Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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USE OF PROTEIN CONFORMATION FOR THE PROTECTION AND
RELEASE OF CHEMICAL COMPOUNDS
FIELD OF THE INVENTION
The present invention is directed to the preparation of synthetic amino acid
polymers and is directed to the use of these polymers to protect chemical
compounds, especially drug substances, from degradation, and to release the
compounds under specific conditions.
BACKGROUND OF THE INVENTION
Under physiologic conditions, proteins (polymer chains of peptide-linked
amino acids) normally do not exist as extended linear polymer chains. A
combination of molecular forces, including hydrogen bonding, hydrophilic and
hydrophobic interactions, promote thermodynamically more stable secondary
structures that can be highly organized (helices, beta pleated sheets, etc.).
These
structures can then combine to form higher order structures with critical
biological
functions. Natural proteins are peptide-linked polymers containing 20
different
amino acids, each with a different side-chain. The details of the folding into
higher
order structures are dependent on the type, frequency and primary sequence of
the
amino acids in the protein. Since each position in the polymer chain can be
occupied by 20 different amino acids, the thermodynamic rules that describe
the
details of protein folding are complex. For example, we are currently unable
to
design a synthetic protein with a substrate-specific enzymatic site that is
predicted
by the primary amino acid sequence. More complete discussions of the structure
and function of proteins are found in Dickerson et al. "The Structure and
Action of
Proteins" Harper and Row, New York, 1970 and Lehninger "Biochemistry" Worth,
New York, 1970, pp. 109-146.
However, some basic rules of protein folding have been discovered. In
general, the side chains of the 20 L-amino acids commonly found in natural
proteins
can be placed in two categories, hydrophobic/non-polar and hydrophilic/polar,
each
playing separate roles in protein conformation. In the standard "oil drop"
model for
protein folding, the amino acids with more hydrophobic side chains (Val, Leu,
Phe,
Met, Ilu) are sequestered to the inside of the protein structure, away from
the
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aqueous environment. Frequently, these hydrophobic side chains form "pockets"
that bind molecules of biological significance. On the other hand, hydrophilic
amino
acids (e.g. Lys, Arg, Asp, Glu) are most frequently distributed on the outer
surface
of natural proteins, providing overall protein solubility and establishing a
superstructure for the internalized hydrophobic domains.
A highly preferred conformation found in many natural proteins is the 3.613
alpha-helix. This right-handed helix contains 3.6 amino acids per turn and is
stabilized by hydrogen bonding (about 3 kcal/mol) involving the amide hydrogen
and a carbonyl oxygen, separated by 13 atoms along the backbone of the polymer
chain. Since the amino acid side chains in the alpha-helix point away from and
perpendicular to the helix axis, any of the amino acids (except Pro) can
participate
in the helix. Other structures can also appear in higher order protein
conformations,
including the 3Ip helix, and the important left-handed, three residue helix
found in
collagen and pleated sheets.
Other amino acids can also be used with predictable results in the preparation
of synthetic proteins. Tyrosine (Tyr) is frequently found internalized, with
its 4-
hydroxy hydrogen, hydrogen-bonded to another amino acid or potential
ligand/enzyme substrate. Thus, Tyr can be utilized to produce hydrophobic
pockets
with a potential for hydrogen bonding. Proline (Pro) has been found to be
sufficient,
but not always necessary, for a sharp turn in the peptide chain, allowing for
cooperative interactions of different sections of the same polymer. At higher
polymer concentrations, Pro can also disrupt helical structure, producing a
"less
organized" protein. Cystine can be utilized to stabilize higher order
structures by
linking polymer chains through high energy (about 50 kcal/mol) disulfide
bonds.
Some amino acids do not have distinct hydrophobic or hydrophilic character and
provide a "place-keeping" function or contribute more subtle effects on the
overall
protein structure.
Some work on synthetic polypeptides has proceeded with the goal of
producing textile products with desirable properties, but the technology has
been
largely too expensive to compete with natural products, and with other
synthetic
polymers. In the pharmaceutical industry, work on synthetic polypeptides has
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focused' again on specific amino acid sequences having intrinsic hormonal or
drug
activities. A more complete discussion of the use of synthetic polymers for
textiles
. ", ".
and pharmaceuticals is provided by Block in "Polymer" Monographs" Gordon and
Breach, Vol 9, 1983. An historical perspective is provided by Watson
"Molecular
Biology of the Cell" W.A. Benjamin, Inc., New York, 1970.
It was not realized, prior to the present invention, that block polymers,
comprised of a limited sub-set of amino acids, would exhibit intrinsic
conformational structures that could be predicted, based on an analysis of
statistical
distributions and ratios of the amino acids in the polymer. Moreover, it was
also
not realized that these synthetic proteins would have utility in protecting
and
releasing sensitive chemical compounds.
Thus, the present invention describes a use of a limited but sufficient sub-
set
of amino acids, to produce prototype synthetic proteins that reproduce certain
conformational aspects of natural proteins. The present invention describes
the use
and combination of only seven amino acids, each with a specific function in
the
resulting synthetic polypeptide: Glutamic Acid (Glu), Lysine (Lys),
Phenylalanine
(Phe), Proline (Pro), Tryptophan (Trp), Tyrosine (Tyr) and Cysteine (Cys). Cys
is
used herein as the disulfide Bis-dimer (CysS-SCys), referred to as cystine by
convention. Block polymers of this amino acid subset are used to produce
synthetic
proteins with predictable conformations and utility. The ability of these
synthetic
polypeptides to organize into higher order structures, and in some
embodiments, to
form hydrophobic domains, are used in the present invention to protect
sensitive
compounds from chemical (e.g. oxidative) and enzymatic degradation and provide
for the engineered release of these compounds under specific conditions.
SUMMARY OF THE INVENTION
The subject of this invention is the utilization of the ability of amino acid
polymers (polypeptides) to form higher order structures. These structures can
bind
to and protect chemical entities (e.g. drugs) from chemical and enzymatic
degradation and provide a mechanism for controlled release of such entities.
Synthetic polypeptides are described that are composed of carefully selected
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combinations and ratios of amino acids, including a hydrophilic/polar
component
(like Glu or Lys), a hydrophobic component (like Tyr, Phe or Benzyl Glu), and
are
designed to promote the formation of internalized domains, to accommodate
chemical entities like drugs.
In one embodiment, the method relates to the protection of a chemical
compound from degradation comprising combining the chemical compound with a
synthetic protein which may be a homo-polymer, containing for example Glu or
Lys, or may be a co-polymer with an amino acid having hydrophobic character,
contributes a hydrogen bonding capacity, or stabilizes higher order
structures.
In another embodiment, the invention relates to cell culture media
comprising a synthetic polypeptide containing Gln that is co-polymerized with
an
amino acid, like Glu. The polymer provides a chemically stable nutritional
source of
Gln in the culture. A related embodiment utilizes a Gln containing synthetic
protein
as a nutritional source of Gln in humans.
In another embodiment, the invention relates to a pharmaceutical
composition comprising an active ingredient that has been combined with a
synthetic
amino acid polymer. The synthetic protein may be a homo-polymer of Glu or Lys,
for example, or may be a co-polymer containing Glu or Lys and Tyr, Phe or
Benzyl
Glu. In specific related embodiments, the active ingredient of such
pharmaceutical
compositions is L-DOPA, aspirin, hydrocortisone, or estrogen. The
protein/active
ingredient combination may also be combined with other pharmaceutically
acceptable excipients to aid in tablet formation and properties, for example.
In yet another embodiment, the invention relates to a method of controlling
the release of a chemical compound based on response to changes in pH. This
embodiment is comprised of manipulating the higher order structure of a
synthetic
protein by choice of amino acid composition and combining said chemical
compound with the protein.
In another embodiment, the invention relates to the release of chemical
compounds by regulating the rate of proteolytic digestion through the
manipulation
of higher order structures of a synthetic protein by choice of amino acid
composition, and combining said chemical compounds with the protein.
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In another embodiment, the invention relates to the release of chemical
compounds by regulating thermal diffusion of said compounds from a synthetic
protein. Regulation of diffusion rate occurs by manipulating the higher order
structure of the synthetic protein, by choice of amino acid composition, and
combining said chemical with the protein.
Other objects, features and advantages of the present invention will become
apparent from the following detailed description. It should be understood,
however, that the detailed description and the specific examples, while
indicating
preferred embodiments of the invention, are given by way of illustration only,
since
various changes and modifications within the spirit and scope of the invention
will
become apparent to those skilled in the art from this detailed description.
Unless otherwise indicated, a parameter that is qualified by "about" may
vary ~ 10 % from the stated value. That is, "about 50 °C" means 45-55
°C.
Further, unless otherwise indicated, all amino acids are in the L-form.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
A number of general methods could be used in the present synthesis of
synthetic polypeptides, but the most suited is the Fuchs-Farthing approach. In
this
method, the parent amino acid is condensed with Phosgene under anhydrous
conditions to form the N-chlorocarbonyl intermediate. Depending on the
specific
amino acid used, the R group functionality may require protection to block
involvement in the reaction. The intermediate loses HCl as it cyclizes to form
the
N-carboxyanhydride (NCA). Currently, it is more convenient and safe to
substitute
triphosgene [Bis(trichloromethyl)carbonate] for neurotoxic Phosgene (gas)
since
triphosgene is a crystalline solid that is easily weighed and added to the NCA
reaction. Thus, triphosgene is a preferred reagent for amino acid NCA
formation.
Additional advantages to the Fuchs-Farthing chemistry are that the NCA's are
generally easy to purify in crystalline form, negligible racemization occurs
at the
alpha carbon and the polymerization reaction yields only the protein polymer
and
non-toxic carbon dioxide.
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The polymerization reaction normally contains an amino acid NCA (for
homopolymers) or mixture of NCA's, for the synthesis of co- or heteropolymers,
together with a polymerization initiator, all dissolved in a compatible
solvent
system. Highly preferred for the polymerization is a non-protic organic
solvent that
has high solubility for the NCA and the polymer. The preferred solvents
include
ethyl acetate, THF, benzene, dichloromethane, DMF and dioxane. Most preferred
solvents include THF dioxane and DMF. In the case of co-polymer synthesis,
solubility of the polymer in the polymerization solvent is important, since
early
precipitates of polymer, prior to complete use of all the NCA monomers, may
favor
the appearance of one amino acid over another, in the first precipitates.
The initiator of the polymerization reaction can be water, a base (organic or
inorganic) or a preformed amino acid polymer. In theory, the average number of
amino acid residues in the final polymer product is a direct result of the
molar ratio
of the monomer NCA's to the initiator. Since initiation may not behave
ideally,
through, for example, partitioning of the initiator into non-polymerizing
compartments in the reaction, care must also be taken to use an initiator that
is
highly soluble in the reaction solvent. Most preferably, a tertiary amine,
like
triethyamine or tert-butylamine is used, since primary and secondary amines
may
stay covalently attached to the polymer, forming stable end-labeled polymer
products .
The preferred average number of residues (N) in the polymer chains is
between 5 and 400. For polymers like PGIu and PLys, where helical structure
may
be desired, an average N between 10 and 400 is most preferred.
Of particular importance to the present invention are the claimed mechanisms
of protection and release of chemical compounds from synthetic protein
polymers.
A useful embodiment of the invention is the formulation of tablets or liquids
intended for oral delivery of an active drug substance. Standard formulations
of
drugs may have limited shelf life (e.g. from oxidation) or be inactivated by
the
acidic conditions of the stomach. In this case, a drug delivery mechanism that
circumvents the stomach would be desirable. Alternatively, rapid release of a
drug
substance in the stomach may be preferred.
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In order to address these desired applications, chemical compounds may be
formulated as defined herein. "Macroformulation" : blending of a powdered
chemical compound and a powdered synthetic polypeptide prior to formulation
and
tableting. "Microformulation": incorporation of a chemical compound into a
synthetic polypeptide, for example by inclusion into hydrophobic pockets,
prior to
formulation and tableting. "Covalent formulation" : incorporation of a
chemical
compound by peptide linkages into a synthetic polypeptide prior to formulation
and
tableting.
A focus of the present invention makes use of a dramatic effect of pH on
secondary and tertiary structures of synthetic protein polymers containing an
ionizable R group (e.g. Glu, Asp, Lys, Arg). At a pH around the pKA, the
ionizable portion makes a transition from uncharged to highly charged. As a
result
of all the closely spaced repulsive charges , higher order polymeric
structures, like
alpha helices, are rapidly converted to "random coils." Random coils are
highly
flexible and dynamic; this form promotes drug release and enhanced proteolytic
cleavage by digestive enzymes.
For example, a drug can be blended in powder form with polyglutamic acid
(PGIu) and tableted by direct compression. Stability of the drug in this
"macro-
formulated" tablet is achieved by internalization of the drug into the
compressed
matrix, an environment that is nearly anhydrous and low in oxygen. The
presence
of water and oxygen is known to be detrimental to drug stability. After
ingestion,
the external surface of the tablet is exposed to the low pH of the stomach
(about
pH=1). Since the pKA of the gamma carboxyl group of Glu is 4.25, the carboxyl
groups remain in the -COOH form, the tablet remains compact, digestion of the-
polymer is slow and release of the co-formulated drug is slow.
However, upon passing the pyloric valve, the pH of the intestinal contents
increases to about 6.5 and the carboxyl groups become de-protonated and highly
charged. The closely spaced, highly charged carboxyl groups repel each other
strongly enough to overcome intrachain bonds (e.g. hydrogen bonds) responsible
for
higher order structure of the PGIu. The drug then diffuses from the loose,
random
coils of the polymer. The enhanced digestability of the random coil structure
also
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aids drug release. Through this mechanism, the drug is released preferentially
in
the small intestine.
Similarly, a drug intended for oral delivery is "macro-formulated" with
polymeric lysine (PLys) by blending and tableting (e.g. by direct
compression). In
this case, the omega amino group of Lys has a pKA of about 10Ø Once entering
the stomach, the omega amino group becomes fully protonated and highly
charged.
The closely spaced amino groups repel each other, releasing the drug substance
by
diffusion and enhanced digestability of the random coil structure.
A further enhancement of drug stability and controlled release properties,
especially in the digestive system, can be realized by incorporation of a
hydrophobic
amino acid, like Phe, to form a synthetic co-polymer. In one embodiment, a
synthetic co-polymer containing Glu and Phe, in a preferred ratio is, "macro-
formulated" with a hydrophobic drug substance. Stability of the drug in the
compressed tablet is again enhanced by sequestration from water and oxygen.
Release of the drug in stomach is slow. However, once in the small intestine,
the
PGIu/Phe becomes less organized due to pH/charge effects and there is an
initial
release of drug substance accompanied by re-partitioning of the drug into
hydrophobic domains in the polymer. Finally, terminal digestive proteolysis
releases the entire store of drug.
Similarly, PLys/Phe can be used for release of a drug substance in the
stomach, except that drug release and digestion of the polymer are enhanced in
the
stomach, and the drug release profile is blunted by successive re-partitioning
of the
drug into the hydrophobic domain of the polymer. Finally, digestive
proteolysis
destroys even the hydrophobic pockets, releasing all the drug.
A further enhancement of drug stability is accomplished by microformulation
involving inclusion of the drug, into the internal matrix of the synthetic
protein prior
to tableting and oral administration. For example, a hydrophobic drug
substance is
combined in solution with a co-polymer of glutamic acid and phenylalanine
(PGIu/Phe) at a pH that favors the random coil form of the polymer (pH > 4).
The
solution is slowly acidified to promote the formation of higher order
structures in
the polymer, with attendant formation of internalized hydrophobic domains
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containing the "dissolved" hydrophobic drug substance. The PGIu/Phe - Drug
Substance combination precipitates at lower pH; precipitation can be enhanced
by
the addition of an organic solvent like acetone. The vacuum or freeze dried
product
is especially stable since the drug substance is partitioned into anhydrous,
hydrophobic domains inside the protein structure.
A similar process of drug inclusion applies to PLys/Phe except that higher
order structures, like alpha helices, occur above pH 10 for this polymer. In
this
case, the polymer/drug combination is adjusted to pH > 11 in solution and
freeze
dried. Again, the product is especially stable due to partitioning of the drug
into
hydrophobic domains inside the protein. The polymer/drug complexes can be
formulated with other excipients that may facilitate tableting.
Certain drug substances, like DOPA and glutamine (Gln), are also amino
acids and are therefore amenable to co-polymerization into the primary
polypeptide
chain, affording drug protection as described above and an additional control
of
drug release, requiring proteolytic digestion. An embodiment demonstrating the
advantage of this covalent formulation is the protection of Gln from
degradation.
Gln is an essential amino acid for most mammalian cells and is therefore an
important nutritional component, for example in cell culture. However,
monomeric
Gln is chemically unstable, degrading to ammonia and pyrrolidonecarboxylic
acid,
under physiologic conditions. Gln is chemically stabilized as a co-polymer
with Glu
by incorporation into the structure of the polymer. As an oral nutritional
supplement in humans, Gln release is regulated by normal proteolytic digestion
of
the polymer. Gln is released for metabolic use by cultured cells by slow
extracellular hydrolysis of the synthetic protein in the culture media or by
pinocytotic mechanisms in which the synthetic protein is internalized by the
cultured
cells and digested by lysozomes to become a metabolic source of Gln.
Other hydrophobic amino acids find special use when co-polymerized with a
hydrophilic component like Glu or Lys. For example, Tyrosine (Tyr) is
moderately
hydrophobic and can also hydrogen bond with potential drug substances via its
4-
OH group. Tryptophan (Trp) is less hydrophobic than Phe and provides
internalized hydrophobic domains that permit relatively enhanced diffusion of
drug
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substances under physiologic conditions. Trp-NCA synthesis is also facilitated
since
the secondary nitrogen does not need protected.
Proline (Pro) is used in the present invention to provide obligatory turns in
structures like helices, providing for enhanced intrachain interactions and
promoting
the formation of more globular synthetic proteins, when preferred. At higher
levels, Pro destabilizes higher order protein structures since it cannot
participate in
helical structures and can be used in a co-polymer to enhance diffusion of a
drug
substance from hydrophobic domains internalized in a synthetic protein. D-
amino
acids also inhibit formation of helical structures of L-amino acids but are
less
desirable due to their possible unwanted metabolic effects as an unnatural
amino
acid.
Cystine is used in the present invention to stabilize higher order structures
via intra- and inter-chain disulfide linkages. This is accomplished in the
present
invention using the bifunctional Bis-disulfide NCA, since the disulfide
linkage
serves as its own thiol protecting group.
An additional object of the present invention is the synthesis of a synthetic
protein for use in the preparation of a synthetic serum. In this capacity, a
globular
protein that is metabolically stable, non-immunogenic and non-allergenic is
highly
desired. As an artificial serum component, a heteropolymer containing Glu,
Pro,
Tyr, and Cys has been prepared with the desired properties.
The synthetic polypeptides in the current application are also referred to as
synthetic proteins or synthetic amino acid polymers. Polypeptides of the
invention
have two or more amino acids linked by a peptide bond. In a preferred
embodiment, polypeptides have five or more peptide linked amino acids.
The present invention, thus generally described, will be understood more
readily by reference to the following examples, which are provided by way of
illustration and are not intended to be limiting of the present invention.
Example 1: Preparation of Microcrystalline L-Glutamic Acid
L-Glutamic acid (200 gm) is dissolved in 2.5 L hot water (T > 95 C). The
hot solution is added slowly to 2.5 L of rapidly stirred, cold (T < 10 C)
acetone to
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form a thick slurry. After cooling, the precipitated solid is separated by
filtration,
washed with 200 ml of acetone and the filter cake compressed to remove excess
solvent. The white filter cake is dried in vacuo at 80 C for 4 hours and is
suitable
for use in Example 2. Yield: 189 gm (94 %). "Microcrystalline" means that the
crystalline nature is not obvious by macroscopic inspection; i. e. , the
resulting L-
Glutamic acid is amorphous.
Example 2: Synthesis of Glutamic N Carboxyarzhydride (Glu-NCA)
Microcrystalline, dry L-Glutamic Acid (73.6 gm, 0.5 mol) from Example 1
is suspended in 2.0 L of anhydrous THF containing triphosgene (98 gm, 1 Eq.)
and
heated with stirring to 50 °C for 4 hours or until the reaction is
homogeneous. The
reaction is then heated to a gentle reflux for about 1 hour, using a condenser
protected with a drying tube. "About 1 hour" means 1 hour ~ 20 min. The
solution is then decanted or filtered from any remaining solids and evaporated
under
oil vacuum using a water bath less than 40 °C, until a precipitate
forms or until a
thick oil remains, with no additional solvent evaporation. The product is
dissolved
in 360 ml of dry ethyl acetate, any insoluble material ( < 5 gm) is filtered
off, and
the crude product is precipitated by the rapid addition of 360 ml of hexanes
with
active stirring. After 20 min., an additional 200 ml of hexanes is added to
fully
precipitate the crude Glu-NCA. The precipitate is collected by filtration
under a dry
carbon dioxide curtain, wash the filter cake with hexanes ( 100 ml) and
compact
under pressure to remove excess solvent. In order to purify the crude Glu-NCA,
the hexane-damp filter cake is dissolved in a combination of 350 ml anhydrous
THF
and 350 ml of anhydrous ethyl acetate. Any insoluble material is filtered off
and is
precipitated with rapid addition of 700 ml of hexanes with stirring. Once
precipitation has commenced, an additional 350 ml hexanes is added to complete
the
precipitation. After 30 min, the precipitate is isolated by filtration under a
dry
carbon dioxide curtain. The product is compacted with pressure to remove
excess
solvent, and it is then washed with hexanes (100 ml) and dried in vacuo (T <
30 °C).
The product is stable when stored under dry carbon dioxide, in the cold
(temperature is less than about 10 °C, where "about 10 °C" means
~ 5 °C).
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Example 3: Synthesis of Polymeric Glutamic Acid (PGIu)
The dry Glu-NCA product (17.3 gm, 100 mmol) of Example 2 is dissolved
in anhydrous THF (86 ml) and polymerization is initiated by the addition of 86
ml
of anhydrous ethyl acetate containing 0.1 gm triethylamine. The reaction is
warmed
to reflux for 15 min then allowed to cool and react at 25 °C for 24
hours with
continuous stirring. The precipitate is isolated by filtration, washed with
anhydrous
diethyl ether and dried in vacuo at 60 °C for 2 hours, to yield a white
powder.
Yield: 10.2 gm (79 % ).
Example 4: Synthesis of Polymeric Lysine (PLys)
Polymeric L-Lysine is prepared as previously described (Seta et. al. ,
Biopolymers 1,517, 1963). The dry polymer is converted to the alpha helical
form,
and contaminant bromine is removed by dissolving in water as a 10 % solution
and
titrating the pH to 12 by the addition on 1.0 N NaOH. The spontaneous
precipitate
is further precipitated by the addition of acetone and collected by filtration
and
vacuum dried to obtain a white powder. Yield: 78 % from the starting polymeric
Lysine. The helical form is confirmed by measuring the optical rotation
[alpha)D of
the pH 12 solution (minimal negative value of -40 degrees optical rotation,
compared to -130 degrees optical rotation for the random coil).
Example 5: Synthesis of Glutamic AcidlPhenylalaniue Co-Polymer (PGIylPhe)
The conditions of Example 3 are repeated except that Glu-NCA (12.46 gm,
72 mmol) and Phe-NCA (5.35 gm, 28 mmol) are co-dissolved in the THF. Crude
Phe-NCA is prepared by the method of Poche (Tet. Letters 29:5859-5862, 1988).
However, the crude Phe-NCA is further purified prior to polymerization by
dissolving 50 gm in a mixture of 100 ml of THF and 100 ml of ethyl acetate
followed by precipitation with 600 ml of hexanes. The fluffy, white product is
isolated by filtration, washed with hexanes and dried in vacuo to yield 36 gm
(72%).
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Example 6: Synthesis of LysinelPhenylalanine Co-Polymer (PLyslPhe)
Example 4 is repeated except that Phe-NCA is included in the polymerization
to for the co-polymer in a final Lys/Phe ratio of 3.6. The polymer is
converted to
the helical form as described in Example 4.
Example 7: Synthesis of Glutamic AcidlGlutamine Co-Polymer (PGIulGln)
The polymer of Example 3 is converted to the Glu/Gln co-polymer.
Polymeric glutamic acid (20 gm) is suspended in 100 ml of water, with
stirring, and
heated to 50 °C. Concentrated aqueous ammonia (30 % ) is added while
monitoring
the pH of the solution. At pH=5.0, the mixture is stirred for an additional 10
min.
while making small adjustments to pH 5.0 and the solution is freeze dried to
obtain
20.4 gm of a white powder. The partial ammonium salt is then heated to 80
°C in a
vacuum oven for conversion to the Glu/Gln co-polymer. Yield: 18.6 gm.
Elemental analysis for N shows this preparation to contain 8 % Gln. Titrations
to
higher pH with ammonia yield higher % Gln in the final co-polymer.
Example 8: Synthesis of Glutamic AcidlProlinelCystinelTyrosine Heteropolymer
(PGIulProlCyslTyr)
Example 3 is repeated except that 4 separate amino acid NCA's: Glu-NCA
10.71 gm, 62 mmol), Pro-NCA (1.42 gm, 10 mmol), Tyr-NCA (3.54 gm, 17
mmol) and Cys-NCA (1.14 gm, 4 mmol) are dissolved in 86 ml THF prior to
polymerization. Glu-NCA is used a synthesized in Example 2. Pro-NCA, Tyr-
NCA and Cys-NCA are synthesized as summarized by Blacklock, Hirschmann and
Veber (The Peptides 9:39-95, 1987). Cys-NCA is prepared and used as the Bis-
Cysteine- NCA (NCA-Cys-S-S-Cys-NCA). Pro-NCA is prepared just prior to use
but the remaining NCA's are stable when stored as described in Example 2.
Yield:
12.2 gm (76 %).
Example 9: Release of Tryptophan (Trp) from PGIu + Trp Blended Tablets
A uniform blend of PGIu ( 19.0 gm) and Trp ( 1.0 gm) is prepared in a ball
mill and 100 mg tablets are prepared as described in Example 13. Tablets are
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subjected to a standard dissolution test under two pH conditions (pH 1.0 and
6.5) in
order to mimic conditions in the stomach and small intestine. In the test, a
tablet is
placed in a jacketed (37 °C) glass beaker containing 100 ml of the test
solution, and
stirred at 100 RPM. Samples (0.1 ml) of the solution are taken at 30, 60 and
120
min, diluted 1 / 10 in water, and the OD measured at a wavelength of 280
nanometers
and compared to the control samples containing 5 mg of Trp dissolved in 100 ml
of
solution. Release of Trp is expressed as % of Control ( 100 % ) .
pH=1.0 pH=6.5
Trp Released ( % Control) Trp Released ( % Control)
Tablet No. 30 min 60 min 120 min 30 min 60 min 120 min
1 0.8 1.1 0.8 98.2 98.1 99.0
2 0.8 0.8 0.7 97.1 98.0 98.2
3 0.7 1.0 0.9 97.5 99.1 98.9
Example 10: Release of Tryptophan (Trp) from PLys + Tip Blended Tablets
Example 9 is repeated except that tablets were formulated using the helical
form of PLys from Example 4.
pH=1.0 pH=6.5
Trp Released as % of Control Trp Released as % of Control
Tablet No. 30 min 60 min 120 min 30 min 60 min 120 min
1 102.5 101.4 98.4 99.1 98.9 98.9
2 99.1 100.1 100.1 99.0 98.6 98.8
3 99.3 99.2 99.1 98.9 99.6 99.3
Example 1l: Hydrophobic Inclusion of Tryptophan in PGIulPhe
Tryptophan is included in hydrophobic sites in PGIu/Phe from Example 5 by
dissolving 10 gm of the polymer in a solution composed of 50 % ethanol and
0.05 M
sodium phosphate buffer at pH 7.2.
The solution is bubbled with nitrogen to remove dissolved oxygen.
Tryptophan ( 1.0 gm) is then added and the pH slowly adjusted to 3 .0 by
titration
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with 1.0 N HCI. The precipitate formed is collected by centrifugation and
washed
by re-suspension in water and the product is freeze dried. Solution of the
product in
0.05 M sodium phosphate and measuring the OD at 280 nanometers is used to
measure the Trp included in the polymer.
Example 12: Protection of L-DOPA by Hydrophobic Inclusion in PGIulPhe
(PGIulPhe + DOPA)
Example 11 is repeated except that L-DOPA is substituted for tryptophan.
Decomposition of DOPA is measured by formation of colored quinone oxidation
products, when compared to DOPA alone. DOPA loss is measured by reverse
phase HPLC as previously described. Gerlach et al., J. Chromat. 380:379-385,
1986.
Example 13: Tableting for Oral Dosage Forms
Oral dosage forms of drugs combined with synthetic polypeptides can be
prepared by direct compression of the polypeptide drug combination.
Alternatively,
the synthetic polypeptide/drug combination can be combined with other
excipients to
enhance tablet properties, as described for 5 mg hydrocortisone in a 200 mg
tablet.
Component mg/Tablet %
1. Lys/Phe-Hydrocortisone 35.0 17.50
2. Microcrystalline Cellulose 25.5 12.75
3. Lactose 135.25 67.63
4. Croscarmellose 3.40 1.70
5. Mgz+ Stearate 0.85 0.42
Total: 200.00 mg 100.00 %
Procedure: A pre-mix of the Plys/Phe-Hydrocortisone combination and
cellulose is blended to uniformity and then blended with the remaining
ingredients,
except stearate, until uniform. Finally, the stearate is added and blended 5
min.
Tablets are formed by direct compression to a hardness of 16 kg.
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Example 14: Blending and Tableting of PGIu + Aspirin
A uniform blend of equal masses of PGIu and aspirin is prepared in a shell
blender and 200 mg tablets containing 100 mg aspirin each are compressed as
described in Example 13 to a hardness of 7 kg.
Example 15: Blending and Tableting of PlyslPhe + Cortisol
Example 11 is repeated except that hydrocortisone ( 17-
hydroxycorticosterone) is substituted for Trp and tableted with other
excipients as
described in Example 13. Hydrocortisone in the tablets is determined by
quantitative
reverse phase HPLC of solutions containing dissolved tablets as previously
described. Waters Corporation, Symmetry Applications Notebook II, August,
1994, p. 19.
Example 16: Stability of Gln in PglulGln Co-Polymer
Stability of Gln in the PGIu/Gln co-polymer of Example 7 is measured by
the production of free ammonia in 0.2 M phosphate buffer, as described
previously
(Gilbert et al., J. Biol. Chem. 180:209, 1949). The Glu/Gln co-polymer yields
no
detectable ammonia in this assay, while the control sample, containing free
Gln is
almost completely de-amidated.
Example 17: Use of PGIulGln in Cell Culture
The co-polymer of Example 7 is dissolved as an 0.8 %o solution (0.8 gm/ 100
ml) in a standard media, devoid of monomeric glutamine (alpha-MEM, without
glutamine) and combined with insulin (5 ug/ul) and transferrin (holo, 5
~.g/~,l). The
solution is adjusted to pH 7.2 with 1.0 N NaOH and filtered through a 0.2
micron
sterilization membrane. This combined media is found to support growth of
cultured
human amniocytes, through multiple passages, with no addition of monomeric L-
glutamine.
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Example 18: Treatment of Gln Deficiency in Humans with Oral Dosage of
PGIulGln
The Glu/Gln co-polymer of Example 7 is formed into tablets by direct
compression of 125 mg of the co-polymer, as described in Example 13. This oral
dose will suffice as an oral preparation to deliver about 10 mg L-glutamine,
in order
to treat a deficiency of this essential amino acid in humans.
Example 19: PGIulProlCyslTyr as a Synthetic Serum Component
The heteropolymer product of example 8 is dissolved in phosphate buffered
saline (PBS) and adjusted to pH 7.2 with 1.0 N NaOH, prior to sterile
filtration.
This sterile solution is intended as a synthetic serum replacement, to
supplement
serum volume in humans and other mammals.
Example 20: Treatment for Inflammation
The product of Example 14 is tableted by direct compression, as in Example
13, to contain about 50 mg of aspirin per tablet. This preparation is intended
as an
oral treatment for inflammation in mammals, especially humans.
Example 21: Treatment for Primary Adrenal Insufficiency
The product of Example 15 is combined with other excipients to formulate
an oral dosage for the treatment of adrenal insufficiency or inflammation in
mammals, especially humans.
Example 22: Treatment for Parkinson's Disease
The product of Example 12 is tableted by direct compression, as in example
13, to contain 50 mg of L-DOPA per tablet, as oral dosage form for the
treatment of
Parkinsons disease in humans.
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