amino acid MW Active agent
Glycine 57 Acetaminophen 151
Alanine 71 Vitamin B6 (Pyroxidine) 169
Valine 99 Vitamin C (Ascorbic acid) 176
Leucine 113 Aspirin 180
Isoleucine 113 Ibuprofen 206
Phenylalanine147 Retinoic acid 300
Tyrosine 163 Vitamin BZ (Riboflavin) 376
Vitamin DZ 397
Vitamin E (Tocopherol) 431
Lipophilic amino acids are preferred because conformational protection through
the
stomach is important for the selected active agents, which were selected based
on ease of
covalent attachment to an oligopeptide. Eighteen was subtracted from the amino
acid's
molecular weight so that their condensation into a polypeptide is considered.
For
example, a decamer of glycine (MW=588) linked to aspirin would have a total
molecular
weight of 750 and aspirin would represent 24% of the total weight of the
active agent
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delivery composition or over two times the maximum drug loading for dextran.
This is
only for an N- or C- terminus application, for those active agents attached to
pendant
groups of decaglutamic acid, for instance, a drug with a molecular weight of
180 could
conceivably have a loading of 58%, although this may not be entirely
practical.
The alcohol, amine or carboxylic acid group of an active agent may be
covalently
attached to the N-terminus, the C-terminus or the side chain of the
oligopeptide or
polypeptide. The location of attachment depends somewhat on the functional
group
selection. For instance, if the active drug is a carboxylic acid (e.g.,
aspirin) then the N-
terminus of the oligopeptide is the preferred point of attachment. If the
active agent is an
amine (e.g., ampicillin), then the C-terminus is the preferred point of
attachment in order
to achieve a stable peptide linked active agent. In both, the C- and N-
terminus examples,
the peptide is, in essence, extended by one monomeric unit forming a new
peptide bond.
If the active agent is an alcohol, then either the C-terminus or the N-
terminus is the
preferred point of attachment in order to achieve a stable composition. As in
the example
t5 above where the alcohol, norethindrone, was covalently attached to
poly(hydroxypropylglutamine), an alcohol can be converted into an
alkylchloroformate
with phosgene. This invention, then, pertains to the reaction of this key
intermediate with
the N-terminus of the peptide carrier. The active ingredient can be released
from the
peptide carrier by intestinal peptidases.
The alcohol can be selectively bound to the gamma carboxylate of glutamic acid
and then this conjugate covalently attached to the C-terminus of the peptide
carrier.
Because the glutamic acid-drug conjugate can be considered a dimer, this
product adds
two monomeric units to the C-terminus of the peptide carrier where the
glutamic acid
moiety serves as a spacer between the peptide and the drug as shown in Fig. 4.
Intestinal
enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drug
moiety
from the peptide carrier. The newly formed free amine of the glutamic acid
residue will
then undergo an intramolecular transamination reaction, thereby, releasing the
active
agent with coincident formation of pyroglutamic acid as shown in Fig. 5.
Alternatively,
the glutamic acid-drug dimer can be converted into the gamma ester of glutamic
acid N-
3o carboxyanhydride. This intermediate can then be polymerized, as described
above, using
~t
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any suitable initiator as shown in Fig. 4. The product of this polymerization
is
polyglutamic acid with active ingredients attached to multiple pendant groups.
Hence,
maximum drug loading of the carrier peptide can be achieved. In addition,
other amino
acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to
impart
specific properties to the drug delivery system.
The invention also provides a method of imparting the same mechanism of action
for other polypeptides containing functional side chains. Examples include,
but are not
limited to, polylysine, polyasparagine, polyarginine, polyserine,
polycysteine,
polytyrosine, polythreonine and polyglutamine. The mechanism can translate to
these
polypeptides through a spacer or linker on the pendant group, which is
terminated,
preferably, by the glutamic acid-drug dimer. This carrier peptide-drug
conjugate is
distinguished from the prior art by virtue of the fact that the primary
release of the drug
moiety relies on peptidases and not on esterases. Alternatively, the active
agent can be
attached directly to the pendant group where some other indigenous enzymes in
the
alimentary tract can affect release.
The active agent can be covalently attached to the N-terminus, the C-terminus
or
the side chain of the polypeptide using known techniques. Examples of linking
organic
compounds to the N-terminus type of a peptide include, but are not limited to,
the
attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides
and 1,3-
dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example,
there are
known techniques for forming peptide linked biotin and peptide linked
acridine.
In the present invention, alendronate is covalently attached to the
polypeptide via
the hydroxyl or phosphate groups.
The polypeptide carrier can be prepared using conventional techniques. A
preferred technique is copolymerization of mixtures of amino acid N-
carboxyanhydrides.
Alternatively, if a specific sequence is desired, a solid state automated
peptide synthesizer
can be used.
12
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The addition of stabilizers to the composition has the potential of
stabilizing the
polypeptide further. Stabilizers such as sugar, amino acids, polyethylene
glycol (PEG)
and salts have been shown to prevent protein unfolding. In another embodiment
of the
invention, a pre-first order release of the active agent is imparted by
microencapsulating
the Garner polypeptide-active agent conjugate in a polysaccharide, amino acid
complex,
PEG or salts.
There is evidence that hydrophilic compounds are absorbed through the
intestinal
epithelia efficiently via specialized transporters. The entire membrane
transport system is
intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one
can expect
1o that excitation of the membrane transport system will involve some sort of
specialized
adjuvant resulting in localized delivery of active agents. There are seven
known
intestinal transport systems classified according to the physical properties
of the
transported substrate. They include the amino acid, oligopeptide, glucose,
monocarboxic
acid, phosphate, bile acid and the P-glycoprotein transport systems and each
has its own
15 associated mechanism of transport. The mechanisms can depend on hydrogen
ions,
sodium ions, binding sites or other cofactors. The invention also allows
targeting the
mechanisms for intestinal epithelial transport systems to facilitate
absorption of active
agents.
In another embodiment of the invention, the composition includes one or more
2o adjuvants to enhance the bioavailability of the active agent. Addition of
an adjuvant is
particularly preferred when using an otherwise poorly absorbed active agent.
Suitable
adjuvants, for example, include: papain, which is a potent enzyme for
releasing the
catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which
activate
enzymes in the BBM; and bile acids, which have been attached to peptides to
enhance
25 absorption of the peptides.
Preferably, the resultant peptide-alendronate conjugate is formulated into a
tablet
using suitable excipients and can either be wet granulated or dry compressed.
Compositions of the invention are, in essence, the formation of amides from
acids
and amines and can be prepared by the following examples.
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Acid/N-terminus conjugation
An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to
0°C. The solution can then be treated with diisopropylcarbodiimide and
hydroxybenzotriazole followed by the amine peptide carrier. The reaction can
then be
stirred for several hours at room temperature, the urea by-product filtered
off, the product
precipitated out in ether and purified using gel permeation chromatography
(GPC) or
dialysis.
Amine/C-terminus coqjugation
The peptide carrier can be dissolved in DMF under nitrogen and cooled to
0°C.
The solution can then be treated with diisopropylcarbodiimide and
hydroxybenzotriazole
followed by the amine bioactive agent. The reaction can then be stirred for
several hours
at room temperature, the urea by-product filtered off, and the product
precipitated out in
ether and purified using GPC or dialysis.
Alcohol/N-Terminus Conjugation
In the following example the combination of the alcohol with triphosgene
produces a chloroformate, which when reacted with the N-terminus of the
peptide
produces a carbamate. Pursuant to this, an alcohol bioactive agent can be
treated with
triphosgene in dry DMF under nitrogen. The suitably protected peptide Garner
is then
added slowly and the solution stirred at room temperature for several hours.
The product
2o is then precipitated out in ether. The crude product is suitably
deprotected and purified
using GPC.
Other solvents, activating agents, cocatalysts and bases can be used. Examples
of
other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or
chlorinated
solvents such as chloroform. Examples of other activating agents include
dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst
is N-
hydroxysuccinimide. Examples of bases include pyrrolidinopyridine,
dimethylaminopyridine, triethylamine or tributylamine.
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Preparation of ~-Alkyl Glutamate
There have been over 30 different 'y alkyl glutamates prepared any one of
which
may be suitable for the drug alcohol of choice. For example, a suspension of
glutamic
acid, the alcohol and concentrated hydrochloric acid can be prepared and
heated for
several hours. The y alkyl glutamate product can be precipitated out in
acetone, filtered,
dried and recrystallized from hot water.
y~Alkyl GlutamateJC-Terminus Conjugation
The peptide carrier can be dissolved in DMF under nitrogen and cooled to
0°C.
The solution can then be treated with diisopropylcarbodiimide and
hydroxybenzotriazole
followed by the 'y alkyl glutamate bioactive agent. The reaction can then be
stirred for
several hours at room temperature, the urea by-product filtered off, and the
product
precipitated out in ether and purified using GPC or dialysis.
Preparation of y~Alkyl Glutamate-NCA
y Alkyl glutamate can be suspended in dry TI-IF where triphosgene is added and
~5 the mixture refluxed under a nitrogen atmosphere until the mixture becomes
homogenous. The solution can be poured into heptane to precipitate the NCA
product,
which is filtered, dried and recrystallized from a suitable solvent.
Preparation of Poly['y Alkyl Glutamate]
y Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of
2o a primary amine can be added to the solution until it becomes viscous
(typically
overnight). The product can be isolated from the solution by pouring it into
water and
filtering. The product can be purified using GPC or dialysis.
Although illustrated and described above with reference to specific
embodiments,
the invention is nevertheless not intended to be limited to the details shown.
Rather,
25 various modifications may be made in the details within the scope and range
of
equivalents of the claims and without departing from the spirit of the
invention.
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CLAIMS
What is claimed is:
1. A pharmaceutical composition comprising:
a polypeptide; and
alendronate covalently attached to said polypeptide.
2. The composition of claim 1 wherein said polypeptide is an oligopeptide.
3. The composition of claim 1 wherein said polypeptide is a homopolymer of a
naturally occurring amino acid.
4. The composition of claim 1 wherein said polypepdde is a heteropolymer of
1o two or more naturally occurring amino acids.
5. The composition of claim 1 wherein said polypeptide is a homopolymer of a
synthetic amino acid.
6. The composition of claim 1 wherein said polypeptide is a heteropolymer of
two or more synthetic amino acids.
7. The composition of claim 1 wherein said polypeptide is a heteropolymer of
one or more naturally occurring amino acids and one or more synthetic amino
acids.
8. The composition of claim 1 wherein alendronate is covalently attached to a
side chain, the N-terminus or the C-terminus of said polypeptide.
9. The composition of claim 1 further comprising a microencapsulating agent.
10. The composition of claim 9 wherein said microencapsulating agent is
selected from the group consisting of polyethylene glycol (PEG), an amino
acid, a sugar
and a salt.
11. The composition of claim 1 further comprising an adjuvant.
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12. The composition of claim 11 wherein said adjuvant activates an intestinal
transporter.
13. The composition of claim 1 further comprising a pharmaceutically
acceptable
excipient.
14. The composition of claim 1 wherein said composition is in the form of an
ingestable tablet.
15. The composition of claim 1 wherein said composition is in the form of an
intravenous preparation.
16. The composition of claim 1 wherein said composition is in the form of an
oral suspension.
17. The composition of claim 1 wherein alendronate is conformadonally
protected by folding of said polypeptide about said active agent.
18. The composition of claim 1 wherein said polypeptide is capable of
releasing
alendronate from said composition in a pH-dependent manner.
19. A method for protecting alendronate from degradation comprising covalently
attaching said active agent to a polypeptide.
20. A method for controlling release of alendronate from a composition wherein
said composition comprises a polypeptide, said method comprising covalently
attaching
alendronate to said polypeptide.
21. A method for delivering alendronate to a patient comprising administering
to
said patient a composition comprising:
a polypeptide; and
alendronate covalently attached to said polypeptide.
22. The method of claim 21 wherein alendronate is released from said
composition by an enzyme-catalyzed release.
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23. The method of claim 21 wherein alendronate is released from said
composition by a pH-dependent unfolding of said polypeptide.
24. The method of claim 21 wherein said active agent is released from said
composition in a sustained release.
25. The method of claim 21 wherein said composition further comprises an
adjuvant covalently attached to said polypeptide and wherein release of said
adjuvant
from said composition is controlled by said polypeptide.
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Abstract
A composition comprising a polypeptide and alendronate covalently attached to
the polypeptide. Also provided is a method for delivery of alendronate to a
patient
comprising administering to the patient a composition comprising a polygeptide
and
alendronate covalently attached to the polypeptide. Also provided is a method
for
protecting alendronate from degradation comprising covalently attaching it to
a
polypeptide. Also provided is a method for controlling release of alendronate
from a
composition comprising covalently attaching it to the polypeptide.
19
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A NOVEL PHARMACEUTICAL COMPOUND CONTAINING ALLOPURINAL
AND METHODS OF MAKING AND USING SAME
FIELD OF THE INVENTION
The present invention relates to a novel pharmaceutical compound that
comprises
a polypeptide that is preferably covalently attached to allopurinal, as well
as methods for
protecting and administering allopurinal. This novel compound, referred to as
a
CARRIERWAVETM Molecular Analogue (CMA), has the benefit of taking a known
effective pharmaceutical agent that is both well studied and occupies a known
segment of
1o the pharmaceutical market, and combining it with a carrier compound that
enhances the
usefulness of the pharmaceutical agent without compromising its pharmaceutical
effectiveness.
BACKGROUND OF THE INVENTION
Allopurinal is a known pharmaceutical agent that is is a xanthine oxidase
inhibitor
used in the treatment of gout and selected hyperuricemias. It is both
commercially
available and readily manufactured using published synthetic schemes by those
of
ordinary skill in the art. Its structure is:
H
I
'N N~
HN I
n
0
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The novel pharmaceutical compound of the present invention is useful in
accomplishing one or more of the following goals: enhancement of the chemical
stability
of the original compound; alteration of the release profile of an orally
administered
product; enhanced digestion or absorption; targeted delivery to particular
tissue/cell type;
and provision for an oral dosage form when none exists. The novel
pharmaceutical
compound may contain one or more of the following: another active
pharmaceutical
agent, an adjuvant, or an inhibitor.
Active agent delivery systems are often critical for the effective delivery of
a
biologically active agent (active agent) to the appropriate target. The
importance of these
systems becomes magnified when patient compliance and active agent stability
are taken
under consideration. For instance, one would expect patient compliance to
increase
markedly if an active agent is administered orally in lieu of an injection or
another
invasive technique. Increasing the stability of the active agent, such as
prolonging shelf
life or survival in the stomach, will assure dosage reproducibility and
perhaps even
reduce the number of dosages required which could improve patient compliance.
Absorption of an orally administered active agent is often blocked by the
harshly
acidic stomach milieu, powerful digestive enzymes in the GI tract,
permeability of
cellular membranes and transport across lipid bilayers. Incorporating
adjuvants such as
resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance
permeability of
2o cellular membranes. Microencapsulating active agents using protenoid
microspheres,
liposomes or polysaccharides have been effective in abating enzyme degradation
of the
active agent. Enzyme inhibiting adjuvants have also been used to prevent
enzyme
degradation. Enteric coatings have been used as a protector of pharmaceuticals
in the
stomach.
Active agent delivery systems also provide the ability to control the release
of the
active agent. For example, formulating diazepam with a copolymer of glutamic
acid and
aspartic acid enables a sustained release of the active agent. As another
example,
copolymers of lactic acid and glutaric acid are used to provide timed release
of human
growth hormone. A wide range of pharmaceuticals purportedly provide sustained
release
2
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through microencapsulation of the active agent in amides of dicarboxylic
acids, modified
amino acids or thermally condensed amino acids. Slow release rendering
additives can
also be intermixed with a large array of active agents in tablet formulations.
Each of these technologies imparts enhanced stability and time-release
properties
to active agent substances. Unfortunately, these technologies suffer from
several
shortcomings. Incorporation of the active agent is often dependent on
diffusion into the
microencapsulating matrix, which may not be quantitative and may complicate
dosage
reproducibility. In addition, encapsulated drugs rely on diffusion out of the
matrix, which
is highly dependant on the water solubility of the active agent. Conversely,
water-soluble
i0 microspheres swell by an infinite degree and, unfortunately, may release
the active agent
in bursts with little active agent available for sustained release.
Furthermore, in some
technologies, control of the degradation process required for active agent
release is
unreliable. For example, an enterically coated active agent depends on pH to
release the
active agent and, as such, is difficult to control the rate of release.
In the past, use has been made of amino acid side chains of polypeptides as
pendant groups to which active agents can be attached. These technologies
typically
require the use of spacer groups between the amino acid pendant group and the
active
agent. The peptide-drug conjugates of this class of drug delivery system rely
on enzymes
in the bloodstream for the release of the drug and, as such, are not used for
oral
2o administration. Examples of timed and targeted release of injectable or
subcutaneous
pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer,
to the
gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a
peptide
spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been
covalently
attached directly to the beta carboxylate of polyaspartic acid without a
spacer group.
This prodrug formulation was designed as a colon-specific drug delivery system
where
the drug is released by bacterial hydrolytic enzymes residing in the large
intestines. The
released dexamethasone active agent, in turn, was targeted to treat large
bowel disorders
and was not intended to be absorbed into the bloodstream. Yet another
technology
combines the advantages of covalent drug attachment with liposome formation
where the
3o active ingredient is attached to highly ordered lipid films (known as HARs)
via a peptide
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linker. Thus, there has been no drug delivery system, heretofore reported,
that
incorporates the concept of attaching an active ingredient to a polypeptide
pendant group
with its targeted delivery into the bloodstream via oral administration.
It is also important to control the molecular weight, molecular size and
particle
size of the active agent delivery system. Variable molecular weights have
unpredictable
diffusion rates and pharmacokinetics. High molecular weight carriers are
digested slowly
or late, as in the case of naproxen-linked dextran, which is digested almost
exclusively in
the colon by bacterial enzymes. High molecular weight microspheres usually
have high
moisture content which may present a problem with water labile active
ingredients.
to Particle size not only becomes a problem with injectable drugs, as in the
HAR
application, but absorption through the brush-border membrane of the
intestines is limited
to less than 5 microns.
SUMMARY OF THE INVENTION
The present invention provides covalent attachment of the active agent
(allopurinal) to a polymer of peptides or amino acids. The invention is
distinguished
from the above-mentioned technologies by virtue of covalently attaching
allopurinal to
the N-terminus, the C-terminus or directly to the amino acid side chain of an
oligopeptide
or polypeptide, also referred to herein as a Garner peptide. In certain
applications, the
polypeptide will stabilize the active agent, primarily in the stomach, through
conformational protection. In these applications, delivery of the active agent
is
controlled, in part, by the kinetics of unfolding of the Garner peptide. Upon
entry into the
upper intestinal tract, indigenous enzymes release the active ingredient for
absorption by
the body by selectively hydrolyzing the peptide bonds of the carrier peptide.
This
enzymatic action introduces a second'order sustained release mechanism.
Alternatively, the present invention provides a pharmaceutical composition
comprising allopurinal microencapsulated by a polypeptide.
The invention provides a composition comprising a polypeptide and allopurinal
covalently attached to the polypeptide. Preferably, the polypeptide is (i) an
oligopeptide,
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(ii) a homopolymer of one of the twenty naturally occurnng amino acids, (iii)
a
heteropolymer of two or more naturally occurring amino acids, (iv) a
homopolymer of a
synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids
or (vi) a
heteropolymer of one or more naturally occurring amino acids and one or more
synthetic
amino acids.
Allopurinal preferably is covalently attached to a side chain, the N-terminus
or the
C-terminus of the polypeptide. In a preferred embodiment, the active agent is
a
carboxylic acid and is covalently attached to the N-terminus of the
polypeptide. In
another preferred embodiment, the active agent is an amine and is covalently
attached to
the C-terminus of the polypeptide. In another preferred embodiment, the active
agent is
an alcohol and is covalently attached to the C-terminus of the polypeptide. In
yet another
preferred embodiment, the active agent is an alcohol and is covalently
attached to the N-
terminus of the polypeptide.
The composition of the invention can also include one or more of a
microencapsulating agent, an adjuvant and a pharmaceutically acceptable
excipient. The
microencapsulating agent can be selected from polyethylene glycol (PEG), an
amino
acid, a sugar and a salt. When an adjuvant is included in the composition, the
adjuvant
preferably activates an intestinal transporter.
Preferably, the composition of the invention is in the form of an ingestable
tablet,
2o an intravenous preparation or an oral suspension. The active agent can be
conformationally protected by folding of the polypeptide about the active
agent. In
another embodiment, the polypeptide is capable of releasing the active agent
from the
composition in a pH-dependent manner.
The invention also provides a method for protecting allopurinal from
degradation
comprising covalently attaching it to a polypeptide.
The invention also provides a method for delivering allopurinal to a patient,
the
patient being a human or a non-human animal, comprising administering to the
patient a
composition comprising a polypeptide and an active agent covalently attached
to the
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polypeptide. In a preferred embodiment, allopurinal is released from the
composition by
an enzyme-catalyzed release. In another preferred embodiment, allopurinal is
released in
a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed
release. In another preferred embodiment, the composition further comprises a
microencapsulating agent and allopurinal is released from the composition by
dissolution
of the microencapsulating agent. In another preferred embodiment, allopurinal
is
released from the composition by a pH-dependent unfolding of the polypeptide.
In
another preferred embodiment, allopurinal is released from the composition in
a sustained
release. In yet another preferred embodiment, the composition further
comprises an
to adjuvant covalently attached to the polypeptide and release of the adjuvant
from the
composition is controlled by the polypeptide. The adjuvant can be
microencapsulated
into a carrier peptide-drug conjugate for biphasic release of active
ingredients.
The invention also provides a method for preparing a composition comprising a
polypeptide and an active agent covalently attached to the polypeptide. The
method
comprises the steps of:
(a) attaching allopurinal to a side chain of an amino acid to form an active
agent/amino acid complex;
(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA)
from the active agendamino acid complex; and
(c) polymerizing the active agentlamino acid complex N-carboxyanhydride
(NCA).
In a preferred embodiment, steps (a) and (b) are repeated prior to step (c)
with a
second active agent. When steps (a) and (b) are repeated prior to step (c)
with a second
agent, allopurinal and a second active agent can be copolymerized in step (c).
In another
preferred embodiment, the amino acid is glutamic acid and the active agent is
released
from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and
wherein the
active agent is released from the glutamic acid by coincident intramolecular
transamination. In another preferred embodiment, the glutamic acid is replaced
by an
amino acid selected from the group consisting of aspartic acid, arginine,
asparagine,
3o cysteine, lysine, threonine, and serine, and wherein the active agent is
attached to the side
chain of the amino acid to form an amide, a thioester, an ester, an ether, a
urethane, a
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carbonate, an anhydride or a carbamate. In yet another preferred embodiment,
the
glutamic acid is replaced by a synthetic amino acid with a pendant group
comprising an
amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.
It is to be understood that both the foregoing general description and the
following detailed description are exemplary, but are not restrictive, of the
invention.
The general applications of this invention to other active pharmaceutical
agents is
described in U.S. Patent Application Serial Number 09/642,820, filed August
22, 2000,
incorporated herein by reference.
DETAILED DESCRIPTION OF INVENTION
l0 The present invention provides several benefits for active agent delivery.
First,
the invention can stabilize allopurinal and prevent its digestion in the
stomach. In
addition, the pharmacologic effect can be prolonged by delayed release of
allopurinal.
Furthermore, active agents can be combined to produce synergistic effects.
Also,
absorption of the active agent in the intestinal tract can be enhanced. The
invention also
15 allows targeted delivery of active agents to specifics sites of action.
The composition of the invention comprises allopurinal covalently attached to
a
polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a
homopolymer of one
of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or
more
naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid,
(v) a
20 heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer
of one or
more naturally occurring amino acids and one or more synthetic amino acids.
Proteins, oligopeptides and polypeptides are polymers of amino acids that have
primary, secondary and tertiary structures. The secondary structure of the
protein is the
local conformation of the polypeptide chain and consists of helices, pleated
sheets and
25 turns. The protein's amino acid sequence and the structural constraints on
the
conformations of the chain determine the spatial arrangement of the molecule.
The
folding of the secondary structure and the spatial arrangement of the side
chains
constitute the tertiary structure.
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Proteins fold because of the dynamics associated between neighboring atoms on
the protein and solvent molecules. The thermodynamics of protein folding and
unfolding
are defined by the free energy of a particular condition of the protein that
relies on a
particular model. The process of protein folding involves, amongst other
things, amino
acid residues packing into a hydrophobic core. The amino acid side chains
inside the
protein core occupy the same volume as they do in amino acid crystals. The
folded
protein interior is therefore more like a crystalline solid than an oil drop
and so the best
model for determining forces contributing to protein stability is the solid
reference state.
The major forces contributing to the thermodynamics of protein folding are Van
1o der Waals interactions, hydrogen bonds, electrostatic interactions,
configurational
entropy and the hydrophobic effect. Considering protein stability, the
hydrophobic effect
refers to the energetic consequences of removing apolar groups from the
protein interior
and exposing them to water. Comparing the energy of amino acid hydrolysis with
protein unfolding in the solid reference state, the hydrophobic effect is the
dominant
force. Hydrogen bonds are established during the protein fold process and
intramolecular
bonds are formed at the expense of hydrogen bonds with water. Water molecules
are
"pushed out" of the packed, hydrophobic protein core. All of these forces
combine and
contribute to the overall stability of the folded protein where the degree to
which ideal
packing occurs determines the degree of relative stability of the protein. The
result of
2o maximum packing is to produce a center of residues or hydrophobic core that
has
maximum shielding from solvent.
Since it is likely that lipophilic drugs would reside in the hydrophobic core
of a
peptide, it would require energy to unfold the peptide before the drug can be
released.
The unfolding process requires overcoming the hydrophobic effect by hydrating
the
amino acids or achieving the melting temperature of the protein. The heat of
hydration is
a destabilization of a protein. Typically, the folded state of a protein is
favored by only
5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at
neutral pH and
at room temperature requires chemical reagents. In fact, partial unfolding of
a protein is
often observed prior to the onset of irreversible chemical or conformation
processes.
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Moreover, protein conformation generally controls the rate and extent of
deleterious
chemical reactions.
Conformational protection of active agents by proteins depends on the
stability of
the protein's folded state and the thermodynamics associated with the agent's
decomposition. Conditions necessary for the agent's decomposition should be
different
than for protein unfolding.
Selection of the amino acids will depend on the physical properties desired.
For
instance, if increase in bulk or lipophilicity is desired, then the Garner
polypeptide will be
enriched in the amino acids in the table provided below. Polar amino acids, on
the other
to hand, can be selected to increase the hydrophilicity of the polypeptide.
Ionizing amino acids can be selected for pH controlled peptide unfolding.
Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the
stomach, but will
ionize upon entry into the intestine. Conversely, basic amino acids, such as
histidine,
lysine and arginine, ionize in the stomach and are neutral in an alkaline
environment.
t5 Other factors such as n-~ interactions between aromatic residues, kinking
of the
peptide chain by addition of proline, disulfide crosslinking and hydrogen
bonding can all
be used to select the optimum amino acid sequence for a given application.
Ordering of
the linear sequence can influence how these interactions can be maximized and
is
important in directing the secondary and tertiary structures of the
polypeptide.
2o Furthermore, amino acids with reactive side chains (e.g., glutamic acid,
lysine,
aspartic acid, serine, threonine and cysteine) can be incorporated for
attaching multiple
active agents or adjuvants to the same carrier peptide. This is particularly
useful if a
synergistic effect between two or more active agents is desired.
As stated above, variable molecular weights of the Garner compound can have
25 profound effects on the active agent release kinetics. As a result, low
molecular weight
active agent delivery systems are preferred. An advantage of this invention is
that chain
length and molecular weight of the polypeptide can be optimized depending on
the level
of conformational protection desired. This property can be optimized in
concert with the
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kinetics of the first order release mechanism. Thus, another advantage of this
invention is
that prolonged release time can be imparted by increasing the molecular weight
of the
carrier polypeptide. Another, significant advantage of the invention is that
the kinetics of
active agent release is primarily controlled by the enzymatic hydrolysis of
the key bond
between the Garner peptide and the active agent.
Dextran is the only polysaccharide known that has been explored as a
macromolecular carrier for the covalent binding of drug for colon specific
drug delivery.
Generally, it was only possible to load up to 1/10 of the total drug-dextran
conjugate
weight with drug. As stated earlier, polysaccharides are digested mainly in
the colon and
1o drug absorption is mainly limited to the colon. As compared to dextran,
this invention
has two major advantages. First, peptides are hydrolyzed by any one of several
aminopeptidases found in the intestinal lumen or associated with the brush-
border
membrane and so active agent release and subsequent absorption can occur in
the
jejunum or the ileum. Second, the molecular weight of the Garner molecule can
be
controlled and, thus, active agent loading can also be controlled.
As a practical example, the following table lists the molecular weights of
lipophilic amino acids (less one water molecule) and selected analgesics and
vitamins.
TABLE
Amino acid MW Active agent
Glycine 57 Acetaminophen 151
Alanine 71 Vitamin B6 (Pyroxidine) 169
Valine 99 Vitamin C (Ascorbic acid) 176
Leucine 113 Aspirin 180
Isoleucine 113 Ibuprofen 206
Phenylalanine147 Retinoic acid 300
Tyrosine 163 Vitamin B2 (Riboflavin) 376
Vitamin DZ 397
Vitamin E (Tocopherol) 431
Lipophilic amino acids are preferred because conformational protection through
the
2o stomach is important for the selected active agents, which were selected
based on ease of
covalent attachment to an oligopeptide. Eighteen was subtracted from the amino
acid's
to
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molecular weight so that their condensation into a polypeptide is considered.
For
example, a decamer of glycine (MW=588) linked to aspirin would have a total
molecular
weight of 750 and aspirin would represent 24% of the total weight of the
active agent
delivery composition or over two times the maximum drug loading for dextran.
This is
only for an N- or C- terminus application, for those active agents attached to
pendant
groups of decaglutamic acid, for instance, a drug with a molecular weight of
180 could
conceivably have a loading.of 58%, although this may not be entirely
practical.
The alcohol, amine or carboxylic acid group of an active agent may be
covalently
attached to the N-terminus, the C-terminus or the side chain of the
oligopeptide or
polypeptide. The location of attachment depends somewhat on the functional
group
selection. For instance, if the active drug is a carboxylic acid (e.g.,
aspirin) then the N-
terminus of the oligopeptide is the preferred point of attachment. If the
active agent is an
amine (e.g., ampicillin), then the C-terminus is the preferred point of
attachment in order
to achieve a stable peptide linked active agent. In both, the C- and N-
terminus examples,
the peptide is, in essence, extended by one monomeric unit forming a new
peptide bond.
If the active agent is an alcohol, then either the C-terminus or the N-
terminus is the
preferred point of attachment in order to achieve a stable composition. As in
the example
above where the alcohol, norethindrone, was covalently attached to
poly(hydroxypropylglutamine), an alcohol can be converted into an
alkylchloroformate
with phosgene. This invention, then, pertains to the reaction of this key
intermediate with
the N-terminus of the peptide carrier. The active ingredient can be released
from the
peptide carrier by intestinal peptidases.
The alcohol can be selectively bound to the gamma carboxylate of glutamic acid
and then this conjugate covalently attached to the C-terminus of the peptide
carrier.
Because the glutamic acid-drug conjugate can be considered a dimer, this
product adds
two monomeric units to the C-terminus of the peptide carrier where the
glutamic acid
moiety serves as a spacer between the peptide and the drug as shown in Fig. 4.
Intestinal
enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drug
moiety
from the peptide carrier. The newly formed free amine of the glutamic acid
residue will
then undergo an intramolecular transamination reaction, thereby, releasing the
active
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agent with coincident formation of pyroglutamic acid as shown in Fig. 5.
Alternatively,
the glutamic acid-drug dimer can be converted into the gamma ester of glutamic
acid N-
carboxyanhydride. This intermediate can then be polymerized, as described
above, using
any suitable initiator as shown in Fig. 4. The product of this polymerization
is
polyglutamic acid with active ingredients attached to multiple pendant groups.
Hence,
maximum drug loading of the Garner peptide can be achieved. In addition, other
amino
acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to
impart
specific properties to the drug delivery system.
The invention also provides a method of imparting the same mechanism of action
to for other polypeptides containing functional side chains. Examples include,
but are not
limited to, polylysine, polyasparagine, polyarginine, polyserine,
polycysteine,
polytyrosine, polythreonine and polyglutamine. The mechanism can translate to
these
polypeptides through a spacer or linker on the pendant group, which is
terminated,
preferably, by the glutamic acid-drug dimer. This carrier peptide-drug
conjugate is
15 distinguished from the prior art by virtue of the fact that the primary
release of the drug
moiety relies on peptidases and not on esterases. Alternatively, the active
agent can be
attached directly to the pendant group where some other indigenous enzymes in
the
alimentary tract can affect release.
The active agent can be covalently attached to the N-terminus, the C-terminus
or
2o the side chain of the polypeptide using known techniques. Examples of
linking organic
compounds to the N-terminus type of a peptide include, but are not limited to,
the
attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides
and 1,3-
dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example,
there are
known techniques for forming peptide linked biotin and peptide linked
acridine.
25 In the present invention, allopurinal is covalently attached to the
polypeptide via
its -NH group.
The polypeptide carrier can be prepared using conventional techniques. A
preferred technique is copolymerization of mixtures of amino acid N-
carboxyanhydrides.
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Alternatively, if a specific sequence is desired, a solid state automated
peptide synthesizer
can be used.
The addition of stabilizers to the composition has the potential of
stabilizing the
polypeptide further. Stabilizers such as sugar, amino acids, polyethylene
glycol (PEG)
and salts have been shown to prevent protein unfolding. In another embodiment
of the
invention, a pre-first order release of the active agent is imparted by
microencapsulating
the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid
complex,
PEG or salts.
There is evidence that hydrophilic compounds are absorbed through the
intestinal
l0 epithelia efficiently via specialized transporters. The entire membrane
transport system is
intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one
can expect
that excitation of the membrane transport system will involve some sort of
specialized
adjuvant resulting in localized delivery of active agents. There are seven
known
intestinal transport systems classified according to the physical properties
of the
15 transported substrate. They include the amino acid, oligopeptide, glucose,
monocarboxic
acid, phosphate, bile acid and the P-glycoprotein transport systems and each
has its own
associated mechanism of transport. The mechanisms can depend on hydrogen ions,
sodium ions, binding sites or other cofactors. The invention also allows
targeting the
mechanisms for intestinal epithelial transport systems to facilitate
absorption of active
20 agents.
In another embodiment of the invention, the composition includes one or more
adjuvants to enhance the bioavailability of the active agent. Addition of an
adjuvant is
particularly preferred when using an otherwise poorly absorbed active agent.
Suitable
adjuvants, for example, include: papain, which is a potent enzyme for
releasing the
25 catalytic domain of aminopeptidase-N into the lumen; glycorecognizers,
which activate
enzymes in the BBM; and bile acids, which have been attached to peptides to
enhance
absorption of the peptides.
Preferably, the resultant peptide-allopurinal conjugate is formulated into a
tablet
using suitable excipients and can either be wet granulated or dry compressed.
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Compositions of the invention are, in essence, the formation of amides from
acids
and amines and can be prepared by the following examples.
Acidl/N-terminus conjugation
An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to
0°C. The solution can then be treated with diisopropylcarbodiimide and
hydroxybenzotriazole followed by the amine peptide carrier. The reaction can
then be
stirred for several hours at room temperature, the urea by-product filtered
off, the product
precipitated out in ether and purified using gel permeation chromatography
(GPC) or
dialysis.
1o Amine/C-terminus conjugation
The peptide Garner can be dissolved in DMF under nitrogen and cooled to
0°C.
The solution can then be treated with diisopropylcarbodiimide and
hydroxybenzotriazole
followed by the amine bioactive agent. The reaction can then be stirred for
several hours
at room temperature, the urea by-product filtered off, and the product
precipitated out in
15 ether and purified using GPC or dialysis.
Alcohol/N-Terminus Conjugation
In the following example the combination of the alcohol with triphosgene
produces a chloroformate, which when reacted with the N-terminus of the
peptide
produces a carbamate. Pursuant to this, an alcohol bioactive agent can be
treated with
20 triphosgene in dry DMF under nitrogen. The suitably protected peptide
Garner is then
added slowly and the solution stirred at room temperature for several hours.
The product
is then precipitated out in ether. The crude product is suitably deprotected
and purified
using GPC.
Other solvents, activating agents, cocatalysts and bases can be used. Examples
of
25 other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or
chlorinated
solvents such as chloroform. Examples of other activating agents include
dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst
is N-
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hydroxysuccinimide. Examples of bases include pyrrolidinopyridine,
dimethylaminopyridine, triethylamine or tributylamine.
Preparation of Alkyl Glutamate
There have been over 30 different y alkyl glutamates prepared any one of which
may be suitable for the drug alcohol of choice. For example, a suspension of
glutamic
acid, the alcohol and concentrated hydrochloric acid can be prepared and
heated for
several hours. The y alkyl glutamate product can be precipitated out in
acetone, filtered,
dried and recrystallized from hot water.
Alkyl Glutamate/C-Terminus Conjugation
The peptide carrier can be dissolved in DMF under nitrogen and cooled to
0°C.
The solution can then be treated with diisopropylcarbodiimide and
hydroxybenzotriazole
followed by the y alkyl glutamate bioactive agent. The reaction can then be
stirred for
several hours at room temperature, the urea by-product filtered off, and the
product
precipitated out in ether and purified using GPC or dialysis.
Preparation of Alkyl Glutamate-NCA
y Alkyl glutamate can be suspended in dry THF where triphosgene is added and
the mixture refluxed under a nitrogen atmosphere until the mixture becomes
homogenous. The solution can be poured into heptane to precipitate the NCA
product,
which is filtered, dried and recrystallized from a suitable solvent.
Preparation of Poly[y Alkyl Glutamate]
y Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of
a primary amine can be added to the solution until it becomes viscous
(typically
overnight). The product can be isolated from the solution by pouring it into
water and
filtering. The product can be purified using GPC or dialysis.
Although illustrated and described above with reference to specific
embodiments,
the invention is nevertheless not intended to be limited to the details shown.
Rather,
is
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various modifications may be made in the details within the scope and range of
equivalents of the claims and without departing from the spirit of the
invention.
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CLAIMS
What is claimed is:
1. A pharmaceutical composition comprising:
a polypeptide; and
allopurinal covalently attached to said polypeptide.
2. The composition of claim 1 wherein said polypeptide is an oligopeptide.
3. The composition of claim 1 wherein said polypeptide is a homopolymer of a
naturally occurring amino acid.
4. The composition of claim 1 wherein said polypeptide is a heteropolymer of
two or more naturally occurnng amino acids.
5. The composition of claim 1 wherein said polypeptide is a homopolymer of a
synthetic amino acid.
6. The composition of claim 1 wherein said polypeptide is a heteropolymer of
two or more synthetic amino acids.
7. The composition of claim 1 wherein said polypeptide is a heteropolymer of
one or more naturally occurring amino acids and one or more synthetic amino
acids.
8. The composition of claim 1 wherein allopurinal is covalently attached to a
side
chain, the N-terminus or the C-terminus of said polypeptide.
9. The composition of claim 1 further comprising a microencapsulating agent.
10. The composition of claim 9 wherein said microencapsulating agent is
selected from the group consisting of polyethylene glycol (PEG), an amino
acid, a sugar
and a salt.
11. The composition of claim 1 further comprising an adjuvant.
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12. The composition of claim 11 wherein said adjuvant activates an intestinal
transporter.
13. The composition of claim 1 further comprising a pharmaceutically
acceptable
excipient.
14. The composition of claim 1 wherein said composition is in the form of an
ingestable tablet.
15. The composition of claim 1 wherein said composition is in the form of an
intravenous preparation.
16. The composition of claim 1 wherein said composition is in the form of an
oral suspension.
17. The composition of claim 1 wherein allopurinal is conformationally
protected
by folding of said polypeptide about said active agent.
18. The composition of claim 1 wherein said polypeptide is capable of
releasing
allopurinal from said composition in a pH-dependent manner.
19. A method for protecting allopurinal from degradation comprising covalently
attaching said active agent to a polypeptide.
20. A method for controlling release of allopurinal from a composition wherein
said composition comprises a polypeptide, said method comprising covalently
attaching
allopurinal to said polypeptide.
21. A method for delivering allopurinal to a patient comprising administering
to
said patient a composition comprising:
a polypeptide; and
allopurinal covalently attached to said polypeptide.
22. The method of claim 21 wherein allopurinal is released from said
composition by an enzyme-catalyzed release.
~8
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23. The method of claim 21 wherein allopurinal is released from said
composition by a pH-dependent unfolding of said polypeptide.
24. The method of claim 21 wherein said active agent is released from said
composition in a sustained release.
25. The method of claim 21 wherein said composition further comprises an
adjuvant covalently attached to said polypeptide and wherein release of said
adjuvant
from said composition is controlled by said polypeptide.
Abstract
1o A composition comprising a polypeptide and allopurinal covalently attached
to
the polypeptide. Also provided is a method for delivery of allopurinal to a
patient
comprising administering to the patient a composition comprising a polypeptide
and
allopurinal covalently attached to the polypeptide. Also provided is a method
for
protecting allopurinal from degradation comprising covalently attaching it to
a
t 5 polypeptide. Also provided is a method for controlling release of
allopurinal from a
composition comprising covalently attaching it to the polypeptide.
l9
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A NOVEL PHARMACEUTICAL COMPOUND CONTAINING ALPHA 1
PROTEINASE INHIBITOR AND METHODS OF MAKING AND USING SAME
FIELD OF THE INVENTION
The present invention relates to a novel pharmaceutical compound that
comprises
a polypeptide that is preferably covalently attached to Alpha 1 proteinase
inhibitor, as
well as methods for protecting and administering Alpha 1 proteinase inhibitor.
This
novel compound, referred to as a CARRIERWAVETM Molecular Analogue (CMA), has
the benefit of taking a known effective pharmaceutical agent that is both well
studied and
occupies a known segment of the pharmaceutical market, and combining it with a
carrier
compound that enhances the usefulness of the pharmaceutical agent without
compromising its pharmaceutical effectiveness.
BACKGROUND OF THE INVENTION
Alpha 1 proteinase inhibitor is a known pharmaceutical agent that is used in
the
treatment of emphysema. It is a natural product isolated from human blood,
using
methods known to those of ordinary skill in the art.
The novel pharmaceutical compound of the present invention is useful in
accomplishing one or more of the following goals: enhancement of the chemical
stability
of the original compound; alteration of the release profile of an orally
administered
product; enhanced digestion or absorption; targeted delivery to particular
tissue/cell type;
and provision for an oral dosage form when none exists. The novel
pharmaceutical
compound may contain one or more of the following: another active
pharmaceutical
agent, an adjuvant, or an inhibitor.
Active agent delivery systems are often critical for the effective delivery of
a
biologically active agent (active agent) to the appropriate target. The
importance of these
systems becomes magnified when patient compliance and active agent stability
are taken
under consideration. For instance, one would expect patient compliance to
increase
markedly if an active agent is administered orally in lieu of an injection or
another
invasive technique. Increasing the stability of the active agent, such as
prolonging shelf
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life or survival in the stomach, will assure dosage reproducibility and
perhaps even
reduce the number of dosages required which could improve patient compliance.
Absorption of an orally administered active agent is often blocked by the
harshly
acidic stomach milieu, powerful digestive enzymes in the GI tract,
permeability of
cellular membranes and transport across lipid bilayers. Incorporating
adjuvants such as
resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance
permeability of
cellular membranes. Microencapsulating active agents using protenoid
microspheres,
liposomes or polysaccharides have been effective in abating enzyme degradation
of the
active agent. Enzyme inhibiting adjuvants have also been used to prevent
enzyme
1o degradation. Enteric coatings have been used as a protector of
pharmaceuticals in the
stomach.
Active agent delivery systems also provide the ability to control the release
of the
active agent. For example, formulating diazepam with a copolymer of glutamic
acid and
aspartic acid enables a sustained release of the active agent. As another
example,
copolymers of lactic acid and glutaric acid are used to provide timed release
of human
growth hormone. A wide range of pharmaceuticals purportedly provide sustained
release
through microencapsulation of the active agent in amides of dicarboxylic
acids, modified
amino acids or thermally condensed amino acids. Slow release rendering
additives can
also be intermixed with a large array of active agents in tablet formulations.
2o Each of these technologies imparts enhanced stability and time-release
properties
to active agent substances. Unfortunately, these technologies suffer from
several
shortcomings. Incorporation of the active agent is often dependent on
diffusion into the
microencapsulating matrix, which may not be quantitative and may complicate
dosage
reproducibility. In addition, encapsulated drugs rely on diffusion out of the
matrix, which
is highly dependant on the water solubility of the active agent. Conversely,
water-soluble
microspheres swell by an infinite degree and, unfortunately, may release the
active agent
in bursts with little active agent available for sustained release.
Furthermore, in some
technologies, control of the degradation process required for active agent
release is
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unreliable. For example, an enterically coated active agent depends on pH to
release the
active agent and, as such, is difficult to control the rate of release.
In the past, use has been made of amino acid side chains of polypeptides as
pendant groups to which active agents can be attached. These technologies
typically
require the use of spacer groups between the amino acid pendant group and the
active
agent. The peptide-drug conjugates of this class of drug delivery system rely
on enzymes
in the bloodstream for the release of the drug and, as such, are not used for
oral
administration. Examples of timed and targeted release of injectable or
subcutaneous
pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer,
to the
to gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard,
via a peptide
spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been
covalently
attached directly to the beta carboxylate of polyaspartic acid without a
spacer group.
This prodrug formulation was designed as a colon-specific drug delivery system
where
the drug is released by bacterial hydrolytic enzymes residing in the large
intestines. The
released dexamethasone active agent, in turn, was targeted to treat large
bowel disorders
and was not intended to be absorbed into the bloodstream. Yet another
technology
combines the advantages of covalent drug attachment with liposome formation
where the
active ingredient is attached to highly ordered lipid films (known as HARs)
via a peptide
linker. Thus, there has been no drug delivery system, heretofore reported,
that
incorporates the concept of attaching an active ingredient to a polypeptide
pendant group
with its targeted delivery into the bloodstream via oral administration.
It is also important to control the molecular weight, molecular size and
particle
size of the active agent delivery system. Variable molecular weights have
unpredictable
diffusion rates and pharmacokinetics. High molecular weight carriers are
digested slowly
or late, as in the case of naproxen-linked dextran, which is digested almost
exclusively in
the colon by bacterial enzymes. High molecular weight microspheres usually
have high
moisture content which may present a problem with water labile active
ingredients.
Particle size not only becomes a problem with injectable drugs, as in the HAR
application, but absorption through the brush-border membrane of the
intestines is limited
to less than 5 microns.
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SUMMARY OF THE INVENTION
The present invention provides covalent attachment of the active agent (Alpha
1
proteinase inhibitor) to a polymer of peptides or amino acids. The invention
is
distinguished from the above-mentioned technologies by virtue of covalently
attaching
Alpha 1 proteinase inhibitor to the N-terminus, the C-terminus or directly to
the amino
acid side chain of an oligopeptide or polypeptide, also referred to herein as
a carrier
peptide. In certain applications, the polypeptide will stabilize the active
agent, primarily
in the stomach, through conformational protection. In these applications,
delivery of the
active agent is controlled, in part, by the kinetics of unfolding of the
Garner peptide.
Upon entry into the upper intestinal tract, indigenous enzymes release the
active
ingredient for absorption by the body by selectively hydrolyzing the peptide
bonds of the
Garner peptide. This enzymatic action introduces a second order sustained
release
mechanism.
Alternatively, the present invention provides a pharmaceutical composition
comprising Alpha 1 proteinase inhibitor microencapsulated by a polypeptide.
The invention provides a composition comprising a polypeptide and Alpha 1
proteinase inhibitor covalently attached to the polypeptide. Preferably, the
polypeptide is
(i) an oligopeptide, (ii) a homopolymer of one of the twenty naturally
occurring amino
acids, (iii) a heteropolymer of two or more naturally occurring amino acids,
(iv) a
homopolymer of a synthetic amino acid, (v) a heteropolymer of two or more
synthetic
amino acids or (vi) a heteropolymer of one or more naturally occurnng amino
acids and
one or more synthetic amino acids.
Alpha 1 proteinase inhibitor preferably is covalently attached to a side
chain, the
N-terminus or the C-terminus of the polypeptide. In a preferred embodiment,
the active
agent is a carboxylic acid and is covalently attached to the N-terminus of the
polypeptide.
In another preferred embodiment, the active agent is an amine and is
covalently attached
to the C-terminus of the polypeptide. In another preferred embodiment, the
active agent
is an alcohol and is covalently attached to the C-terminus of the polypeptide.
In yet
4
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another preferred embodiment, the active agent is an alcohol and is covalently
attached to
the N-terminus of the polypeptide.
The composition of the invention can also include one or more of a
microencapsulating agent, an adjuvant and a pharmaceutically acceptable
excipient. The
microencapsulating agent can be selected from polyethylene glycol (PEG), an
amino
acid, a sugar and a salt. When an adjuvant is included in the composition, the
adjuvant
preferably activates an intestinal transporter.
Preferably, the composition of the invention is in the form of an ingestable
tablet,
an intravenous preparation or an oral suspension. The active agent can be
~o conformationally protected by folding of the polypeptide about the active
agent. In
another embodiment, the polypeptide is capable of releasing the active agent
from the
composition in a pH-dependent manner.
The invention also provides a method for protecting Alpha 1 proteinase
inhibitor
from degradation comprising covalently attaching it to a polypeptide.
15 The invention also provides a method for delivering Alpha 1 proteinase
inhibitor
to a patient, the patient being a human or a non-human animal, comprising
administering
to the patient a composition comprising a polypeptide and an active agent
covalently
attached to the polypeptide. In a preferred embodiment, Alpha 1 proteinase
inhibitor is
released from the composition by an enzyme-catalyzed release. In another
preferred
2o embodiment, Alpha 1 proteinase inhibitor is released in a time-dependent
manner based
on the pharmacokinetics of the enzyme-catalyzed release. In another preferred
embodiment, the composition further comprises a microencapsulating agent and
Alpha 1
proteinase inhibitor is released from the composition by dissolution of the
microencapsulating agent. In another preferred embodiment, Alpha 1 proteinase
inhibitor
25 is released from the composition by a pH-dependent unfolding of the
polypeptide. In
another preferred embodiment, Alpha 1 proteinase inhibitor is released from
the
composition in a sustained release. In yet another preferred embodiment, the
composition further comprises an adjuvant covalently attached to the
polypeptide and
release of the adjuvant from the composition is controlled by the polypeptide.
The
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adjuvant can be microencapsulated into a carrier peptide-drug conjugate for
biphasic
release of active ingredients.
The invention also provides a method for preparing a composition comprising a
polypeptide and an active agent covalently attached to the polypeptide. The
method
comprises the steps of:
(a) attaching Alpha 1 proceinase inhibitor to a side chain of an amino acid to
form an
active agent/amino acid complex;
(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA)
from the active agent/amino acid complex; and
(c) polymerizing the active agent/amino acid complex N-carboxyanhydride
(NCA).
In a preferred embodiment, steps (a) and (b) are repeated prior to step (c)
with a
second active agent. When steps (a) and (b) are repeated prior to step (c)
with a second
agent, Alpha 1 proteinase inhibitor and a second active agent can be
copolymerized in
step (c). In another preferred embodiment, the amino acid is glutamic acid and
the active
agent is released from the glutamic acid as a dimer upon a hydrolysis of the
polypeptide
and wherein the active agent is released from the glutamic acid by coincident
intramolecular transamination. In another preferred embodiment, the glutamic
acid is
replaced by an amino acid selected from the group consisting of aspartic acid,
arginine,
2o asparagine, cysteine, lysine, threonine, and serine, and wherein the active
agent is
attached to the side chain of the amino acid to form an amide, a thioester, an
ester, an
ether, a urethane, a carbonate, an anhydride or a carbamate. In yet another
preferred
embodiment, the glutamic acid is replaced by a synthetic amino acid with a
pendant
group comprising an amine, an alcohol, a sulfhydryl, an amide, a urea, or an
acid
functionality.
It is to be understood that both the foregoing general description and the
following detailed description are exemplary, but are not restrictive, of the
invention.
The general applications of this invention to other active pharmaceutical
agents is
described in U.S. Patent Application Serial Number 09/642,820, filed August
22, 2000,
incorporated herein by reference.
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DETAILED DESCRIPTION OF INVENTION
The present invention provides several benefits for active agent delivery.
First,
the invention can stabilize Alpha 1 proteinase inhibitor and prevent its
digestion in the
stomach. In addition, the pharmacologic effect can be prolonged by delayed
release of
Alpha 1 proteinase inhibitor. Furthermore, active agents can be combined to
produce
synergistic effects. Also, absorption of the active agent in the intestinal
tract can be
enhanced. The invention also allows targeted delivery of active agents to
specifics sites
of action.
The composition of the invention comprises Alpha 1 proteinase inhibitor
to covalently attached to a polypeptide. Preferably, the polypeptide is (i) an
oligopeptide,
(ii) a homopolymer of one of the twenty naturally occurring amino acids, (iii)
a
heteropolymer of two or more naturally occurnng amino acids, (iv) a
homopolymer of a
synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids
or (vi) a
heteropolymer of one or more naturally occurnng amino acids and one or more
synthetic
15 amino acids.
Proteins, oligopeptides and polypeptides are polymers of amino acids that have
primary, secondary and tertiary structures. The secondary structure of the
protein is the
local conformation of the polypeptide chain and consists of helices, pleated
sheets and
turns. The protein's amino acid sequence and the structural constraints on the
20 conformations of the chain determine the spatial arrangement of the
molecule. The
folding of the secondary structure and the spatial arrangement of the side
chains
constitute the tertiary structure
Proteins fold because of the dynamics associated between neighboring atoms on
the protein and solvent molecules. The thermodynamics of protein folding and
unfolding
25 are defined by the free energy of a particular condition of the protein
that relies on a
particular model. The process of protein folding involves, amongst other
things, amino
acid residues packing into a hydrophobic core. The amino acid side chains
inside the
protein core occupy the same volume as they do in amino acid crystals. The
folded
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protein interior is therefore more like a crystalline solid than an oil drop
and so the best
model for determining forces contributing to protein stability is the solid
reference state.
The major forces contributing to the thermodynamics of protein folding are Van
der Waals interactions, hydrogen bonds, electrostatic interactions,
configurational
entropy and the hydrophobic effect. Considering protein stability, the
hydrophobic effect
refers to the energetic consequences of removing apolar groups from the
protein interior
and exposing them to water. Comparing the energy of amino acid hydrolysis with
protein unfolding in the solid reference state, the hydrophobic effect is the
dominant
force. Hydrogen bonds are established during the protein fold process and
intramolecular
l0 bonds are formed at the expense of hydrogen bonds with water. Water
molecules are
"pushed out" of the packed, hydrophobic protein core. All of these forces
combine and
contribute to the overall stability of the folded protein where the degree to
which ideal
packing occurs determines the degree of relative stability of the protein. The
result of
maximum packing is to produce a center of residues or hydrophobic core that
has
15 maximum shielding from solvent.
Since it is likely that lipophilic drugs would reside in the hydrophobic core
of a
peptide, it would require energy to unfold the peptide before the drug can be
released.
The unfolding process requires overcoming the hydrophobic effect by hydrating
the
amino acids or achieving the melting temperature of the protein. The heat of
hydration is
20 a destabilization of a protein. Typically, the folded state of a protein is
favored by only
5-15 kcal/mole over the unfolded state. Nonetheless, protein unfolding at
neutral pH and
at room temperature requires chemical reagents. In fact, partial unfolding of
a protein is
often observed prior to the onset of irreversible chemical or conformation
processes.
Moreover, protein conformation generally controls the rate and extent of
deleterious
25 chemical reactions.
Conformational protection of active agents by proteins depends on the
stability of
the protein's folded state and the thermodynamics associated with the agent's
decomposition. Conditions necessary for the agent's decomposition should be
different
than for protein unfolding.
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Selection of the amino acids will depend on the physical properties desired.
For
instance, if increase in bulk or lipophilicity is desired, then the carrier
polypeptide will be
enriched in the amino acids in the table provided below. Polar amino acids, on
the other
hand, can be selected to increase the hydrophilicity of the polypeptide.
Ionizing amino acids can be selected for pH controlled peptide unfolding.
Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the
stomach, but will
ionize upon entry into the intestine. Conversely, basic amino acids, such as
histidine,
lysine and arginine, ionize in the stomach and are neutral in an alkaline
environment.
Other factors such as n-n interactions between aromatic residues, kinking of
the
peptide chain by addition of proline, disulfide crosslinking and hydrogen
bonding can all
be used to select the optimum amino acid sequence for a given application.
Ordering of
the linear sequence can influence how these interactions can be maximized and
is
important in directing the secondary and tertiary structures of the
polypepdde.
Furthermore, amino acids with reactive side chains (e.g., glutamic acid,
lysine,
aspartic acid, serine, threonine and cysteine) can be incorporated for
attaching multiple
active agents or adjuvants to the same carrier peptide. This is particularly
useful if a
synergistic effect between two or more active agents is desired.
As stated above, variable molecular weights of the Garner compound can have
profound effects on the active agent release kinetics. As a result, low
molecular weight
active agent delivery systems are preferred. An advantage of this invention is
that chain
length and molecular weight of the polypeptide can be optimized depending on
the level
of conformational protection desired. This property can be optimized in
concert with the
kinetics of the first order release mechanism. Thus, another advantage of this
invention is
that prolonged release time can be imparted by increasing the molecular weight
of the
carrier polypeptide. Another, significant advantage of the invention is that
the kinetics of
active agent release is primarily controlled by the enzymatic hydrolysis of
the key bond
between the carrier peptide and the active agent.
9
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Dextran is the only polysaccharide known that has been explored as a
macromolecular carrier for the covalent binding of drug for colon specific
drug delivery.
Generally, it was only possible to load up to 1/10 of the total drug-dextran
conjugate
weight with drug. As stated earlier, polysaccharides are digested mainly in
the colon and
drug absorption is mainly limited to the colon. As compared to dextran, this
invention
has two major advantages. First, peptides are hydrolyzed by any one of several
aminopeptidases found in the intestinal lumen or associated with the brush-
border
membrane and so active agent release and subsequent absorption can occur in
the
jejunum or the ileum. Second, the molecular weight of the carrier molecule can
be
controlled and, thus, active agent loading can also be controlled.
As a practical example, the following table lists the molecular weights of
lipophilic amino acids (less one water molecule) and selected analgesics and
vitamins.
TABLE
Amino acidMW Active went MW
Glycine 57 Acetaminophen 151
Alanine 71 Vitamin B6 (Pyroxidine) 169
Valine 99 Vitamin C (Ascorbic acid) 176
Leucine 113 Aspirin 180
Isoleucine113 Ibuprofen 206
Phenylalanine147 Retinoic acid 300
Tyrosine 163 Vitamin BZ (Riboflavin) 376
Vitamin D2 397
Vitamin E (Tocopherol) 431
Lipophilic amino acids are preferred because conformational protection through
the
stomach is important for the selected active agents, which were selected based
on ease of
covalent attachment to an oligopeptide. Eighteen was subtracted from the amino
acid's
molecular weight so that their condensation into a polypeptide is considered.
For
example, a decamer of glycine (MW=588) linked to aspirin would have a total
molecular
weight of 750 and aspirin would represent 24% of the total weight of the
active agent
delivery composition or over two times the maximum drug loading for dextran.
This is
only for an N- or C- terminus application, for those active agents attached to
pendant
t0
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groups of decaglutamic acid, for instance, a drug with a molecular weight of
180 could
conceivably have a loading of 58%, although this may not be entirely
practical.
The alcohol, amine or carboxylic acid group of an active agent may be
covalently
attached to the N-terminus, the C-terminus or the side chain of the
oligopeptide or
polypeptide. The location of attachment depends somewhat on the functional
group
selection. For instance, if the active drug is a carboxylic acid (e.g.,
aspirin) then the N-
terminus of the oligopeptide is the preferred point of attachment. If the
active agent is an
amine (e.g., ampicillin), then the C-terminus is the preferred point of
attachment in order
to achieve a stable peptide linked active agent. In both, the C- and N-
terminus examples,
the peptide is, in essence, extended by one monomeric unit forming a new
peptide bond.
If the active agent is an alcohol, then either the C-terminus or the N-
terminus is the
preferred point of attachment in order to achieve a stable composition. As in
the example
above where the alcohol, norethindrone, was covalently attached to
poly(hydroxypropylglutamine), an alcohol can be converted into an
alkylchloroformate
with phosgene. This invention, then, pertains to the reaction of this key
intermediate with
the N-terminus of the peptide carrier. The active ingredient can be released
from the
peptide carrier by intestinal peptidases.
The alcohol can be selectively bound to the gamma carboxylate of glutamic acid
and then this conjugate covalently attached to the C-terminus of the peptide
Garner.
Because the glutamic acid-drug conjugate can be considered a dimer, this
product adds
two monomeric units to the C-terminus of the peptide carrier where the
glutamic acid
moiety serves as a spacer between the peptide and the drug as shown in Fig. 4.
Intestinal
enzymatic hydrolysis of the key peptide bond releases the glutamic acid-drug
moiety
from the peptide carrier. The newly formed free amine of the glutamic acid
residue will
then undergo an intramolecular transamination reaction, thereby, releasing the
active
agent with coincident formation of pyroglutamic acid as shown in Fig. 5.
Alternatively,
the glutamic acid-drug dimer can be converted into the gamma ester of glutamic
acid N-
carboxyanhydride. This intermediate can then be polymerized, as described
above, using
any suitable initiator as shown in Fig. 4. The product of this polymerization
is
polyglutamic acid with active ingredients attached to multiple pendant groups.
Hence,
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maximum drug loading of the carrier peptide can be achieved. In addition,
other amino
acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to
impart
specific properties to the drug delivery system.
The invention also provides a method of imparting the same mechanism of action
for other polypeptides containing functional side chains. Examples include,
but are not
limited to, polylysine, polyasparagine, polyarginine, polyserine,
polycysteine,
polytyrosine, polythreonine and polyglutamine. The mechanism can translate to
these
polypeptides through a spacer or linker on the pendant group, which is
terminated,
preferably, by the glutamic acid-drug dimer. This carrier peptide-drug
conjugate is
1o distinguished from the prior art by virtue of the fact that the primary
release of the drug
moiety relies on peptidases and not on esterases. Alternatively, the active
agent can be
attached directly to the pendant group where some other indigenous enzymes in
the
alimentary tract can affect release.
The active agent can be covalently attached to the N-terminus, the C-terminus
or
15 the side chain of the polypeptide using known techniques. Examples of
linking organic
compounds to the N-terminus type of a peptide include, but are not limited to,
the
attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides
and 1,3-
dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example,
there are
known techniques for forming peptide linked biotin and peptide linked
acridine.
20 The polypeptide carrier can be prepared using conventional techniques. A
preferred technique is copolymerization of mixtures of amino acid N-
carboxyanhydrides.
Alternatively, if a specific sequence is desired, a solid state automated
peptide synthesizer
can be used.
The addition of stabilizers to the composition has the potential of
stabilizing the
25 polypeptide further. Stabilizers such as sugar, amino acids, polyethylene
glycol (PEG)
and salts have been shown to prevent protein unfolding. In another embodiment
of the
invention, a pre-first order release of the active agent is imparted by
microencapsulating
the carrier polypeptide-active agent conjugate in a polysaccharide, amino acid
complex,
PEG or salts.
12
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There is evidence that hydrophilic compounds are absorbed through the
intestinal
epithelia efficiently via specialized transporters. The entire membrane
transport system is
intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one
can expect
that excitation of the membrane transport system will involve some sort of
specialized
adjuvant resulting in localized delivery of active agents. There are seven
known
intestinal transport systems classified according to the physical properties
of the
transported substrate. They include the amino acid, oligopeptide, glucose,
monocarboxic
acid, phosphate, bile acid and the P-glycoprotein transport systems and each
has its own
associated mechanism of transport. The mechanisms can depend on hydrogen ions,
sodium ions, binding sites or other cofactors. The invention also allows
targeting the
mechanisms for intestinal epithelial transport systems to facilitate
absorption of active
agents.
In another embodiment of the invention, the composition includes one or more
adjuvants to enhance the bioavailability of the active agent. Addition of an
adjuvant is
particularly preferred when using an otherwise poorly absorbed active agent.
Suitable
adjuvants, for example, include: papain, which is a potent enzyme for
releasing the
catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which
activate
enzymes in the BBM; and bile acids, which have been attached to peptides to
enhance
absorption of the peptides.
2o Preferably, the resultant peptide-Alpha 1 proteinase inhibitor conjugate is
formulated into a tablet using suitable excipients and can either be wet
granulated or dry
compressed.
Compositions of the invention are, in essence, the formation of amides from
acids
and amines and can be prepared by the following examples.
Acid/N-terminus conjugation
An acid bioactive agent can be dissolved in DMF under nitrogen and cooled to
0°C. The solution can then be treated with diisopropylcarbodiimide and
hydroxybenzotriazole followed by the amine peptide carrier. The reaction can
then be
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stirred for several hours at room temperature, the urea by-product filtered
off, the product
precipitated out in ether and purified using gel permeation chromatography
(GPC) or
dialysis.
AminelC-terminus conjugation
The peptide carrier can be dissolved in DMF under nitrogen and cooled to
0°C.
The solution can then be treated with diisopropylcarbodiimide and
hydroxybenzotriazole
followed by the amine bioactive agent. The reaction can then be stirred for
several hours
at room temperature, the urea by-product filtered off, and the product
precipitated out in
ether and purified using GPC or dialysis.
to AlcohoUN-Terminus Conjugation
In the following example the combination of the alcohol with triphosgene
produces a chloroformate, which when reacted with the N-terminus of the
peptide
produces a carbamate. Pursuant to this, an alcohol bioactive agent can be
treated with
triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier
is then
15 added slowly and the solution stirred at room temperature for several
hours. The product
is then precipitated out in ether. The crude product is suitably deprotected
and purified
using GPC.
Other solvents, activating agents, cocatalysts and bases can be used. Examples
of
other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or
chlorinated
2o solvents such as chloroform. Examples of other activating agents include
dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst
is N-
hydroxysuccinimide. Examples of bases include pyrrolidinopyridine,
dimethylaminopyridine, triethylamine or tributylamine.
Preparation of ~ Alkyl Glutamate
25 There have been over 30 different y alkyl glutamates prepared any one of
which
may be suitable for the drug alcohol of choice. For example, a suspension of
glutamic
acid, the alcohol and concentrated hydrochloric acid can be prepared and
heated for
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several hours. The y alkyl glutamate product can be precipitated out in
acetone, filtered,
dried and recrystallized from hot water.
~-Alkyl GlutamatelC-Terminus Conjugation
The peptide carrier can be dissolved in DMF under nitrogen and cooled to
0°C.
The solution can then be treated with diisopropylcarbodiimide and
hydroxybenzotriazole
followed by the y alkyl glutamate bioactive agent. The reaction can then be
stirred for
several hours at room temperature, the urea by-product filtered off, and the
product
precipitated out in ether and purified using GPC or dialysis.
Preparation of y~Alkyl Glutamate-NCA
y Alkyl glutamate can be suspended in dry THF where triphosgene is added and
the mixture refluxed under a nitrogen atmosphere until the mixture becomes
homogenous. The solution can be poured into heptane to precipitate the NCA
product,
which is filtered, dried and recrystallized from a suitable solvent.
Preparation of Poly[Alkyl Glutamate]
'y Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of
a primary amine can be added to the solution until it becomes viscous
(typically
overnight). The product can be isolated from the solution by pouring it into
water and
filtering. The product can be purified using GPC or dialysis.
Although illustrated and described above with reference to specific
embodiments,
the invention is nevertheless not intended to be limited to the details shown.
Rather,
various modifications may be made in the details within the scope and range of
equivalents of the claims and without departing from the spirit of the
invention.
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CLAIMS
What is claimed is:
1. A pharmaceutical composition comprising:
a polypeptide; and
Alpha 1 proteinase inhibitor covalently attached to said polypeptide.
2. The composition of claim 1 wherein said polypeptide is an oligopeptide.
3. The composition of claim 1 wherein said polypeptide is a homopolymer of a
naturally occurring amino acid.
4. The composition of claim 1 wherein said polypeptide is a heteropolymer of
to two or more naturally occurnng amino acids.
5. The composition of claim 1 wherein said polypeptide is a homopolymer of a
synthetic amino acid.
6. The composition of claim 1 wherein said polypeptide is a heteropolymer of
two or more synthetic amino acids.
7. The composition of claim 1 wherein said polypeptide is a heteropolymer of
one or more naturally occurring amino acids and one or. more synthetic amino
acids.
8. The composition of claim 1 wherein Alpha 1 proteinase inhibitor is
covalently
attached to a side chain, the N-terminus or the C-terminus of said
polypeptide.
9. The composition of claim 1 further comprising a microencapsulating agent.
10. The composition of claim 9 wherein said microencapsulating agent is
selected from the group consisting of polyethylene glycol (PEG), an amino
acid, a sugar
and a salt.
11. The composition of claim 1 further comprising an adjuvant.
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12. The composition of claim 11 wherein said adjuvant activates an intestinal
transporter.
13. The composition of claim 1 further comprising a pharmaceutically
acceptable
excipient.
14. The composition of claim 1 wherein said composition is in the form of an
ingestable tablet.
15. The composition of claim 1 wherein said composition is in the form of an
intravenous preparation.
16. The composition of claim 1 wherein said composition is in the form of an
oral suspension.
17. The composition of claim 1 wherein Alpha 1 proteinase inhibitor is
conformationally protected by folding of said polypeptide about said active
agent.
18. The composition of claim 1 wherein said polypeptide is capable of
releasing
Alpha 1 proteinase inhibitor from said composition in a pH-dependent manner.
19. A method for protecting Alpha 1 proteinase inhibitor from degradation
comprising covalently attaching said active agent to a polypeptide.
20. A method for controlling release of Alpha 1 proteinase inhibitor from a
composition wherein said composition comprises a polypeptide, said method
comprising
covalently attaching Alpha 1 proteinase inhibitor to said polypeptide.
21. A method for delivering Alpha 1 proteinase inhibitor to a patient
comprising
administering to said patient a composition comprising:
a polypeptide; and
Alpha 1 proteinase inhibitor covalently attached to said polypeptide.
22. The method of claim 21 wherein Alpha 1 proteinase inhibitor is released
from
said composition by an enzyme-catalyzed release.
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23. The method of claim 21 wherein Alpha 1 proteinase inhibitor is released
from
said composition by a pH-dependent unfolding of said polypeptide.
24. The method of claim 21 wherein said active agent is released from said
composition in a sustained release.
25. The method of claim 21 wherein said composition further comprises an
adjuvant covalently attached to said polypeptide and wherein release of said
adjuvant
from said composition is controlled by said polypeptide.
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Abstract
A composition comprising a polypeptide and Alpha 1 proteinase inhibitor
covalently attached to the polypeptide. Also provided is a method for delivery
of Alpha 1
proteinase inhibitor to a patient comprising administering to the patient a
composition
comprising a polypeptide and Alpha 1 proteinase inhibitor covalently attached
to the
polypeptide. Also provided is a method for protecting Alpha 1 proteinase
inhibitor from
degradation comprising covalently attaching it to a polypeptide. Also provided
is a
method for controlling release of Alpha 1 proteinase inhibitor from a
composition
comprising covalently attaching it to the polypeptide.
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A NOVEL PHARMACEUTICAL COMPOUND CONTAINING ALPRAZALOM
AND METHODS OF MAKING AND USING SAME
FIELD OF THE INVENTION
The present invention relates to a novel pharmaceutical compound that
comprises
a polypeptide that is preferably covalently attached to alprazalom, as well as
methods for
protecting and administering alprazalom. This novel compound, referred to as a
CARRIERWAVET~" Molecular Analogue (CMA), has the benefit of taking a known
effective pharmaceutical agent that is both well studied and occupies a known
segment of
the pharmaceutical market, and combining it with a carrier compound that
enhances the
usefulness of the pharmaceutical agent without compromising its effectiveness.
BACKGROUND OF THE INVENTION
Alprazalom is a known pharmaceutical agent that is used in the treatment of
anxiety disorders. It is both commercially available and readily manufactured
using
published synthetic schemes by those of ordinary skill in the art. Its
structure is:
H3C~ NON
N
CI
\'
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The novel pharmaceutical compound of the present invention is useful in
accomplishing one or more of the following goals: enhancement of the chemical
stability
of the original compound; alteration of the release profile of an orally
administered
product; enhanced digestion or absorption; targeted delivery to particular
tissue%ell type;
and provision for an oral dosage form when none exists. The novel
pharmaceutical
compound may contain one or more of the following: another active
pharmaceutical
agent, an adjuvant, or an inhibitor.
Active agent delivery systems are often critical for the effective delivery of
a
biologically active agent (active agent) to the appropriate target. The
importance of these
to systems becomes magnified when patient compliance and active agent
stability are taken
under consideration. For instance, one would expect patient compliance to
increase
markedly if an active agent is administered orally in lieu of an injection or
another
invasive technique. Increasing the stability of the active agent, such as
prolonging shelf
life or survival in the stomach, will assure dosage reproducibility and
perhaps even
15 reduce the number of dosages required which could improve patient
compliance.
Absorption of an orally administered active agent is often blocked by the
harshly
acidic stomach milieu, powerful digestive enzymes in the GI tract,
permeability of
cellular membranes and transport across lipid bilayers. Incorporating
adjuvants such as
resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance
permeability of
2o cellular membranes. Microencapsulating active agents using protenoid
microspheres,
liposomes or polysaccharides have been effective in abating enzyme degradation
of the
active agent. Enzyme inhibiting adjuvants have also been used to prevent
enzyme
degradation. Enteric coatings have been used as a protector of pharmaceuticals
in the
stomach.
25 Active agent delivery systems also provide the ability to control the
release of the
active agent. For example, formulating diazepam with a copolymer of glutamic
acid and
aspartic acid enables a sustained release of the active agent. As another
example,
copolymers of lactic acid and glutaric acid are used to provide timed release
of human
growth hormone. A wide range of pharmaceuticals purportedly provide sustained
release
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through microencapsulation of the active agent in amides of dicarboxylic
acids, modified
amino acids or thermally condensed amino acids. Slow release rendering
additives can
also be intermixed with a large array of active agents in tablet formulations.
Each of these technologies imparts enhanced stability and time-release
properties
to active agent substances. Unfortunately, these technologies suffer from
several
shortcomings. Incorporation of the active agent is often dependent on
diffusion into the
microencapsulating matrix, which may not be quantitative and may complicate
dosage
reproducibility. In addition, encapsulated drugs rely on diffusion out of the
matrix, which
is highly dependant on the water solubility of the active agent. Conversely,
water-soluble
microspheres swell by an infinite degree and, unfortunately, may release the
active agent
in bursts with little active agent available for sustained release.
Furthermore, in some
technologies, control of the degradation process required for active agent
release is
unreliable. For example, an enterically coated active agent depends on pH to
release the
active agent and, as such, is difficult to control the rate of release.
In the past, use has been made of amino acid side chains of polypeptides as
pendant groups to which active agents can be attached. These technologies
typically
require the use of spacer groups between the amino acid pendant group and the
active
agent. The peptide-drug conjugates of this class of drug delivery system rely
on enzymes
in the bloodstream for the release of the drug and, as such, are not used for
oral
administration. Examples of timed and targeted release of injectable or
subcutaneous
pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer,
to the
gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a
peptide
spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been
covalently
attached directly to the beta carboxylate of polyaspartic acid without a
spacer group.
This prodrug formulation was designed as a colon-specific drug delivery system
where
the drug is released by bacterial hydrolytic enzymes residing in the large
intestines. The
released dexamethasone active agent, in turn, was targeted to treat large
bowel disorders
and was not intended to be absorbed into the bloodstream. Yet another
technology
combines the advantages of covalent drug attachment with liposome formation
where the
3o active ingredient is attached to highly ordered lipid films (known as HARs)
via a peptide
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linker. Thus, there has been no drug delivery system, heretofore reported,
that
incorporates the concept of attaching an active ingredient to a polypeptide
pendant group
with its targeted delivery into the bloodstream via oral administration.
It is also important to control the molecular weight, molecular size and
particle
size of the active agent delivery system. Variable molecular weights have
unpredictable
diffusion rates and pharmacokinetics. High molecular weight carriers are
digested slowly
or late, as in the case of naproxen-linked dextran, which is digested almost
exclusively in
the colon by bacterial enzymes. High molecular weight microspheres usually
have high
moisture content which may present a problem with water labile active
ingredients.
Particle size not only becomes a problem with injectable drugs, as in the HAR
application, but absorption through the brush-border membrane of the
intestines is limited
to less than 5 microns.
SUMMARY OF THE INVENTION
The present invention provides covalent attachment of the active agent
(alprazalom) to a polymer of peptides or amino acids. The invention is
distinguished
from the above-mentioned technologies by virtue of covalently attaching
alprazalom to
the N-terminus, the C-terminus or directly to the amino acid side chain of an
oligopeptide
or polypeptide, also referred to herein as a Garner peptide. In certain
applications, the
polypeptide will stabilize the active agent, primarily in the stomach, through
2o conformational protection. In these applications, delivery of the active
agent is
controlled, in part, by the kinetics of unfolding of the Garner peptide. Upon
entry into the
upper intestinal tract, indigenous enzymes release the active ingredient for
absorption by
the body by selectively hydrolyzing the peptide bonds of the carrier peptide.
This
enzymatic action introduces a second order sustained release mechanism.
Alternatively, the present invention provides a pharmaceutical composition
comprising alprazalom microencapsulated by a polypeptide.
The invention provides a composition comprising a polypeptide and alprazalom
covalently attached to the polypeptide. Preferably, the polypeptide is (i) an
oligopeptide,
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(ii) a homopolymer of one of the twenty naturally occurnng amino acids, (iii)
a
heteropolymer of two or more naturally occurnng amino acids, (iv) a
homopolymer of a
synthetic amino acid, (v) a heteropolymer of two or more synthetic amino acids
or (vi) a
heteropolymer of one or more naturally occurring amino acids and one or more
synthetic
amino acids.
Alprazalom preferably is covalently attached to a side chain, the N-terminus
or
the C-terminus of the polypeptide. In a preferred embodiment, the active agent
is a
carboxylic acid and is covalently attached to the N-terminus of the
polypeptide. In
another preferred embodiment, the active agent is an amine and is covalently
attached to
to the C-terminus of the polypeptide. In another preferred embodiment, the
active agent is
an alcohol and is covalently attached to the C-terminus of the polypeptide. In
yet another
preferred embodiment, the active agent is an alcohol and is covalently
attached to the N-
terminus of the polypeptide.
The composition of the invention can also include one or more of a
15 microencapsulating agent, an adjuvant and a pharmaceutically acceptable
excipient. The
microencapsulating agent can be selected from polyethylene glycol (PEG), an
amino
acid, a sugar and a salt. When an adjuvant is included in the composition, the
adjuvant
preferably activates an intestinal transporter.
Preferably, the composition of the invention is in the form of an ingestable
tablet,
2o an intravenous preparation or an oral suspension. The active agent can be
conformationally protected by folding of the polypeptide about the active
agent. In
another embodiment, the polypeptide is capable of releasing the active agent
from the
composition in a pH-dependent manner.
The invention also provides a method for protecting alprazalom from
degradation
25 comprising covalently attaching it to a polypeptide.
The invention also provides a method for delivering alprazalom to a patient,
the
patient being a human or a non-human animal, comprising administering to the
patient a
composition comprising a polypeptide and an active agent covalently attached
to the
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polypeptide. In a preferred embodiment, alprazalom is released from the
composition by
an enzyme-catalyzed release. In another preferred embodiment, alprazalom is
released in
a time-dependent manner based on the pharmacokinetics of the enzyme-catalyzed
release. In another preferred embodiment, the composition further comprises a
microencapsulating agent and alprazalom is released from the composition by
dissolution
of the microencapsulating agent. In another preferred embodiment, alprazalom
is
released from the composition by a pH-dependent unfolding of the polypeptide.
In
another preferred embodiment, alprazalom is released from the composition in a
sustained release. In yet another preferred embodiment, the composition
further
1o comprises an adjuvant covalently attached to the polypeptide and release of
the adjuvant
from the composition is controlled by the polypeptide. The adjuvant can be
microencapsulated into a carrier peptide-drug conjugate for biphasic release
of active
ingredients.
The invention also provides a method for preparing a composition comprising a
15 polypeptide and an active agent covalently attached to the polypeptide. The
method
comprises the steps of:
(a) attaching alprazalom to a side chain of an amino acid to form an active
agentlamino acid complex;
(b) forming an active agent/amino acid complex N-carboxyanhydride (NCA)
20 from the active agent/amino acid complex; and
(c) polymerizing the active agent/amino acid complex N-carboxyanhydride
(NCA).
In a preferred embodiment, steps (a) and (b) are repeated prior to step (c)
with a
second active agent. When steps (a) and (b) are repeated prior to step (c)
with a second
25 agent, alprazalom and a second active agent can be copolymerized in step
(c). In another
preferred embodiment, the amino acid is glutamic acid and the active agent is
released
from the glutamic acid as a dimer upon a hydrolysis of the polypeptide and
wherein the
active agent is released from the glutamic acid by coincident intramolecular
transamination. In another preferred embodiment, the glutamic acid is replaced
by an
3o amino acid selected from the group consisting of aspartic acid, arginine,
asparagine,
cysteine, lysine, threonine, and serine, and wherein the active agent is
attached to the side
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chain of the amino acid to form an amide, a thioester, an ester, an ether, a
urethane, a
carbonate, an anhydride or a carbamate. In yet another preferred embodiment,
the
glutamic acid is replaced by a synthetic amino acid with a pendant group
comprising an
amine, an alcohol, a sulfhydryl, an amide, a urea, or an acid functionality.
It is to be understood that both the foregoing general description and the
following detailed description are exemplary, but are not restrictive, of the
invention.
The general applications of this invention, to other active pharmaceutical
agents is
described in U.S. Patent Application Serial Number 09/642,820, filed August
22, 2000,
incorporated herein by reference.
1o DETAILED DESCRIPTION OF INVENTION
The present invention provides several benefits for active agent delivery.
First,
the invention can stabilize alprazalom and prevent its digestion in the
stomach. In
addition, the pharmacologic effect can be prolonged by delayed release of
alprazalom.
Furthermore, active agents can be combined to produce synergistic effects.
Also,
15 absorption of the active agent in the intestinal tract can be enhanced. The
invention also
allows targeted delivery of active agents to specifics sites of action.
In the present invention, alprazalom is covalently attached to the polypeptide
via a
linker. This linker may be a small molecule containing 2-6 carbons and one or
more
functional groups (such as amines, amides, alcohols, or acids) or may be made
up of a
2o short chain of either amino acids or carbohydrates.
The composition of the invention comprises alprazalom covalently attached to a
polypeptide. Preferably, the polypeptide is (i) an oligopeptide, (ii) a
homopolymer of one
of the twenty naturally occurring amino acids, (iii) a heteropolymer of two or
more
naturally occurring amino acids, (iv) a homopolymer of a synthetic amino acid,
(v) a
25 heteropolymer of two or more synthetic amino acids or (vi) a heteropolymer
of one or
more naturally occurring amino acids and one or more synthetic amino acids.
Proteins, oligopeptides and polypeptides are polymers of amino acids that have
primary, secondary and tertiary structures. The secondary structure of the
protein is the
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local conformation of the polypeptide chain and consists of helices, pleated
sheets and
turns. The protein's amino acid sequence and the structural constraints on the
conformations of the chain determine the spatial arrangement of the molecule.
The
folding of the secondary structure and the spatial arrangement of the side
chains
constitute the tertiary structure.
Proteins fold because of the dynamics associated between neighboring atoms on
the protein and solvent molecules. The thermodynamics of protein folding and
unfolding
are defined by the free energy of a particular condition of the protein that
relies on a
particular model. The process of protein folding involves, amongst other
things, amino
acid residues packing into a hydrophobic core. The amino acid side chains
inside the
protein core occupy the same volume as they do in amino acid crystals. The
folded
protein interior is therefore more like a crystalline solid than an oil drop
and so the best
model for determining forces contributing to protein stability is the solid
reference state.
The major forces contributing to the thermodynamics of protein folding are Van
der Waals interactions, hydrogen bonds, electrostatic interactions,
configurational
entropy and the hydrophobic effect. Considering protein stability, the
hydrophobic effect
refers to the energetic consequences of removing apolar groups from the
protein interior
and exposing them to water. Comparing the energy of amino acid hydrolysis with
protein unfolding in the solid reference state, the hydrophobic effect is the
dominant
2o force. Hydrogen bonds are established during the protein fold process and
intramolecular
bonds are formed at the expense of hydrogen bonds with water. Water molecules
are
"pushed out" of the packed, hydrophobic protein core. All of these forces
combine and
contribute to the overall stability of the folded protein where the degree to
which ideal
packing occurs determines the degree of relative stability of the protein. The
result of
maximum packing is to produce a center of residues or hydrophobic core that
has
maximum shielding from solvent.
Since it is likely that lipophilic drugs would reside in the hydrophobic core
of a
peptide, it would require energy to unfold the peptide before the drug can be
released.
The unfolding process requires overcoming the hydrophobic effect by hydrating
the
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amino acids or achieving the melting temperature of the protein. The heat of
hydration is
a destabilization of a protein. Typically, the folded state of a protein is
favored by only
5-15 kcaUmole over the unfolded state. Nonetheless, protein unfolding at
neutral pH and
at room temperature requires chemical reagents. In fact, partial unfolding of
a protein is
often observed prior to the onset of irreversible chemical or conformation
processes.
Moreover, protein conformation generally controls the rate and extent of
deleterious
chemical reactions.
Conformational protection of active agents by proteins depends on the
stability of
the protein's folded state and the thermodynamics associated with the agent's
decomposition. Conditions necessary for the agent's decomposition should be
different
than for protein unfolding.
Selection of the amino acids will depend on the physical properties desired.
For
instance, if increase in bulk or lipophilicity is desired, then the carrier
polypeptide will be
enriched in the amino acids in the table provided below. Polar amino acids, on
the other
hand, can be selected to increase the hydrophilicity of the polypeptide.
Ionizing amino acids can be selected for pH controlled peptide unfolding.
Aspartic acid, glutamic acid and tyrosine carry a neutral charge in the
stomach, but will
ionize upon entry into the intestine. Conversely, basic amino acids, such as
histidine,
lysine and arginine, ionize in the stomach and are neutral in an alkaline
environment.
Other factors such as ~-~ interactions between aromatic residues, kinking of
the
peptide chain by addition of proline, disulfide crosslinking and hydrogen
bonding can all
be used to select the optimum amino acid sequence for a given application.
Ordering of
the linear sequence can influence how these interactions can be maximized and
is
important in directing the secondary and tertiary structures of the
polypeptide.
Furthermore, amino acids with reactive side chains (e.g., glutamic acid,
lysine,
aspartic acid, serine, threonine and cysteine) can be incorporated for
attaching multiple
active agents or adjuvants to the same carrier peptide. This is particularly
useful if a
synergistic effect between two or more active agents is desired.
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As stated above, variable molecular weights of the carrier compound can have
profound effects on the active agent release kinetics. As a result, low
molecular weight
active agent delivery systems are preferred. An advantage of this invention is
that chain
length and molecular weight of the polypeptide can be optimized depending on
the level
of conformational protection desired. This property can be optimized in
concert with the
kinetics of the first order release mechanism. Thus, another advantage of this
invention is
that prolonged release time can be imparted by increasing the molecular weight
of the
Garner polypeptide. Another, significant advantage of the invention is that
the kinetics of
active agent release is primarily controlled by the enzymatic hydrolysis of
the key bond
between the carrier peptide and the active agent.
Dextran is the only polysaccharide known that has been explored as a
macromolecular carrier for the covalent binding of drug for colon specific
drug delivery.
Generally, it was only possible to load up to 1/10 of the total drug-dextran
conjugate
weight with drug. As stated earlier, polysaccharides are digested mainly in
the colon and
drug absorption is mainly limited to the colon. As compared to dextran, this
invention
has two major advantages. First, peptides are hydrolyzed by any one of several
aminopeptidases found in the intestinal lumen or associated with the brush-
border
membrane and so active agent release and subsequent absorption can occur in
the
jejunum or the ileum. Second, the molecular weight of the carrier molecule can
be
controlled and, thus, active agent loading can also be controlled.
As a practical example, the following table lists the molecular weights of
lipophilic amino acids (less one water molecule) and selected analgesics and
vitamins.
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TABLE
Amino acid MW Active agent MW
Glycine 57 Acetaminophen 151
Alanine 71 Vitamin B6 (Pyroxidine) 169
Valine 99 Vitamin C (Ascorbic acid) 176
Leucine 113 Aspirin 180
Isoleucine 113 Ibuprofen 206
Phenylalanine147 Retinoic acid 300
Tyrosine 163 Vitamin B2 (Riboflavin) 376
Vitamin D2 397
Vitamin E (Tocopherol) 431
Lipophilic amino acids are preferred because conformational protection through
the
stomach is important for the selected active agents, which were selected based
on ease of
covalent attachment to an oligopeptide. Eighteen was subtracted from the amino
acid's
molecular weight so that their condensation into a polypeptide is considered.
For
example, a decamer of glycine (MW=588) linked to aspirin would have a total
molecular
weight of 750 and aspirin would represent 24% of the total weight of the
active agent
delivery composition or over two times the maximum drug loading for dextran.
This is
only for an N- or C- terminus application, for those active agents attached to
pendant
to groups of decaglutamic acid, for instance, a drug with a molecular weight
of 180 could
conceivably have a loading of 58%, although this may not be entirely
practical.
The alcohol, amine or carboxylic acid group of an active agent may be
covalently
attached to the N-terminus, the C-terminus or the side chain of the
oligopeptide or
polypeptide. The location of attachment depends somewhat on the functional
group
selection. For instance, if the active drug is a carboxylic acid (e.g.,
aspirin) then the N-
terminus of the oligopeptide is the preferred point of attachment. If the
active agent is an
amine (e.g., ampicillin), then the C-terminus is the preferred point of
attachment in order
to achieve a stable peptide linked active agent. In both, the C- and N-
terminus examples,
the peptide is, in essence, extended by one monomeric unit forming a new
peptide bond.
2o If the active agent is an alcohol, then either the C-terminus or the N-
terminus is the
preferred point of attachment in order to achieve a stable composition. As in
the example
above where the alcohol, norethindrone, was covalently attached to
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poly(hydroxypropylglutamine), an alcohol can be converted into an
alkylchloroformate
with phosgene. This invention, then, pertains to the reaction of this key
intermediate with
the N-terminus of the peptide Garner. The active ingredient can be released
from the
peptide carrier by intestinal peptidases.
The alcohol can be selectively bound to the gamma carboxylate of glutamic acid
and then this conjugate covalently attached to the C-terminus of the peptide
carrier.
Because the glutamic acid-drug conjugate can be considered a dimer, this
product adds
two monomeric units to the C-terminus of the peptide carrier where the
glutamic acid
moiety serves as a spacer between the peptide and the drug as shown in Fig. 4.
Intestinal
1o enzymatic hydrolysis of the key peptide bond releases the glutamic acid-
drug moiety
from the peptide carrier. The newly formed free amine of the glutamic acid
residue will
then undergo an intramolecular transamination reaction, thereby, releasing the
active
agent with coincident formation of pyroglutamic acid as shown in Fig. 5.
Alternatively,
the glutamic acid-drug dimer can be converted into the gamma ester of glutamic
acid N-
15 carboxyanhydride. This intermediate can then be polymerized, as described
above, using
any suitable initiator as shown in Fig. 4. The product of this polymerization
is
polyglutamic acid with active ingredients attached to multiple pendant groups.
Hence,
maximum drug loading of the carrier peptide can be achieved. In addition,
other amino
acid-NCA's can be copolymerized with the gamma ester glutamic acid NCA to
impart
20 specific properties to the drug delivery system.
The invention also provides a method of imparting the same mechanism of action
for other polypeptides containing functional side chains. Examples include,
but are not
limited to, polylysine, polyasparagine, polyarginine, polyserine,
polycysteine,
polytyrosine, polythreonine and polyglutamine. The mechanism can translate to
these
25 polypeptides through a spacer or linker on the pendant group, which is
terminated,
preferably, by the glutamic acid-drug dimer. This carrier peptide-drug
conjugate is
distinguished from the prior art by virtue of the fact that the primary
release of the drug
moiety relies on peptidases and not on esterases. Alternatively, the active
agent can be
attached directly to the pendant group where some other indigenous enzymes in
the
3o alimentary tract can affect release.
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The active agent can be covalently attached to the N-terminus, the C-terminus
or
the side chain of the polypeptide using known techniques. Examples of linking
organic
compounds to the N-terminus type of a peptide include, but are not limited to,
the
attachment of naphthylacetic acid to LH-RH, coumarinic acid to opioid peptides
and 1,3-
dialkyl-3-acyltriazenes to tetragastrin and pentagastrin. As another example,
there are
known techniques for forming peptide linked biotin and peptide linked
acridine.
In the present invention, alprazalom is covalently attached to the polypeptide
via
the zzzzzzz.
The polypeptide carrier can be prepared using conventional techniques. A
1o preferred technique is copolymerization of mixtures of amino acid N-
carboxyanhydrides.
Alternatively, if a specific sequence is desired, a solid state automated
peptide synthesizer
can be used.
The addition of stabilizers to the composition has the potential of
stabilizing the
polypeptide further. Stabilizers such as sugar, amino acids, polyethylene
glycol (PEG)
15 and salts have been shown to prevent protein unfolding. In another
embodiment of the
invention, a pre-first order release of the active agent is imparted by
microencapsulating
the Garner polypeptide-active agent conjugate in a polysaccharide, amino acid
complex,
PEG or salts.
There is evidence that hydrophilic compounds are absorbed through the
intestinal
2o epithelia efficiently via specialized transporters. The entire membrane
transport system is
intrinsically asymmetric and responds asymmetrically to cofactors. Thus, one
can expect
that excitation of the membrane transport system will involve some sort of
specialized
adjuvant resulting in localized delivery of active agents. There are seven
known
intestinal transport systems classified according to the physical properties
of the
25 transported substrate. They include the amino acid, oligopeptide, glucose,
monocarboxic
acid, phosphate, bile acid and the P-glycoprotein transport systems and each
has its own
associated mechanism of transport. The mechanisms can depend on hydrogen ions,
sodium ions, binding sites or other cofactors. The invention also allows
targeting the
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mechanisms for intestinal epithelial transport systems to facilitate
absorption of active
agents.
In another embodiment of the invention, the composition includes one or more
adjuvants to enhance the bioavailability of the active agent. Addition of an
adjuvant is
particularly preferred when using an otherwise poorly absorbed active agent.
Suitable
adjuvants, for example, include: papain, which is a potent enzyme for
releasing the
catalytic domain of aminopeptidase-N into the lumen; glycorecognizers, which
activate
enzymes in the BBM; and bile acids, which have been attached to peptldes to
enhance
absorption of the peptides.
to Preferably, the resultant peptide-alprazalom conjugate is formulated into a
tablet
using suitable excipients and can either be wet granulated or dry compressed.
Compositions of the invention are, in essence, the formation of amides from
acids
and amines and can be prepared by the following examples.
Acid/N-terminus coqjugation
15 An acid bioactive agent can be dissolved in DMF under nitrogen and cooled
to
0°C. The solution can then be treated with diisopropylcarbodiimide and
hydroxybenzotriazole followed by the amine peptide Garner. The reaction can
then be
stirred for several hours at room temperature, the urea by-product filtered
off, the product
precipitated out in ether and purified using gel permeation chromatography
(GPC) or
2o dialysis.
Amine/C-terminus conjugation
The peptide Garner can be dissolved in DMF under nitrogen and cooled to
0°C.
The solution can then be treated with diisopropylcarbodiimide and
hydroxybenzotriazole
followed by the amine bioactive agent. The reaction can then be stirred for
several hours
25 at room temperature, the urea by-product filtered off, and the product
precipitated out in
ether and purified using GPC or dialysis.
Alcohol/N-Terminus Conjugation
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In the following example the combination of the alcohol with triphosgene
produces a chloroformate, which when reacted with the N-terminus of the
peptide
produces a carbamate. Pursuant to this, an alcohol bioactive agent can be
treated with
triphosgene in dry DMF under nitrogen. The suitably protected peptide carrier
is then
added slowly and the solution stirred at room temperature for several hours.
The product
is then precipitated out in ether. The crude product is suitably deprotected
and purified
using GPC.
Other solvents, activating agents, cocatalysts and bases can be used. Examples
of
other solvents include dimethylsulfoxide, ethers such as tetrahydrofuran or
chlorinated
solvents such as chloroform. Examples of other activating agents include
dicyclohexylcarbodiimide or thionyl chloride. An example of another cocatalyst
is N-
hydroxysuccinimide. Examples of bases include pyrrolidinopyridine,
dimethylaminopyridine, triethylamine or tributylamine.
Preparation of ~-Alkyl Glutamate
There have been over 30 different y alkyl glutamates prepared any one of which
may be suitable for the drug alcohol of choice. For example, a suspension of
glutamic
acid, the alcohol and concentrated hydrochloric acid can be prepared and
heated for
several hours. The y alkyl glutamate product can be precipitated out in
acetone, filtered,
dried and recrystallized from hot water.
Alkyl Glutamate/C-Terminus Conjugation
The peptide carrier can be dissolved in DMF under nitrogen and cooled to
0°C.
The solution can then be treated with diisopropylcarbodiimide and
hydroxybenzotriazole
followed by the y alkyl glutamate bioactive agent. The reaction can then be
stirred for
several hours at room temperature, the urea by-product filtered off, and the
product
precipitated out in ether and purified using GPC or dialysis.
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Preparation of Alkyl Glutamate-NCA
y Alkyl glutamate can be suspended in dry THF where triphosgene is added and
the mixture refluxed under a nitrogen atmosphere until the mixture becomes
homogenous. The solution can be poured into heptane to precipitate the NCA
product,
which is filtered, dried and recrystallized from a suitable solvent.
Preparation of Poly[Alkyl Glutamate]
y Alkyl glutamate-NCA can be dissolved in dry DMF where a catalytic amount of
a primary amine can be added to the solution until it becomes viscous
(typically
overnight). The product can be isolated from the solution by pouring it into
water and
filtering. The product can be purified using GPC or dialysis.
Although illustrated and described above with reference to specific
embodiments,
the invention is nevertheless not intended to be limited to the details shown.
Rather,
various modifications may be made in the details within the scope and range of
equivalents of the claims and without departing from the spirit of the
invention.
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CLAIMS
What is claimed is:
1. A pharmaceutical composition comprising:
a polypeptide; and
alprazalom covalently attached to said polypeptide.
2. The composition of claim 1 wherein said polypeptide is an oligopeptide.
3. The composition of claim 1 wherein said polypeptide is a homopolymer of a
naturally occurring amino acid.
4. The composition of claim 1 wherein said polypeptide is a heteropolymer of
two or more naturally occurring amino acids.
5. The composition of claim 1 wherein said polypepdde is a homopolymer of a
synthetic amino acid.
6. The composition of claim 1 wherein said polypeptide is a heteropolymer of
two or more synthetic amino acids.
7. The composition of claim 1 wherein said polypeptide is a heteropolymer of
one or more naturally occurring amino acids and one or more synthetic amino
acids.
8. The composition of claim 1 wherein alprazalom is covalently attached to a
side
chain, the N-terminus or the C-terminus of said polypeptide.
9. The composition of claim 1 further comprising a microencapsulating agent.
10. The composition of claim 9 wherein said microencapsulating agent is
selected from the group consisting of polyethylene glycol (PEG), an amino
acid, a sugar
and a salt.
11. The composition of claim 1 further comprising an adjuvant.
~7
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12. The composition of claim 11 wherein said adjuvant activates an intestinal
transporter.
13. The composition of claim 1 further comprising a pharmaceutically
acceptable
excipient.
14. The composition of claim 1 wherein said composition is in the form of an
ingestable tablet.
15. The composition of claim 1 wherein said composition is in the form of an
intravenous preparation.
16. The composition of claim 1 wherein said composition is in the form of an
1o oral suspension.
17. The composition of claim 1 wherein alprazalom is conformationally
protected
by folding of said polypeptide about said active agent.
18. The composition of claim 1 wherein said polypeptide is capable of
releasing
alprazalom from said composition in a pH-dependent manner.
19. A method for protecting alprazalom from degradation comprising ~covalently
attaching said active agent to a polypeptide.
20. A method for controlling release of alprazalom from a composition wherein
said composition comprises a polypeptide, said method comprising covalently
attaching
alprazalom to said polypeptide.
2o 21. A method for delivering alprazalom to a patient comprising
administering to
said patient a composition comprising:
a polypeptide; and
alprazalom covalently attached to said polypeptide.
22. The method of claim 21 wherein alprazalom is released from said
composition by an enzyme-catalyzed release.
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23. The method of claim 21 wherein alprazalom is released from said
composition by a pH-dependent unfolding of said polypeptide.
24. The method of claim 21 wherein said active agent is released from said
composition in a sustained release.
25. The method of claim 21 wherein said composition further comprises an
adjuvant covalently attached to said polypeptide and wherein release of said
adjuvant
from said composition is controlled by said polypeptide.
1o Abstract
A composition comprising a polypeptide and alprazalom covalently attached to
the polypeptide. Also provided is a method for delivery of alprazalom to a
patient
comprising administering to the patient a composition comprising a polypeptide
and
alprazalom covalently attached to the polypeptide. Also provided is a method
for
protecting alprazalom from degradation comprising covalently attaching it to a
polypeptide. Also provided is a method for controlling release of alprazalom
from a
composition comprising covalently attaching it to the polypeptide.
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A NOVEL PHARMACEUTICAL COMPOUND CONTAINING ALPROSTADIL
AND METHODS OF MAKING AND USING SAME
FIELD OF THE INVENTION
The present invention relates to a novel pharmaceutical compound that
comprises
a polypeptide that is preferably covalently attached to alprostadil, as well
as methods for
protecting and administering alprostadil. This novel compound, referred to as
a
CARRIERWAVETM Molecular Analogue (CMA), has the benefit of taking a known
effective pharmaceutical agent that is both well studied and occupies a known
segment of
to the pharmaceutical market, and combining it with a Garner compound that
enhances the
usefulness of the pharmaceutical agent without compromising its pharmaceutical
effectiveness.
BACKGROUND OF THE INVENTION
Alprostadil is a known pharmaceutical agent that is used in the treatment of
male
~5 erectile dysfunction. Its chemical name is (llalpha,13E,15S)-11,15-
dihydroxy-9-
oxoprost-13-en-1-oic acid. Its structure is:
0
- - ri
0
0
U
O
The novel pharmaceutical compound of the present invention is useful in
accomplishing one or more of the following goals: enhancement of the chemical
stability
20 of the original compound; alteration of the release profile of an orally
administered
product; enhanced digestion or absorption; targeted delivery to particular
tissue/cell type;
and provision for an oral dosage form when none exists. The novel
pharmaceutical
compound may contain one or more of the following: another active
pharmaceutical
agent, an adjuvant, or an inhibitor.
25 Active agent delivery systems are often critical for the effective delivery
of a
biologically active agent (active agent) to the appropriate target. The
importance of these
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systems becomes magnified when patient compliance and active agent stability
are taken
under consideration. For instance, one would expect patient compliance to
increase
markedly if an active agent is administered orally in lieu of an injection or
another
invasive technique. Increasing the stability of the active agent, such as
prolonging shelf
life or survival in the stomach, will assure dosage reproducibility and
perhaps even
reduce the number of dosages required which could improve patient compliance.
Absorption of an orally administered active agent is often blocked by the
harshly
acidic stomach milieu, powerful digestive enzymes in the GI tract,
permeability of
cellular membranes and transport across lipid bilayers. Incorporating
adjuvants such as
resorcinol, surfactants, polyethylene glycol (PEG) or bile acids enhance
permeability of
cellular membranes. Microencapsulating active agents using protenoid
microspheres,
liposomes or polysaccharides have been effective in abating enzyme degradation
of the
active agent. Enzyme inhibiting adjuvants have also been used to prevent
enzyme
degradation. Enteric coatings have been used as a protector of pharmaceuticals
in the
stomach.
Active agent delivery systems also provide the ability to control the release
of the
active agent. For example, formulating diazepam with a copolymer of glutamic
acid and
aspartic acid enables a sustained release of the active agent. As another
example,
copolymers of lactic acid and glutaric acid are used to provide timed release
of human
2o growth hormone. A wide range of pharmaceuticals purportedly provide
sustained release
through microencapsulation of the active agent in amides of dicarboxylic
acids, modified
amino acids or thermally condensed amino acids. Slow release rendering
additives can
also be intermixed with a large array of active agents in tablet formulations.
Each of these technologies imparts enhanced stability and time-release
properties
to active agent substances. Unfortunately, these technologies suffer from
several
shortcomings. Incorporation of the active agent is often dependent on
diffusion into the
microencapsulating matrix, which may not be quantitative and may complicate
dosage
reproducibility. In addition, encapsulated drugs rely on diffusion out of the
matrix, which
is highly dependant on the water solubility of the active agent. Conversely,
water-soluble
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microspheres swell by an infinite degree and, unfortunately, may release the
active agent
in bursts with little active agent available for sustained release.
Furthermore, in some
technologies, control of the degradation process required for active agent
release is
unreliable. For example, an enterically coated active agent depends on pH to
release the
active agent and, as such, is difficult to control the rate of release.
In the past, use has been made of amino acid side chains of polypeptides as
pendant groups to which active agents can be attached. These technologies
typically
require the use of spacer groups between the amino acid pendant group and the
active
agent. The peptide-drug conjugates of this class of drug delivery system rely
on enzymes
to in the bloodstream for the release of the drug and, as such, are not used
for oral
administration. Examples of timed and targeted release of injectable or
subcutaneous
pharmaceuticals include: linking of norethindrone, via a hydroxypropyl spacer,
to the
gamma carboxylate of polyglutamic acid; and linking of nitrogen mustard, via a
peptide
spacer, to the gamma carbamide of polyglutamine. Dexamethasone has been
covalently
15 attached directly to the beta carboxylate of polyaspartic acid without a
spacer group.
This prodrug formulation was designed as a colon-specific drug delivery system
where
the drug is released by bacterial hydrolytic enzymes residing in the large
intestines. The
released dexamethasone active agent, in turn, was targeted to treat large
bowel disorders
and was not intended to be absorbed into the bloodstream. Yet another
technology
2o combines the advantages of covalent drug attachment with liposome formation
where the
active ingredient is attached to highly ordered lipid films (known as HARs)
via a peptide
linker. Thus, there has been no drug delivery system, heretofore reported,
that
incorporates the concept of attaching an active ingredient to a polypeptide
pendant group
with its targeted delivery into the bloodstream via oral administration.
25 It is also important to control the molecular weight, molecular size and
particle
size of the active agent delivery system. Variable molecular weights have
unpredictable
diffusion rates and pharmacokinetics. High molecular weight carriers are
digested slowly
or late, as in the case of naproxen-linked dextran, which is digested almost
exclusively in
the colon by bacterial enzymes. High molecular weight microspheres usually
have high
3o moisture content which may present a problem with water labile active
ingredients.
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