Note: Descriptions are shown in the official language in which they were submitted.
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PHARMACEUTICAL FORMULATIONS FOR THE DELIVERY
OF DRUGS HAVING LOW AQUEOUS SOLUBILITY
TECHNICAL FIELD
The present invention relates generally to pharmaceutical formulations, and
more.
particularly relates to pharmaceutical formulations for the delivery of water-
insoluble or
sparingly water-soluble drugs. The invention additionally relates to methods
for using the
novel formulations to administer such drugs in the treatment of disease. The
invention has
1o utility in the fields of pharmaceutical formulation, drug delivery, and
medicine.
BACKGROUND ART
The formulation and administration of water-insoluble or sparingly water-
soluble
drugs is problematic because of the difficulty of achieving sufficient
systemic bioavailability.
15 Low aqueous solubility results not only in decreased bioavailability, but
also in formulations
that are insufficiently stable over extended storage periods. A classic
example in this regard
is paclitaxel, available commercially as Taxol° from Bristol-Myers
Squibb. Although
paclitaxel has been shown to exhibit powerful antineoplastic efficacy,
particularly for cancers
of the breast, ovaries and prostate gland, its use is limited in large part by
the side effects of
2o the solvent generally used for clinical administration, a mixture of
Cremophor EL°
(polyethoxylated castor oil) and ethanol. The amount of solvent that is
required to deliver an
effective dose of paclitaxel is substantial, and Cremophor has been shown to
result in serious
or fatal hypersensitivity episodes in laboratory animals (see, e.g., Lorenz et
al. (1977) Agents
Actions 7:63-67) as well as in humans (Weiss et al. (1990) J. Clin. Oncol.
8:1263-1268).
25 Because of the undesirable physiologic reactions associated with paclitaxel-
Cremophor
formulations, patients are generally premeditated with corticosteroids and/or
antihistamines.
While premeditation has proven to be somewhat effective, mild to moderate
hypersensitivity
is still a problem in a significant number of patients. Weiss et al., supra;
see also Runowicz et
al. (1993) Cancer 71:1591-1596.
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Thus, extensive research has been conducted with the aim of producing an
improved paclitaxel formulation having reduced toxicity. In particular,
efforts have been
directed toward (1) modifying the chemistry of the drug itself to make it more
hydrophilic
and (2) combining the drug with agents that produce water-soluble dispersions.
Chemically
modified paclitaxel analogs include sulfonated paclitaxel derivatives (see
U.S. Patent No.
5,059,699), amino acid esters (Mathew et al. (1992) J. Med. Chen2. 3B:145-151)
as well as
covalent conjugates of paclitaxel and polyethylene glycol (U.S. Patent No.
5,648,506 to Desai
et al.; Liu et al. (1999) J. Polymer Sci.,Part A - Polymer Chem. 37:3492-
3503). For the most
part, however, research has focused on entrapment of the drug in vesicles or
liposomes, and
l0 on the incorporation of surfactants into paclitaxel formulations.
Representative liposomal drug delivery systems are described in U.S. Patent
Nos.
5,395,619, 5,340,588 and 5,154,930. Liposomes, as is well known in the art,
are vesicles
comprised of concentrically ordered lipid bilayers that encapsulate an aqueous
phase.
Liposomes form when phospholipids, amphipathic compounds having a polar
(hydrophilic)
head group covalently bound to a long-chain aliphatic (hydrophobic) tail, are
exposed to
water. That is, in an aqueous medium, phospholipids aggregate to form a
structure in which
the long-chain aliphatic tails are sequestered within the interior of a shell
formed by the polar
head groups. Unfortunately, use of liposomes for delivering many drugs has
proven
unsatisfactory, in part because liposome compositions are, as a general rule,
rapidly cleared
2o from the bloodstream. Finally, even if satisfactory liposomal formulations
could be
prepared, it may still be necessary to use some sort of physical release
mechanism so that the
vesicle releases the active agent in the body before it is taken up by the
liver and spleen.
Encasement of paclitaxel microcrystals in shells of biocompatible polymeric
materials is described in U.S. Patent No. 6,096,331 to Desai et al. However,
as crystals of
hydrophobic drugs may be difficult to dissolve, the rate of drug release in
these formulations
is hard to control.
Incorporation of surfactants into paclitaxel formulations as described, for
example,
in International Patent Publication No. WO 97/30695, is also problematic.
Surfactants tend
to alter the chemistry of a pharmaceutical formulation such that the effective
ratio of drug to
3o inactive ingredients is lowered, resulting in the need to increase dosage
volume and/or
administration time. Additionally, formulations that employ surfactants
readily dissociate
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upon dilution, e.g., following intravenous injection, resulting in premature
drug release.
Also, many surfactants are considered unsuitable for parenteral drug
administration because
of their interaction with cellular membranes.
Accordingly, there is a need in the art for a pharmaceutical formulation that
is
suitable for administration of a water-insoluble or sparingly water-soluble
drug such as
paclitaxel or the like, wherein (1) the formulation is optimized such that the
amount of drug
administered is maximized while undesirable side effects are minimized, (2)
the rate of drug
release can be precisely controlled, (3) no surfactants are necessary, (4) no
liposomes or other
vesicles are required, (5) premeditation is unnecessary, and (6) the
formulation displays
to excellent stability over extended storage periods.
DISCLOSURE OF THE INVENTION
It is accordingly a primary object of the invention to address the above-
mentioned
needs in the art by providing a pharmaceutical formulation effective to
deliver a drug having
15 low aqueous solubility.
It is another object of the invention to provide a therapeutic method wherein
the
aforementioned formulation is administered to a patient to treat a condition,
disease or
disorder for which the drug is indicated.
It is an additional object of the invention to provide such a method wherein
the
20 drug is an anticancer agent and the patient is suffering from cancer.
It is a further object of the invention to provide a method for administering
a drug
so as to enhance the systemic bioavailability thereof.
Additional objects, advantages and novel features of the invention will be set
forth
in part in the description which follows, and in part will become apparent to
those skilled in
25 the art upon examination of the following, or may be learned by practice of
the invention.
In one aspect of the invention, then, a pharmaceutical formulation is provided
that
comprises: (a) a matrix of a spatially stabilized hydrophilic polymer that is
optionally
covalently bound (or "conjugated") to a phospholipid moiety; (b) a drug that
is physically
entrapped within the matrix but not covalently bound thereto, wherein the drug
has greater
30 solubility in polyethylene glycol 400 than in water; (c) an optional
stabilizing agent, (d) an
optional targeting ligand, and (e) an optional excipient. A variety of
hydrophilic polymers
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may be employed, although polyethylene glycol and polyethylene oxide-co-
propylene oxide)
are preferred. Preferred drugs are those for which systemic bioavailability
can be enhanced
by increasing the solubility of the drug in an aqueous vehicle; generally,
although not
necessarily, such drugs are hydrophobic, i.e., water insoluble or sparingly
water-soluble.
Also preferred are drugs for which a sustained release drug delivery system is
desirable, i.e.,
drugs that are administered to patients on an ongoing, scheduled basis. The
stabilizing agents
are generally polymers containing hydrophobic and hydrophilic areas, proteins
are preferred.
Suitable excipients include free phospholipids, which may or may not be the
same as the
phospholipid moieties conjugated to the hydrophilic polymer. The formulation
may be in
to lyophilized form, which is advantageous for storage stability.
In another aspect of the invention, a pharmaceutical formulation is provided
that
comprises an aqueous suspension of (a) drug-containing particles having an
average size in
the range of approximately 1 nm to 500 ~,m, comprised of (i) a matrix of a
spatially
stabilized hydrophilic polymer that is optionally covalently bound to a
phospholipid moiety,
(ii) a drug that is physically entrapped within the matrix but not covalently
bound thereto,
wherein the drug has greater solubility in polyethylene glycol 400 than in
water, optionally
(iii) a stabilizing agent, optionally (iv) a targeting ligand, and optionally
(v) an excipient, in
(b) an aqueous vehicle. The aqueous vehicle may be, for example, water,
isotonic saline
solution or phosphate buffer, and may be instilled with an acoustically active
gas to facilitate
ultrasound imaging and ultrasonic cavitation for local drug release with
ultrasound.
In still another aspect of the invention, a method is provided for delivering
a drug
to a mammalian individual to achieve a desired therapeutic effect, wherein the
method
involves administering to the individual a therapeutically effective amount of
a formulation of
the invention, e.g., orally or parenterally.
z5 In a related aspect of the invention, a method is provided fox treating an
individual
suffering from cancer, comprising parenterally administering to the patient a
surfactant-free
formulation of: (a) drug-containing particles comprised of (i) a matrix of a
spatially stabilized
hydrophilic polymer that is optionally covalently bound to a phospholipid
moiety, (ii) an
anticancer agent that is entrapped by but not covalently bound to the
hydrophilic polymer,
30 wherein the anticancer agent is selected from the group consisting
paclitaxel, docetaxel,
camptothecin, and derivatives and analogs thereof, (iii) a stabilizing agent,
optionally (iv) a
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stabilizing agent; optionally (v) a targeting ligand; and optionally (vi) an
excipient selected
from the group consisting of a free phospholipid, a saccharide, a liquid
polyethylene glycol,
propylene glycol, glycerol, ethyl alcohol, other polyhydroxyalcohols, and
combinations
thereof; in (b) an aqueous vehicle suitable for parenteral drug
administration.
In another related aspect of the invention, an alternative method is provided
for
treating an individual suffering from cancer, comprising orally administering
to the individual
a pharmaceutical formulation of (a) drug-containing particles comprised of (i)
a matrix of a
spatially stabilized hydrophilic polymer that is optionally covalently bound
to a phospholipid
moiety, (ii) an anticancer agent that is entrapped by but not covalently bound
to the
l0 hydrophilic polymer, wherein the anticancer agent is selected from the
group consisting of
paclitaxel, docetaxel, camptothecin, and derivatives and analogs thereof,
(iii) an effective
amount of a P-glycoprotein inhibitor, optionally (iv) a stabilizing agent,
optionally (v) a
targeting ligand, and optionally (vi) an excipient selected from the group
consisting of a free
phospholipid, a saccharide, a liquid polyethylene glycol, propylene glycol,
glycerol, ethyl
15 alcohol, other polyhydroxyalcohols, and combinations thereof; in (b) an
aqueous vehicle
suitable for oral drug administration.
In yet another aspect of the invention, an improved method is provided for
administering a drug so as to enhance the bioavailability thereof, wherein the
improvement
comprises administering the drug in a pharmaceutical formulation comprised of
(a) a matrix
of a spatially stabilized hydrophilic polymer that is optionally covalently
bound to a
phospholipid moiety, (b) a drug that is physically entrapped within the matrix
but not
covalently bound thereto, wherein the drug is water insoluble or sparingly
water soluble,
optionally (c) a stabilizing agent, optionally a targeting ligand, and
optionally (e) an excipient
selected from the group consisting of a free phospholipid, a saccharide, a
liquid polyethylene
z5 glycol, propylene glycol, glycerol, ethyl alcohol, other
polyhydroxyalcohols, and
combinations thereof, wherein the formulation is free of surfactants.
The present invention is based on the formation of a noncovalent complex of
drug
molecules with a hydrophilic polymer and an optional biocompatible stabilizing
agent. This
drug/polymer complex allows for the formation of an aqueous suspension of
nanoparticles of
30 the complex without requiring chemical modification of the drug. This
technology can be
applied to many drugs having poor solubility in water, e.g., paclitaxel.
Problems related to
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stability, toxicity of the carrier, and large injection volume of currently
available formulations
of paclitaxel are well documented. Nanoparticle solubilization technology
enables the
preparation of paclitaxel formulations with decreased toxicity and improved
efficacy.
We have discovered that a unique class of nanoparticles ranging from about 1
nm
to about 500-1000 p.m, preferably from about 1 nm to about 500 ~,m, that can
be stabilized
with a stabilizing agent to form nanoparticles having diameters ranging from
about lnm to
about 300 nm, preferably from about 20 nm to about 100 nm. The resulting
nanoparticles are
biocompatible and highly useful for drug delivery. The drug delivery is
preferably via IV
injection but the technology has applications for oral, subcutaneous, e.g.,
sustained release,
to and pulmonary delivery. For IV delivery, the nanoparticles decrease
toxicity of the
therapeutic agents such as paclitaxel. Larger doses of the active agents can
therefore be
administered via IV, allowing for higher blood levels of the therapeutic agent
yielding greater
efficacy. For oral applications, the nanoparticles improve dispersal of
insoluble drugs and
increase uptake from the gastrointestinal tract. For sustained release
applications, the
nanoparticles can be formulated into gels, powders or suspensions. For
pulmonary
applications, the nanoparticles' small effective hydrodynamic radii improves
delivery of
therapeutic agents into the distal airways, such as the alveoli, thereby
allowing systemic
delivery of bioactive agents via the pulmonary route.
The size of the particles within the formulation helps to control dispersal of
the
z0 drug and drug release. Surprisingly, stabilized and unstabilized
drug/polymer complexes
have improved solubility and drug release properties compared to crystalline
forms of the
drug. The rate of release of drug/polymer complexes can be fine-tuned by
optionally
including a stabilizing agent and by varying the nature of the drug complex.
For example,
branched polyethylene glycol (PEG) is a soluble polymer that is capable of
forming
z5 complexes with certain hydrophobic drugs. Once in the body, the PEG will
eventually
dissolve, releasing the complexed drug. The rate of drug release can be
modified by varying
the conditions and parameters of complex formation, e.g., ratios of PEG to
drug, chemical
structure of the PEG, and the amount and type of stabilizing agent.
Hydrolyzable bonds may
also be incorporated into the hydrophilic polymer and/or the stabilizing agent
to accelerate
3o drug release, and pH-responsive groups may be used to increase drug release
at a desired pH.
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BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 schematically illustrates a formulation of the invention in which a
drug
molecule (paclitaxel) is "entrapped" within a spatially stabilized matrix of
phospholipid-
conjugated polyethylene glycol (e.g., dipalmitoyl phosphatidylethanolamine
[DPPE]
conjugated to polyethylene glycol).
FIG. 2 schematically illustrates an alternative formulation of the invention
in which
a drug is entrapped within a spatially stabilized matrix of a highly
branchedpolyethylene
glycol molecule.
FIG. 3 schematically illustrates another alternative formulation of the
invention in
l0 which a drug is entrapped within a spatially stabilized matrix formed by
star polyethylene
glycol.
FIG. 4 schematically illustrates still another alternative formulation of the
invention
in which a drug is entrapped within a spatially stabilized matrix of lower
molecular weight,
branched polyethylene glycol.
FIG. 5 presents sizing data of paclitaxel nanoparticles stabilized with human
serum
albumin.
FIG. 6 presents the body weights of nude mice treated with nanoparticulate
formulations of paclitaxel in an MTD (maximally tolerated dose study).
FIG. 7 relates tumor growth in nude mice following single doses at 2/3ras MTD
of
Taxol and the formulation of the invention.
FIG. 8 relates tumor growth in nude mice following single doses at full MTD of
Taxol and the formulation of the invention.
FIG. 9 shows a branched PEG molecule with 4 arms stabilizing 4 molecules of a
therapeutic agent. The number of therapeutic molecules stabilized per molecule
of branched
PEG will vary depending upon the molecular weight of each arm the PEG, the
molecular
weight of the drug, the formulation and the intended application. In the
figure, the numeral
"1" refers to a drug molecule, while the numeral "2" refers to the polymer.
FIG. 10 shows a modified branched PEG with 4 arms stabilizing 4 molecules of a
therapeutic agent. In this case the core of the branched molecule has been
substituted with
another polymer more hydrophobic than PEG such as polypropylene glycol. The
hydrophobic core favors partitioning of hydrophobic drugs into the core and
stabilization of
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drug within the interior of the branched molecule. Depending upon the ratio of
drug to
branched molecule, the PEG comprised outer arms may be free to move as would a
hydrated
PEG molecule. In this case, a single branched molecule may form a stable
association with a
plurality of drug molecules, and do not aggregate to form a particle. In the
figure, the
numeral "1" refers to a drug molecule, the numeral "2" refers to hydrophilic
polymer in outer
part of arms and the numeral "3" refers to the more hydrophobic core polymer.
FIG. 11 shows a modified branched PEG molecule similar to Figure 10, except in
this case the termini of the outermost PEG groups have been modified to
covalently bind
targeting ligands. As shown in this Figure, the branched PEG molecule may bind
more than
l0 one type of targeting ligand. The bound targeting ligands facilitate drug
delivery to specific
cells bearing receptors for the particular targeting ligands. In the figure,
the numeral "1"
refers to a drug molecule, the numeral "2" refers to hydrophilic polymer in
outer part of arms,
the numeral "3" refers to the more hydrophobic core polymer and the numeral
"4" refers to
the targeting ligands (two different types are shown here).
MODES FOR CARRYING OUT THE INVENTION
I. DEFINITIONS AND OVERVIEW:
It is to be understood that unless otherwise indicated, this invention is not
limited
to specific active agents, hydrophilic polymers, phospholipids, excipients,
methods of
manufacture or the like, as such may vary. It is also to be understood that
the terminology
used herein is for the purpose of describing particular embodiments only and
is not intended
to be limiting.
It must be noted that, as used in the specification and the appended claims,
the
singular forms "a," "an" and "the" include plural referents unless the context
clearly dictates
otherwise. Thus, for example, reference to "an active agent" or "a drug" in a
formulation
means that more than one active agent can be present, reference to "a
hydrophilic polymer"
includes combinations of hydrophilic polymers, reference to "a phospholipid"
includes
mixtures of phospholipids, and the like.
In this specification and in the claims that follow, reference will be made to
a
number of terms that shall be defined to have the following meanings:
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By "pharmaceutically acceptable" is meant a material that is not biologically
or
otherwise undesirable, i.e., the material may be administered to an individual
along with the
selected active agent without causing any undesirable biological effects or
interacting in a
deleterious manner with any of the other components of the pharmaceutical
composition in
which it is contained.
"Pharmaceutically or therapeutically effective dose or amount" refers to a
dosage
level sufficient to induce a desired biological result. That result can be
alleviation of the
signs, symptoms, or causes of a disease, or any other desired alteration of a
biological system.
The term "treat" as in "to treat a disease" is intended to include any means
of
treating a disease in a mammal, including (1) preventing the disease, i.e.,
avoiding any
clinical symptoms of the disease, (2) inhibiting the disease, that is,
arresting the development
or progression of clinical symptoms, and/or (3) relieving the disease, i.e.,
causing regression
of clinical symptoms.
The terms "disease," "disorder" and "condition" are used interchangeably
herein to
refer to a physiological state that may be treated using the formulations of
the invention.
The terms "drug," "active agent" and "therapeutic agent" are used
interchangeably
herein to refer to a chemical material or compound which, when administered to
an organism
(human or animal), induces a desired pharmacologic effect. Included are
analogs and
derivatives (including salts, esters, prodrugs, and the like) of those
compounds or classes of
compounds specifically mentioned which also induce the desired pharmacologic
effect.
The number given as the "molecular weight" of a compound, as in the molecular
weight of a hydrophilic polymer such as polyethylene glycol, refers to weight
average
molecular weight MW.
The "solubility" of a compound refers to its solubility in the indicated
liquid
determined under standard conditions, e.g., at room temperature (typically
about 25 °C),
atmospheric pressure, and neutral pH.
The term "hydrophobic" is used to refer to a compound having an octanol:water
partition coefficient (at room temperature, generally about 23 °C) of
at least about 8:1,
preferably at least about I0:1, more preferably 20:I or higher. "Hydrophobic"
drugs are
sometimes referred to herein as "water insoluble" or "sparingly water
soluble," or as having
"low aqueous solubility." The term "hydrophilic" refers to a material that is
not hydrophobic.
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In referring to chemical compounds herein,, the following definitions apply:
The term "alkyl" refers to a branched or unbranched saturated hydrocarbon
group
of 1 to 24, typically I to 18, carbon atoms, such as methyl, ethyl, n-propyl,
isopropyl, n-butyl,
isobutyl, t-butyl, octyl, decyl, tetradecyl, hexadecyl, eicosyl, tetracosyl
and the like, as well as'
cycloalkyl groups such as cyclopentyl, cyclohexyl and the like.
The term "aryl" refers to an aromatic species containing 1 to 3 aromatic
rings,
either fused or linked, and either unsubstituted or substituted with one or
more substituents.
Preferred aryl substituents contain one aromatic ring or two fused aromatic
rings.
The term "acyl" refers to a group having the structure R(CO)- wherein R is
alkyl or
l0 aryl as defined above.
"Optional" or "optionally" means that the subsequently described circumstance
may or may not occur, so that the description includes instances where the
circumstance
occurs and instances where it does not.
15 II. FORMULATIONS:
The pharmaceutical formulations of the invention are advantageously used to
deliver any drug whose systemic bioavailability (including oral
bioavailability) can be
enhanced by increasing the solubility of the drug in water. Thus, the drugs
that are preferred
for use in conjunction with the present invention are generally hydrophobic in
nature, tending
2o toward low aqueous solubility. The invention incorporates such drugs in a
composition
comprised of a matrix of a spatially stabilized hydrophilic polymer that
physically entraps
and thereby immobilizes the drug, but does not covalently bind thereto. The
composition
may additionally comprise a stabilizing agent that further stabilizes the
hydrophilic polymer/
drug complex and is useful in forming nanoparticulate-stabilized complexes.
A. THE HYDROPHILIC POLYMER
The hydrophilic polymer of the present formulations is spatially stabilized so
as to
facilitate physical entrapment and thus immobilization of the active agent;
that is, the
"spatially stabilized" hydrophilic polymer forms a matrix or three-dimensional
structure in
3o which discrete regions of drug are dispersed. By "spatially stabilized" is
meant that the
relative orientation of the drug in the polymer matrix is fixed in three-
dimensional space
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without directional specification. Generally, although not necessarily, the
"spatially
stabilized" matrix is sterically constrained. Any polymer that can form such a
matrix can be
used in conjunction with the invention, providing that the polymer is
sufficiently hydrophilic
to increase the aqueous solubility of the entrapped drug. It is preferred that
the polymer is not
cross-linked.
Examples of suitable hydrophilic polymers include, but are not limited to,
polyethylene glycol, polypropylene glycol, polyvinyl alcohol, polyvinyl
pyrrolidone,
polylactide, poly(lactide-co-glycolide), polysorbate, polyethylene oxide,
polypropylene
oxide, polyethylene oxide-co-propylene oxide), poly(oxyethylated) glycerol,
l0 poly(oxyethylated) sorbitol, poly(oxyethylated) glucose), and derivatives,
mixtures and
copolymers thereof. Examples of suitable derivatives include those in which
one or more C-
H bonds, e.g., in alkylene linking groups, are replaced with C-F bonds, such
that the polymers
are fluorinated or even perfluorinated.
The preferred hydrophilic polymer for use in the present formulations is
polyethylene glycol (PEG) or a copolymer thereof, e.g., polyethylene glycol
containing some
fraction of other monomer units (e.g., other alkylene oxide segments such as
propylene
oxide), with polyethylene glycol itself most preferred. In order to form the
spatially
stabilized matrix, the polyethylene glycol used is either branched PEG
(including
"dendrimeric" PEG, i.e., higher molecular weight, highly branched PEG) or star
PEG,
optionally conjugated to a phospholipid moiety as will be discussed below.
Covalent
conjugates of linear PEG and phospholipids may also be used, since such
conjugates can give
rise to a spatially stabilized matrix, as the hydrophobic chains of the
phospholipids will tend
to associate in an aqueous medium. See FIG. l, which schematically illustrates
a formulation
in which a drug is entrapped within a spatially stabilized matrix of
phospholipid-conjugated
linear PEG. Combinations of different types of PEG (e.g., branched PEG and
linear PEG,
star PEG and linear PEG, branched PEG and phospholipid-conjugated linear PEG,
etc.) may
also be employed.
Branched PEG molecules will generally although not necessarily have a
molecular
weight in the range of approximately 1000 to 600,000 Daltons, more typically
in the range of
approximately 2000 to 10,000 Daltons, preferably in the range of approximately
20,000 to
40,000 Daltons. Branched PEG is commercially available, such as from Nippon
Oil and Fat
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(NOF Corporation, Tokyo, Japan) and from Shearwater Polymers (Huntsville,
Alabama), or
may be readily synthesized by polymerizing lower molecular weight linear PEG
molecules
(i.e., PEG 2000 or smaller) functionalized at one or both termini with a
reactive group. For
example, branched PEG can be synthesized by solution polymerization of lower
molecular
weight PEG acrylates (i.e., PEG molecules in which a terminal hydroxyl group
is replaced by
an acrylate functionality -O-(CO)-CH=CHZ) or methacrylates (similarly, PEG
molecules in
which a hydroxyl group is replaced by a methacrylate functionality -O-(CO)-
C(CH3)=CH2) in
the presence of a free radical polymerization initiator such as 2,2'-
azobisisobutyronitrile
(AIBN). If desired, mixtures of PEG monoacrylates or monomethacrylates having
different
l0 molecular weights can be used in order to synthesize a branched polymer
having "branches"
or "arms" of differing lengths. Branched PEGS have 2 or more arms but may have
as many as
1000 arms. The branched PEGS herein preferably have about 4 to 40 arms, more
preferably
about 4 to 10 arms, and most preferably about 4 to 8 arms. Higher molecular
weight, highly
branched PEG, e.g., branched PEG having a molecular weight of greater than
about 10,000
and at least about 1 arm (i.e., one branch point) per 5000 Daltons, will
sometimes be referred
to herein as "dendrimeric" PEG. Such PEG is preferably formed by reaction of a
hydroxyl-
substituted amine such as triethanolamine with lower molecular weight PEG that
may be
linear, branched or star, to form a molecular lattice that serves as the
spatially stabilized
matrix and entraps the active agent to be delivered. Dendrimeric structures
including
2o dendrimeric PEG are described, inter alia, by Liu et al. (1999) PSTT
2(10):393-401.
Formulations of the invention prepared with highly branched, high molecular
weight
dendrimeric PEG and with lower molecular weight branched PEG are schematically
illustrated in FIGS. 2 and 4, respectively.
Star molecules of PEG are available commercially (e.g., from Shearwater
Polymers, Huntsville, AL) or may be readily synthesized using living free
radical
polymerization techniques as described, for example, by Gnanou et al. (1988)
Makromol.
Chem. 189:2885-2892 and Desai et al., U.S. Patent No. 5,648,506. Star PEG
generally has a
central core of divinyl benzene or glycerol. Preferred molecular weights for
star molecules of
PEG useful herein are typically in the range of about 1000 to 500,000 Daltons,
although
molecular weights in the range of about 10,000 to 200,000 are preferred. A
formulation of
the invention that employs star PEG is schematically illustrated in FIG. 3.
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As explained above, conjugates of hydrophilic polymers and phospholipids,
particularly PEG-phospholipid conjugates (also termed "PEGylated"
phospholipids), are also'
useful in the present formulations. The polyethylene glycol in the PEGylated
phospholipids
may be branched, star or linear. Conjugates of linear PEG and phospholipids,
if used, will
generally although not necessarily,employ PEG have a molecular weight in the
range of
approximately 1000 to 50,000 Daltons, preferably in the range of approximately
1000 to
40,000 Daltons. It will be appreciated by those skilled in the art that the
aforementioned
molecular weight ranges correspond to a polymer containing approximately 20 to
1000
ethylene oxide units, preferably about 20 to 2000 ethylene oxide units. The
phospholipid
to moiety that is conjugated to the PEG may be anionic, neutral or cationic,
of naturally
occurring or synthetic origin, and normally comprises a diacyl
phosphatidylcholine, a diacyl
phosphatidylethanolamine, a diacyl phosphatidylserine, a diacyl
phosphatidylinositol, a
diacyl phosphatidylglycerol, or a diacyl phosphatidic acid, wherein each acyl
moiety can be
saturated or unsaturated and will generally be in the range of about 10 to 22
carbon atoms in
15 length. Preferred compounds are polymer-conjugated diacyl phosphatidyl-
ethanolamines
having the structure of formula (I)
H2-O-R~
20 (I) CH-O-R2
O
CH2-O-p-O-CHzCHaNH-L-R3
I
OH
wherein R' and RZ are the acyl groups, R3 represents the hydrophilic polymer,
e.g., a
polyalkylene oxide moiety such as polyethylene oxide) (i.e., polyethylene
glycol),
polypropylene oxide), polyethylene oxide-co-propylene oxide) or the like (for
linear PEG,
R3 is -O-(CHzCH20)n H), and L is an organic linking moiety such as
a.caibamate, an ester, or
3o a diketone having the structure of formula (II)
0 0
(II) II (CH2)n-II
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wherein n is 1, 2, 3 or 4. Preferred unsaturated acyl moieties are esters
formed from oleic and
linoleic acids, and preferred saturated acyl moieties are palmitate, myristate
and stearate.
Particularly preferred phospholipids for conjugation to linear, branched or
star PEG herein are
dipalmitoyl phosphatidylethanolamine (DPPE) and 1-palmitoyl-2-oleyl
phosphatidylethanolamine (POPE).
The conjugates may be synthesized using art-known methods such as described,
for
example, in U.S. Patent No. 4,534,899 to Sears. That is, synthesis of a PEG-
phospholipid
conjugate or a conjugate of a phospholipid and an alternative hydrophilic
polymer may be
carried out by activating the polymer to prepare an activated derivative
thereof, having a
functional group suitable for reaction with an alcohol, a phosphate group, a
carboxylic acid,
an amino group or the like. For example, a polyalkylene oxide such as PEG may
be activated
by the addition of a cyclic polyacid, particularly an anhydride such as
succinic or glutaric
hydride (ultimately resulting in the linker of formula (II) wherein n is 2 or
3, respectively).
The activated polymer may then be covalently coupled to the selected
phosphatidylalkanolamine, such as phosphatidylethanolamine, to give the
desired conjugate.
The hydrophilic polymer may be modif ed in one or more ways. For drugs that
are
ionized at physiological pH, charged groups may be inserted into
the~ydrophilic polymer in
2o order to modify the sustained release profile of the formulation. To reduce
the rate of drug
release and thereby provide sustained delivery over a longer time period,
negatively charged
groups such as phosphates and carboxylates are used for cationic drugs, while
positively
charged groups such as quaternary ammonium groups are used in combination with
anionic
drugs. A terminal hydroxyl group of a hydrophilic polymer such as PEG may be
converted to
Z5 a carboxylic acid or phosphate moiety by using a mild oxidizing agent such
as chromic (VI)
acid, nitric acid or potassium permanganate; a preferred oxidizing agent is
molecular oxygen
used in conjunction with a platinum catalyst. Introduction of phosphate groups
may be
carried out using a phosphorylating reagent such as phosphorous oxychloride
(POC13) (see
Example 11). Terminal quaternary ammonium salts may be synthesized, for
example, by
3o reaction with a moiety such as
+I
R-N-(CH2)~-C-X
R
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wherein R is H or lower alkyl (e.g., methyl or ethyl), n is typically 1 to 4,
and X is an
activating group such as Br, Cl, I or an -NHS ester. If desired, such charged
polymers may be
used to form higher molecular weight aggregates by reaction with a polyvalent
counter ion.
Other possible modifications to the hydrophilic polymer include, but are not
limited to, the following. A terminal hydroxyl group of a PEG molecule may be
replaced by
a thiol group using conventional means, e.g., reacting hydroxyl-containing PEG
with a sulfur-
containing amino acid such as cysteine, using a protected and activated amino
acid. Such
"PEG-SH" is also commercially available, for example from Shearwater Polymers.
l0 Alternatively, a mono(lower alkoxy)-substituted PEG such as monomethoxy
polyethylene
glycol (MPEG) may be used instead of polyethylene glycol pe~~ se, so that the
polymer
terminates with a lower alkoxy substituent (such as a methoxy group) rather
than with a
hydroxyl group. Similarly, PEG amine may be used in lieu of PEG so that a
terminal amine
moiety -NHZ is present instead of a terminal hydroxyl group.
In addition, as discussed above, the polymer may contain two or more types of
monomers, as in a copolymer wherein propylene oxide groups (-CHZCHzCHzO-) have
been
substituted for some fraction of ethylene oxide groups (-CHZCHZO-) in
polyethylene glycol.
Incorporating propylene oxide groups will tend to increase the stability of
the spatially
stabilized matrix that entraps the drug, thus decreasing the rate at which the
drug is released
in the body. The more hydrophobic the drug and the larger the fraction of
propylene oxide
blocks, the slower the drug release rate will be.
The hydrophilic polymer may also contain hydrolyzable linkages to enable
hydrolytic degradation within the body and thus facilitate drug release from
the polymeric
matrix. Suitable hydrolyzable linkages include any intramolecular bonds that
can be cleaved
by hydrolysis, typically in the presence of acid or base. Examples of
hydrolyzable linkages
include, but are not limited to, those disclosed in International Patent
Publication No. WO
99/22770 to Harris, such as carboxylate esters, phosphate esters, acetals,
imines, ortho esters
and amides. Other suitable hydrolyzable linkages include, for example, enol
ethers, diketene
acetals, ketals, anhydrides and cyclic diketenes. Formation of such
hydrolyzable linkages
within the hydrophilic polymer is conducted using routine chemistry known to
those skilled
in the art of organic synthesis and/or described in the pertinent texts and
literature. For
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example, carboxylate linkages may be synthesized by reaction of a carboxylic
acid with an
alcohol, phosphate ester linkages may be synthesized by reaction of a
phosphate group with
an alcohol, acetal linkages may be synthesized by reaction of an aldehyde and
an alcohol, and
the like. Thus, polyethylene glycol containing hydrolyzable linkages "X" might
have the
structure -PEG-X-PEG- or alternatively might be a matrix having the structure
PEG
X
PEG X----f Core---X-PEG
X
PEG
wherein the core is hydrophobic molecule such as pentaerythritol, may be
synthesized by
l0 reaction of various -PEG-Y molecules with -Core-Z or PEG-Z molecules
wherein Z and Y
represent groups located at the terminus of individual PEG molecules and are
capable of
reacting with each other to form the hydrolyzable linkage X.
Accordingly, it will be appreciated that the rate of drug release from the
polymeric
matrix can be controlled by adjusting the degree of branching of the
hydrophilic polymer, by
15 incorporating different types of monomer units in the polymer structure, by
functionalizing
the hydrophilic polymer with different terminal species (which may or may not
be charged),
and/or by varying the density of hydrolyzable linkages present within the
polymeric structure
As illustrated above, the branched PEG molecule may be modified to have a
hydrophobic core. For example, if the central core is pentaerythritol, the
innermost arms
2o bound to the pentaerythritol may comprise a polymer more hydrophobic than
PEG. Useful
polymers to accomplish this include polypropylene glycol and polybutylene
glycol. Useful
monomers for constructing the inner, hydrophobic core structures of the arms
include
propylene oxide, butylene oxide, copolymers of the two, lactic acid and
copolymers of lactic
acid with glycolide (polylactide-co-glycolide and copolymers of the foregoing
with
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polyethylene glycol). The preferred materials for constructing an inner
hydrophobic core
include polypropylene glycol and copolymers of propylene oxide with ethylene
oxide.
Useful polymers for constructing the outer, peripheral parts of the arms
include polyethylene
glycol, polysialic acid and other hydrophilic polymers, with PEG most
preferred. It is
possible that a fraction of the monomers in the outer portion of a given arm
of the carrier
molecule may be replaced with PEG, but in this case, there will be
substantially more of the
hydrophilic monomer (e.g. ethylene oxide) than the hydrophobic monomer (e.g.
propylene
oxide).
The relative proportion of hydrophobic polymer within the branched polymer may
l0 vary from about 10 wt.% to about 90 wt.% on a weight/weight ratio,
preferably from about 40
wt.% to about 60 wt.%. When more hydrophobic polymer is used this may increase
the drug
loading capacity of the branched molecule for hydrophobic drugs. A most
preferred ratio is
about 50 wt.% weight of hydrophobic polymer, e.g. polypropylene glycol, and 50
wt.%
weight ratio of hydrophilic polymer (e.g. PEG) in the outer arms.
The branched molecules in the hydrophobic core and peripheral hydrophilic arms
are thought to have a number of advantages for drug delivery. The hydrophobic
core may
better stabilize hydrophobic drugs within the branched molecule and, as the
drug is stabilized
within the core, the free arms of the PEG may be better able to maintain a
random state in
which the PEG molecules move freely within solution. The outer, hydrophilic
PEG layer
may act as a steric barrier, inhibiting or decreasing the aggregation of
individual branched
molecules into particles. Additionally, in instances when targeting ligands
are attached to the
termini of the peripheral hydrophilic arms, targeting is facilitated by the
unencumbered and
exposed nature of the outer PEG arms. As will be discussed further on, a wide
variety of
targeting ligands can be covalently bound to the free ends of the PEG. The
hydrophobic and
hydrophilic components of the arms may be linked together by a variety of
different. Such
linkers include ethers, amides, esters, carbamates, thioesters, disulfide
bonds. In general, the
linker employed is used to attain the desired drug delivery properties of the
pharmaceutical
formulation. Metabolizable bonds can be selected to improve excretion of the
carrier
molecule as well as to improve drug release.
3o As previously mentioned, the free ends of the hydrophilic portions of the
branches
can be substituted with one or more targeting ligands per carrier molecule.
More than one
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kind of targeting ligand may be bound to each carrier molecule to facilitate
binding to a target
cell bearing more than one kind of receptor. A wide variety of ligands may be
used in this
regard. Exemplary targeting ligands include, for example, proteins, peptides,
polypeptides,
antibodies, antibody fragments, glycoproteins, carbohydrates, hormones,
hormone analogs,
lectins, amino acids, sugars, saccharides, vitamins, steroids, steroid
analogs, enzyme
cofactors, bioactive agents, and genetic material.
Generally speaking, peptides that are particularly useful as targeting Iigands
include natural, modified natural, or synthetic peptides that incorporate
additional modes of
resistance to degradation by vasculaxly circulating esterases, amidases, or
peptidases. While
1o many targeting ligands may be derived from natural sources, some may be
synthesized by
molecular biological recombinant techniques and other ligands may be synthetic
in origin.
Peptides may be prepared by a variety of different combinatorial chemistry
techniques as are
now known in the art. One very useful method of stabilizing a peptide moiety
incorporates
the use of cyclization techniques. For example, end-to-end cyclization,
whereby the carboxy
terminus is covalently linked to the amine terminus via an amide bond, may be
useful to
inhibit peptide degradation and increase circulating half life. Side chain-to-
side chain
cyclization may also be particularly useful in inducing stability. In
addition, an end-to-side
chain cyclization may be a useful modification as well. The substitution of an
L-amino acid
for a D-amino acid in a strategic region of the peptide may also provide
resistance to
biological degradation. Suitable targeting ligands, and methods for their
preparation will be
readily apparent to one skilled in the art. Although the lengths of the
peptides utilized as
targeting ligands may vary, peptides having from about 5 to about 15 amino
acid residues are
generally preferred.
Antibodies may be used as whole antibodies or as antibody fragments, e.g., Fab
or
Fab'2, either of natural or recombinant origin. The antibodies of natural
origin may be of
animal or human origin, or may be chimeric (e.g., mouse/human). Human
recombinant or
chimeric antibodies are preferred and fragments are preferred to whole
antibodies.
Immunoglobulins typically comprise a flexible "hinge" region. See, e.g.,
"Concise
Encyclopedia of Biochemistry," Second Edition, Walter de Gruyter & Co., pp.
282-283
(1988). Antibodies may be linked to the termini of the outer hydrophilic arms
using the thiols
of this "hinge" region. This is a preferred region for coupling antibodies, as
the potential
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binding site may be remote from the antigen-recognition site. Generally
speaking, it may be
difficult to utilize the thiols of the hinge group unless they are adequately
prepared. As
described in Shahinian and Salvias (1995) Biochimica et Biophysica Acta
1239:157-167, it
may be desirable to reduce the thiol groups so that they are available for
coupling, e.g., to
s maleimide derivatized linking groups. Examples of reducing agents that may
be used include
ethanedithiol, mercaptoethanol, mercaptoethylamine or the more commonly used
dithiothreitol, commonly referred to as Cleland's reagent. However, care
should be exercised
when utilizing certain reducing agents, such as dithiothreitol, as
overreduction may
compromise the activity or binding capacity of the targeting ligand. See,
e.g., Shahinian and
l0 Salvias, supra.
Antibody fragments, such as F(ab')2, may be pxepared by incubating the
antibodies
with pepsin (60 ~g/ml) in 0.1 M sodium acetate (pH 4.2) for 4 h at
37°C. Digestion may be
terminated by the addition of 2 M Tris (pH 8.8) to a final concentration of 80
mM. The
fragments may then be obtained by centrifugation. The supernatant may be
dialyzed at 4°C
15 against 150 mM NaCI, 20 mM phosphate at pH 7Ø Undigested IgG may be
removed by
chromatographic methods. The antibody fragments may then be extensively
degassing the
solutions and purging with nitrogen prior to use. The F(ab')2 fragments may be
provided at a
concentration of 5 mg/ml and reduced under argon in 30 mM cysteine.
Alternatively,
cysteamine may be employed; 100 mM Tris, pH 7.6 may be used as a buffer for 15
min at
ZO 37°C. The solutions may then be diluted 2-fold with an equal volume
of the appropriate
experimental buffer and spun through a 0.4 ml spin column of Bio-Gel P-6DG.
The resulting
antibody fragments may be more efficient in their coupling to the outer arms.
The same procedure may also be employed with other macromolecules containing
cysteine residues for coupling to the termini of the PEG arms. Also, peptides
may be utilized,
zs especially if they contain a cysteine residue. If the peptides have not
been made fresh and
there is a possibility of oxidation of cysteine residues within the peptide
structure, it may be
necessary to regenerate the thiol group using the approach outlined above.
In one embodiment of the invention, the attached targeting ligands may be
directed
toward lymphocytes that may be T--cells or B-cells, with T-cells being the
preferred target.
3o To select a class of targeted lymphocytes, a targeting ligand having
specific affinity for that
class may be preferably employed. For example, an anti CD-4 antibody may be
used for
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selecting the class of T-cells harboring CD-4 receptors, an anti CD-8 antibody
may be used
for selecting the class of T-cells harboring CD-8 receptors, an anti CD-34
antibody may be
used for selecting the class of T-cells harboring CD-34 receptors, and so on.
A lower
molecular weight ligand may preferably be employed, e.g., Fab or a peptide
fragment. For
example, an OI~T3 antibody or OI~T3 antibody fragment may be used.
Another major area for targeted delivery preferably involves the interleukin-2
(IL-
2) system. IL 2 is a T-cell growth factor generally produced following antigen-
or mitogen-
induced stimulation of lymphoid cells. Cell types that typically produce IL-2
include, for
example, CD4+ and CD8+ T-cells and large granular lymphocytes, as well as
certain T--cell
tumors. Generally speaking, IL-2 receptors are glycoproteins that are
expressed on
responsive cells. They are notable in connection with the present invention
because they are
generally readily endocytosed into lysosomal inclusions when bound to IL-2.
In addition to IL-2 receptors, preferred targets include the anti-IL-2
receptor
antibody, natural IL-2 and an IL-2 fragment of a 20-mer peptide or smaller
generated by
phage display that binds to the IL-2 receptor. In use, for example, IL-2 may
be conjugated to
stabilizing materials, for example, in the form of vesicles, and thus may
mediate the targeting
of cells bearing IL-2 receptors. Endocytosis of the ligand-receptor complex
may then deliver
the compound to be delivered to the targeted cell. Additionally, an IL-2
peptide fragment
which has binding affinity for IL-2 receptors may be incorporated, for
example, by
2o attachment to the termini of a different outer arm either directly to a
reactive moiety or via a
spacer or linker molecule with a reactive end such as an amine, hydroxyl, or
carboxylic acid
functional group. Such linkers are well known in the art and may comprise from
3 to 20
amino acid residues. In addition, D-amino acids or derivatized amino acids may
be used
which avoids proteolysis in the target tissue.
Still other systems which may be used in the present invention include IgM-
mediated endocytosis in B-cells or a variant of the ligand-receptor
interactions described
above wherein the T--cell receptor is CD2 and the ligand is lymphocyte
function-associated
antigen 3 (LFA-3), as described, for example, in Wallner et al. (1987) J.
Experimental Med.
166:923-932. Targeting ligands derived or modified from human leukocyte
origin, such as
3o CD 11 a/CD 18 and leukocyte cell surface glycoprotein (LFA-1 ), may also be
used as these
may bind to the endothelial cell receptor ICAM-1. The cytokine inducible
member of the
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immunoglobulin superfamily, VCAM-l, which is mononuclear leukocyte-selective,
may also
be used as a targeting ligand. VLA-4, derived from human monocytes, may be
used to target
VCAM-1.
Preferred targeting ligands in accordance with the present invention include,
for
example, Sialyl Lewis X, mucin, hyaluronic acid, LFA-1, N-formal peptide; CSa,
leukotriene
B4, platelet activating factor, IL-8/NAP-l, CTAP-III, RANTES, and I-309. In
addition, the
integrins may be used as targeting ligands for targeting VLA-4, fibrinogen,
von Willebrand
factor, fibronectin, vitronectin, VCAM-1 and CD49dlCD29. A particularly
preferred
targeting ligand may be Sialyl Lewis X which binds to P-selectin and which has
the following
to sequence: aNeuSAc(2-~3)(3Ga1(I~4)[aFuc(1-~3)]-(3GlcNAc-OR wherein R is an
aglycone
having at least one carbon atom.. P-selectin may be a preferred target because
it typically
localizes on the luminal side of endothelium during inflammation, but
generally not in non-
inflammatory synovia where it is generally cytoplasmic only.
Other preferred targeting ligands include, for example, antibodies directed to
autoantigens on T-cell receptors. Peptides having the amino acid sequences Leu
Leu Ile Tyr
Phe Asn Asn Asn Val Pro Ile Asp Asp Ser Gly Met (SEQ ID NO:1) and Lys Ile Gln
Pro Ser
Glu Pro Arg Asp Ser Ala Val Tyr Phe Cys Ala (SEQ ID N0:2) can be used to
produce
antibodies that may bind to the autoantigen portions of T-cell receptors. In
addition,
antibodies to additional T-cell and B-cell receptors may be used as targeting
ligands. T- and
B-cell receptors involved in inflammation and rheumatoid arthritis are
described in Struyk et
al. (1995), "T Cell Receptors in Rheumatoid Arthritis," Arthritis & Rheumatism
38:577-89;
Marchalonis et al. (1994), "Naturally Occurring Human Autoantibodies to
Defined T-Cell
Receptor and Light Chain Peptides," Immunobiol. Proteins Peptides, pp. 135-45;
Dedeoglu et
al. (1993), "Lack of Preferential Usage in Synovial T Cells of Rheumatoid
Arthritis Patients,"
z5 Inamuhol. Res. 12:12-20; " Marchalonis, et al. (1993), "Human
Autoantibodies to a Synthetic
Putative T Cell Receptor Beta-Chain Regulatory Idiotype: Expression in
Autoimmunity and
Aging," Exp. Clin. Immunogehet. 10:1-15; Dehghanpisheh et al. (1996), "Peptide
Epitope
Binding Specificity and VK and VH Gene Usage in a Monoclonal IgM Natural
Autoantibody
to T Cell Receptor CDR1 from a Viable Motheaten Mouse," Immunological Invest.
25:241-
52; Schluter et al. (1995), "Autoregulation of TCR V Region Epitopes in
Autoimmune
Disease," Immunobiol. Proteins Peptides VIII, pp.231-36; Lake, et al. (1995),
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WO 01/49268 PCT/US00/35322
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"Characterization of Autoantibodies Directed Against T Cell Receptors,"
Immunobiol.
P~oteihs Peptides VIII, pp.223-29; Lake et al. (1994), "Construction and
Serological
Characterization of a Recombinant Human Single Chain T Cell Receptor,"
Biochem. Biophys.
Res. Comm. 201:1502-1509; Lanchbury et al. (1995), "T Cell Receptor Usage in
Rheumatoid
Arthritis," British Med. Bull. 51:346-S8; Marchalonis et al. (1994);
"Synthetic Autoantigens
of Immunoglobulins and T-Cell Receptors: Their Recognition in Aging,
Infection, and
Autoimmunity," Autoantibodies Immunoglobulins T Cell Receptors, pp. 129-47;
Marchalonis
et al. (1993), "Natural Human Antibodies to Synthetic Peptide Autoantigens:
Correlations
with Age and Autoimmune Disease," Gerontology, 39:65-79; Marchalonis et al.
(1992),
to "Human Autoantibodies Reactive with Synthetic Autoantigens from T-Cell
Receptor ~3
Chain," Proc. Natl. Acad. Sci. USA 89:3325-29; "Sakkas, et al. (1994), "T-Cell
Antigen
Receptors in Rheumatoid Arthritis," Immunol. Res. 13:117-38; and
Theofilpooulos et al.
(1989), "B and T Cell Antigen Repertoires in Lupus/Arthritis Murine Models,"
Sp~ihger
Semin Immunopathol. 11:335-68; Cronstein et al. (1994) Curr. Opin. Rheum.
6:300-304;
is Szekanecz et al. (1996) J. Invest. Med. 44:124-135; Liao et al. (1995)
Rheum. Arth. 21:715-
740; Veale et al. (1996) Drugs & Aging 9:87-92; Cronstein et al. (1994) Curr.
Opin. Rheum.
6:300-304; Haskard (1995) Curr. Opiu. Rheum. 7:229-234: Cronstein et al.
(1994) Curr.
Opih. Rheum. 6:300-304; Remy et al. (1995) Proc. Natl. Acad. Sci. USA 92:1744-
1748;
Ashkenas et al. (1996) Dev. Biol. 180:433-444; Springer (1994) Cell 76:301-
314; Hynes
20 (1992) Cell 69:11-2S; and Schwartz et al. (1995) Annu. Rev. Cell Dev. Biol.
11:549-599.
Additional targeting ligands which may be employed in the compositions and
methods of the present invention are described in Schwarzenberger et al.
(1996), "Targeting
Gene Transfer to Human Hematopoietic Progenitor Cell Lines Through the c-kit
Receptor,"
Blood. 87:472-8; Prokopova et al. (1993), "Methyl-a-D-Mannopyranoside,
25 Monooligosaccharides and Yeast Mannans Inhibit Development of Rat Adjuvant
Arthritis,"
J. Rheumatol. 20:673-7; U.S. Patent No. 5,627,263; ," Chen, et al. (1987),
"The Platelt
Glycoprotein IIb/IIIa-Like Protein in Human Endothelial Cells Promotes
Adhesion but not
Initial Attachment to Extracellular Matrix," J. Cell. Biol. l OS:188S-92; and
Wallner et al.
(1987), "Primary Structure of Lymphocyte Function-Associated Antigen 3 (LFA-
3)," J. Exp.
30 Med.166:923-32.
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Still additional targeting ligands that may be employed in the compositions
and
methods of the present invention are described in PCT publication WO 96/37194,
and include
fetuin and asialofetuin, hexamine, spermine and spermidine, N-glutaryl DOPE,
IgA, IgM,
IgG and IgD, MHC and HLA markers, and CD1, CD4, CD8-11, CD15, Cdwl7, CD18,
CD21-25, CD27, CD30-45, CD46-48, Cdw49, Cdw50, CD51, CD53-54, Cdw60, CD61-64,
Cdw65, CD66-69, Cdw70, CD71, CD73-74, Cdw75, CD76-77, LAMP-l and LAMP-2.
Exemplary covalent bonds through which the targeting ligands may be covalently
linked to the termini of the outer arms include, for example: amide (-CONH-);
thioamide
(-CSNH-); ether (ROR', where R and R' may be the same or different and are
other than
l0 hydrogen); ester (-COO-); thioesters (-COS-); -O-; -S-; -Sn-, where n is
greater than l,
preferably about 2 to about 8, and more preferably about 2; carbamates; -NH-; -
NR-, where R
is alkyl, for example, alkyl of from 1 to about 4 carbons; urethane;
substituted imidate; and
combinations of two or more of these. Covalent bonds between targeting ligands
and
stabilizing materials, for example, lipids, may be achieved through the use of
molecules that
may act as spacers to increase the conformational and topographical
flexibility of the ligand.
Examples of such spacers include, for example, succinic acid, 1,6-hexanedioic
acid, 1,8-
octanedioic acid, and the like, as well as modified amino acids, such as, for.
example, 6-
aminohexanoic acid, 4-aminobutanoic acid, and the like. In addition, in the
case of targeting
ligands that comprise peptide moieties, sidechain-to-sidechain crosslinking
may be
complemented with sidechain-to-end crosslinking and/or end-to-end
crosslinlcing. Also,
small spacer molecules, such as dimethylsuberimidate, may be used to
accomplish similar
objectives. The use of agents, including those used in Schiff s base-type
reactions, such as
gluteraldehyde, may also be employed. The Schiff s base linkages, which may be
reversible
linkages, can be rendered more permanent covalent linkages via the use of
reductive
amination procedures. This may involve, for example, chemical reducing agents,
such as
lithium aluminum hydride reducing agents or their milder analogs, including
lithium
aluminum diisobutyl hydride (DIBAL), sodium borohydride (NaBH4) or sodium
cyanoborohydride (NaBH3CN).
The covalent linking of the targeting ligands to the stabilizing materials in
the
present compositions may be accomplished using synthetic organic techniques
that would be
readily apparent to one of ordinary skill in the art in view of the present
disclosure. For
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example, the targeting ligands may be linked to the materials via the use of
well-known
coupling or activation agents. As known to the skilled artisan, activating
agents are generally
electrophilic, which can be employed to elicit the formation of a covalent
bond. Exemplary
activating agents that may be used include, for example, carbonyldiimidazole
(CDI),
dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), methyl sulfonyl
chloride,
Castro's Reagent, and diphenyl phosphoryl chloride.
The covalent bonds may involve crosslinking and/or polymerization.
Crosslinking
preferably refers to the attachment of two chains of polymer molecules by
bridges, composed
of an element, a group, or a compound, which join certain carbon atoms of the
chains by
covalent chemical bonds. For example, crosslinking may occur in polypeptides
that are
joined by the disulfide bonds of the cysteine residue. Crosslinking may be
achieved by any
number of methods including the addition of a chemical substance (crosslinking
agent) and
exposing the mixture to heat, and the exposure of the polymer to high energy
radiation. A
variety of crosslinking agents, or "tethers", of different lengths and/or
functionalities are
described, for example, in R.L. Lunbland (1995) Techniques in Protein
Modification, CRC
Press, Inc., Ann Arbor, MI, pp. 249-68. Exemplary crosslinkers include, for
example, 3,3'-
dithiobis(succinimidylpropionate), dimethyl suberimidate, and its variations
thereof, based on
hydrocarbon length, and bis-N-maleimido-1,8-octane.
In accordance with preferred embodiments, the targeting ligands may be linked
or
z0 attached via a linking group. Preferably, the targeting ligand is attached
via a linker that is
also attached to the arms of the polymer. A variety of linking groups are
available and would
be apparent to one skilled in the art in view of the present disclosure.
Preferably, the linking
group comprises a hydrophilic polymer. Suitable hydrophilic linker polymers
include, for
example, polyalkyleneoxides such as, for example, PEG and polypropylene glycol
(PPG),
z5 polyvinylpyrrolidones, polyvinylmethylethers, polyacrylamides, such as, for
example,
polymethacrylamides, polydimethylacrylamides and
polyhydroxypropylmethacrylamides,
polyhydroxyethyl acrylates, polyhydroxypropyl methacrylates,
polymethyloxazolines,
polyethyloxazolines, polyhydroxyethyloxazolines,
polyhyhydroxypropyloxazolines,
polyvinyl alcohols, polyphosphazenes, poly(hydroxyalkylcarboxylic acids),
polyoxazolidines,
30 polyaspartamide, and polymers of sialic acid (polysialics). The hydrophilic
polymers are
preferably selected from the group consisting of PEG, PPG, polyvinylalcohol
and
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polyvinylpyrrolidone and copolymers thereof, with PEG and PPG polymers being
more
preferred and PEG polymers being even more preferred. Preferred among the PEG
polymers
are, for example, bifunctional PEG having a molecular weight of about 1,000
Daltons to
about 10,000 Daltons, preferably about 5,000 Daltons. Preferably, the polymer
is
bifunctional with the targeting ligand bound to a terminus of the polymer.
Generally, the
targeting ligand may be incorporated into the stabilizing agent at
concentrations of from
about 0.1 mole % to about 25 mole %, preferably from about 1 mole % to about
10 mole %.
Of course, the particular ratio employed may depend upon the particular
targeting ligand,
linker group, and stabilizing agents.
l0 Standard peptide methodology may be used to link the targeting ligand to
the
stabilizing materials when utilizing linker groups having two unique terminal
functional
groups. Bifunctional hydrophilic polymers, and especially bifunctional PEGs,
may be
synthesized using standard organic synthetic methodologies. In addition, many
of these
materials are available commercially, such as, for example, oc-amino-~-carboxy-
PEG which
is commercially available from Shearwater Polymers (Huntsville, AL). An
advantage of
using a PEG material as the linking group is that the size of the PEG may be
varied such that
the number of monomeric subunits of ethylene glycol may be as few as about 5,
or as many
as about 500 or even greater. Accordingly, the "tether" or length of the
linkage may be
varied, as desired. This may be important depending on the particular
targeting ligand
employed. For example, a targeting ligand that comprises a large protein
molecule may
require a short tether, and thereby simulate a membrane-bound protein. A short
tether may
also allow for a delivery polymer to maintain a close proximity to the target.
This may be
used advantageously in connection with vesicles that also comprise a
~bioactive agent in that
the concentration of bioactive agent that may be delivered to the cell may be
advantageously
increased. Another suitable linking group that may provide a short tether is
glyceraldehyde.
Glyceraldehyde may be bound to DPPE via a Schiff s base reaction. Subsequent
Amadori
rearrangement can provide a substantially short linking group. The gamma
carbonyl of the
Schiff s base may then react with a lysine or arginine of the targeting
protein or peptide to
form the targeted lipid.
3o In certain embodiments, the targeting ligands may be incorporated in the
present
polymers via non-covalent associations. As known to those skilled in the art,
non-covalent
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association is generally a function of a variety of factors, including, for
example, the polarity
of the involved molecules, the charge (positive or negative), if any, of
the.involved
molecules, the extent of hydrogen bonding through the molecular network, and
the like.
Non-covalent bonds are preferably selected from the group consisting of ionic
interaction,
dipole-dipole interaction, hydrogen bonds, hydrophilic interactions, van der
Waal's forces,
and any combinations thereof. Non-covalent interactions may be employed to
bind the
targeting ligand to the stabilizing agent. Additional techniques that may be
adapted for
incorporating the targeting ligand into the present compositions are
disclosed, for example, in
U.S. Application Serial No. 09/218,660, filed December 28, 1998.
l0
C. THE ACTIVE AGENT
The drug in the formulation, as noted above, is any active agent whose
systemic
bioavailability can be enhanced by increasing the solubility of the agent in
water. Generally,
such drugs will be at least about one-and-one-half times as soluble in the
hydrophilic polymer
as in water, and preferably at least about ten times as soluble in the
hydrophilic polymer as in
water. The latter group of drugs is generally "hydrophobic" as defined in
section (A). Any
number of drugs may be incorporated into the formulations of the invention,
i.e., any
compounds that fit the aforementioned solubility criteria and induce a desired
systemic effect.
Such substances include the broad classes of compounds normally administered
systemically. In general, this includes: analgesic agents; antiarthritic
agents; respiratory
drugs, including antiasthmatic agents and drugs for preventing reactive airway
disease;
antibiotics; anticancer agents, including antineoplastic drugs;
anticholinergics;
anticonvulsants; antidepressants; antidiabetic agents; antidiarrheals;
antihelminthics;
antihistamines; antihyperlipidemic agents; antihypertensive agents;
antiinflammatory agents;
antimetabolic agents; antimigraine preparations; antinauseants;
antiparkinsonism drugs;
antipruritics; antipsychotics; antipyretics; antispasmodics; antiviral agents;
anxiolytics;
attention deficit disorder (ADD) and attention deficit hyperactivity disorder
(ADHD) drugs;
cardiovascular preparations including cardioprotective agents; central nervous
system
stimulants; cough and cold preparations, including decongestants; diuretics;
genetic
materials; gonadotropin releasing hormone (GnIZH) inhibitors; herbal remedies;
hormonolytics; hypnotics; immunosuppressive agents; leukotriene inhibitors;
mitotic
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inhibitors; muscle relaxants; parasympatholytics; peptide drugs;
psychostimulants; sedatives;
steroids; sympathomimetics; tranquilizers; vasodilators, including peripheral
vascular
dilators; and vitamins.
It will be appreciated that the invention is particularly useful for
delivering active
agents for which chronic administration may be required, as the present
formulations provide
for sustained release. The invention is thus advantageous insofar as patient
compliance with
regard to forgotten or mistimed dosages is substantially improved. Any agent
that is typically
incorporated into a capsule, tablet, troche, liquid, suspension or emulsion,
wherein
administration is on a regular (i.e., daily, more than once daily, every other
day, or any other
l0 regular schedule) can be advantageously delivered using the formulations of
the invention.
Examples of drugs for which a sustained release formulation is particularly
desirable include, but are not limited to, the following:
analgesic agents--hydrocodone, hydromorphone, levorphanol, oxycodone,
oxymorphone, codeine,, morphine, alfentanil, fentanyl, meperidine and
sufentanil,
diphenylheptanes such as levomethadyl, methadone and propoxyphene, and
anilidopiperidines such as remifentanil;
ntiandrogens--bicalutamide, flutamide, hydroxyflutamide, zanoterine and
' nilutamide;
anxiolytic agents and tranquilizers--diazepam, alprazolam, chlordiazepoxide,
2o clonazepam, halazepam, lorazepam, oxazepam and clorazepate;
antiarthritic agents--hydroxychloroquine, gold-based compounds such as
auranofin,
aurothioglucose and gold thiomalate, and COX-2 inhibitors such as celecoxib
and rofecoxib;
antibiotics (including antineoplastic antibiotics)--vancomycin, bleomycin,
pentostatin, mitoxantrone, mitomycin, dactinomycin, plicamycin and amikacin;
2S anticancer agents, including antineoplastic agents--paclitaxel, docetaxel,
camptothecin and its analogues and derivatives (e.g., 9-aminocamptothecin, 9-
nitrocamptothecin, 10-hydroxy-camptothecin, irinotecan, topotecan, 20-O-~3-
glucopyranosyl
camptothecin), taxanes (baccatins, cephalornannine and their derivatives),
carboplatin,
cisplatin, interferon-azA, interferon-azB, interferon-aN3 and other agents of
the interferon
30 family, levamisole, altretamine, cladribine, bovine-calmette-guerin (BCG),
aldesleukin,
tretinoin, procarbazine, dacarbazine, gemcitabine, rnitotane, asparaginase,
porfimer, mesna,
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amifostine, mitotic inhibitors including podophyllotoxin derivatives such as
teniposide and
etoposide and vinca alkaloids such as vinorelbine, vincristine and
vinblastine;
antidepressant drugs--selective serotonin reuptake inhibitors such as
sertraline,
paroxetine, fluoxetine, fluvoxamine, citalopram, venlafaxine and nefazodone;
tricyclic anti-
depressants such as amitriptyline, doxepin, nortriptyline, imipramine,
trimipramine,
amoxapine, desipramine, protriptyline, clomipramine, mirtazapine and
maprotiline; other
anti-depressants such as trazodone, buspirone and bupropion;
antiestrogens--tamoxifen, clomiphene and raloxifene;
antifungals-amphotericin B, imidazoles, triazoles, and griesofulvin;
l0 antihyperlipidemic agents--HMG-CoA reductase inhibitors such as
atorastatin,
simvastatin, pravastatin, lovastatin and cerivastatin sodium, and.other lipid-
lowering agents
such as clofibrate, fenofibrate, gemfibrozil and tacrine;
antimetabolic agents--methotrexate, fluorouracil, floxuridine, cytarabine,
mercaptopurine and fludarabine phosphate;
antimigraine preparations--zolmitriptan, naratriptan, sumatriptan,
rizatriptan,
methysergide, ergot alkaloids and isometheptene;
antipsychotic'agents--chlorpromazine, prochlorperazine, trifluoperazine,
promethazine, promazine, thioridazine, mesoridazine, perphenazine,
acetophenazine,
clozapine, fluphenazine, chlorprothixene, thiothixene, haloperidol,
droperidol, molindone,
loxapine, risperidone, pimozide and domepezil;
aromatase inhibitors--anastrozole and letrozole;
attention deficit disorder and attention deficit hyperactivity disorder drugs--
methylphenidate and pemoline;
cardiovascular preparations--angiotensin converting enzyme (ACE) inhibitors;
diuretics; pre- and afterload reducers; cardiac glycosides such as digoxin and
digitoxin;
inotropes such as amrinone and milrinone; calcium channel blockers such as
verapaxnil,
nifedipine, nicardipene, felodipine, isradipine, nimodipine, bepridil,
amlodipine and
diltiazem; beta-blockers such as pindolol, propafenone, propranolol, esmolol,
sotalol and
acebutolol; antiarrhythmics such as moricizine, ibutilide, procainamide,
quinidine,
disopyramide, lidocaine, phenytoin, tocainide, mexiletine, flecainide,
encainide, bretylium
and amiodaxone; caxdioprotective agents such as dexrazoxane and leucovorin;
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GnRH inhibitors and other hormonolytics and hormones--leuprolide, goserelin,
chlorotrianisene, dinestrol and diethylstilbestrol;
herbal remedies--melatonin;
immunosuppressive agents--6-thioguanine, 6-aza-guanine, azathiopurine,
cyclosporin and methotrexate;
lipid-soluble vitamins--tocopherols and retinols;
leukotriene inhibitors--zafirlukast, zileuton and montelukast sodium;
nonsteroidal anti-inflammatory drugs (NSAIDs)--diclofenac, flurbiprofen,
ibuprofen, ketoprofen, piroxicam, naproxen, indomethacin, sulindac, tolmetin,
l0 meclofenamate, mefenamic acid, etodolac, ketorolac and bromfenac;
peptide drugs--leuprolide, somatostatin, oxytocin, calcitonin and insulin;
peripheral vascular dilator--cyclandelate, isoxsuprine and papaverine;
respiratory drugs--such as theophylline, oxytriphylline, aminophylline and
other
xanthine derivatives;
15 steroids--progestogens such as flurogestone acetate, hydroxyprogesterone,
hydroxyprogesterone acetate, hydroxyprogesterone caproate, medroxyprogesterone
acetate,
megestrol, norethindrone, norethindrone acetate, norethisterone,
norethynodrel, desogestrel,
3-keto desogestrel, gestadene and levonorgestrel; estrogens such as estradiol
and its esters
(e.g., estradiol benzoate, valerate, cyprionate, decanoate and acetate),
ethynyl estradiol,
20 estriol, estrone, mestranol and polyestradiol phosphate; corticosteroids
such as
betamethasone, betamethasone acetate, cortisone, hydrocortisone,
hydrocortisone acetate,
corticosterone, fluocinolone acetonide, flunisolide, fluticasone,
prednisolone, prednisone and
triamcinolone; androgens and anabolic agents such as aldosterone,
androsterone, testosterone
and methyl testosterone; and
25 topoimerase inhibitors-camptothecin, anthraquinones, anthracyclines,
teniposide,
etoposide, topotecan and irinotecan.
Genetic material may also be delivered using the present formulation, e.g., a
nucleic acid, RNA, DNA, recombinant RNA, recombinant DNA, antisense RNA,
antisense
DNA, hammerhead RNA, a ribozyme, a hammerhead ribozyme, an antigene nucleic
acid, a
3o ribooligonucleotide, a deoxyribonucleotide, an antisense
ribooligonucleotide, and an
antisense deoxyribooligonucleotide. Representative genes include vascular
endothelial
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growth factor, fibroblast growth factor, BCl-2, cystic fibrosis transmembrane
regulator, nerve
growth factor, human growth factor, erythropoeitin, tumor necrosis factor,
interleukin-2 and
histocompatibility genes such as HLA-B7.
The foregoing list is merely illustrative and is not intended to be limiting.
A wide
variety of drugs and drug types can be effectively administered using the
present
formulations, although the invention is most advantageous with hydrophobic
drugs.
It may be desirable to include one or more P-glycoprotein inhibitors in the
formulation along with the active agent to be administered. It has been
established that
intestinal absorption of certain drugs, of which paclitaxel is exemplary, is
controlled by P-
IO glycoprotein (P-gp). With such drugs, then, the present formulations
preferably include a P-
gp inhibitor for oral administration in order to increase intestinal
absorption and thus oral
bioavailability. A particularly preferred P-gp inhibitor is cyclosporin A~
although other P-gp
inhibitors may also be used. When a P-gp inhibitor is included in the
formulation, the weight
ratio of drug to P-gp inhibitor (e.g., the ratio of paclitaxel to cyclosporin
A) will generally be
in the range of about 1:5 to 5:1, preferably in the range of about 1:2 to
2:l,.more preferably in
the range of about 1:1.5 to 1.5:1, and optimally about 1:1. With paclitaxel,
it may also be
desirable to co-administer a folate (i.e., a salt or ester of folic acid),
which has been found to
increase paclitaxel absorption.
The amount of drug in the formulation should be such that the weight ratio of
drug
to all other components of the formulation is in the range of about 1:1 to
1:50, preferably in
the range of about 1:1 to 1:20, more preferably in the range of about 1:2 to
1:10, and
optimally about 1:5.
D. OTHER COMPONENTS OF THE FORMULATION
Free phospholipids, i.e., phospholipids not conjugated to PEG or other
moieties,
may be incorporated into the present formulations as excipients in order to
reduce the particle
size of the polyrner/drug matrix. For intravenous administration in
particular, particle size is
critical, and is generally in the range of about 1 nm to 10 Vim, preferably in
the range of about
5 nm to 500 nm, most preferably in the range of about 30 nm to 250 nm (the
values given are
3o number average). The free phospholipid, like the phospholipid that may be
conjugated to the
hydrophilic polymer, can be anionic, neutral or cationic, of naturally
occurring or synthetic
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origin, and will generally comprise a diacyl phosphatidylcholine, a diacyl
phosphatidylethanolamine, a diacyl phosphatidylserine, a diacyl
phosphatidylinositol, a
diacyl phosphatidylglycerol or a diacyl phosphatidic acid, wherein each acyl
moiety can be
saturated or unsaturated and typically contains about 8 to 20 carbon atoms. As
with the
conjugated phospholipids, the preferred unsaturated acyl moieties of the free
phospholipids
are oleic and linoleic acid esters, and preferred saturated acyl moieties are
palmitate,
myristate and stearate; particularly preferred phospholipids are DPPE and
POPE. The
amount of free phospholipid should be just sufficient to reduce the particle
size as desired.
Preferably, any free phospholipid that is included in the formulation
represents less than
l0 about 25% of the total phospholipid present, and optimally represents less
than about 10% of
the total phospholipid present.
Stabilizing agents may also be added to the formulation and are useful for
reducing
particle size. The polymers may be used in addition or in lieu of free
phospholipids.
Preferably, the stabilizing agent acts to stabilize the surface of the complex
by virtue of a
combination of hydrophilic and hydrophobic interactions. Thus, it is preferred
that the
stabilizing agentpolymer contains both hydrophilic and hydrophobic groups or
domains thus
allowing this interaction to occur. It is also preferred that the stabilizing
agent contain a
sufficient amount of hydrophilic surfaces that post stabilization
nanoparticles remain
suspended within water and avoid clumping.
The preferred stabilizing agent is a polymer having a molecular weight ranging
from about 400 Daltons to about 400,000 Daltons, more preferably from about
1,000 Daltons
to about 200,000 Daltons, and still more preferably from about 3,000 Daltons
to about
100,000 Daltons. The stabilizing agent may be derived from natural,
recombinant, synthetic
or semisynthetic sources. Most preferably the stabilizing agent will be a
protein or a peptide.
Useful preferred proteins include albumin, collagen, fribrin, immunoglobulins,
hemoglobin,
vascular endothelial growth factor, vascular permeability factor, epidermal
growth factor,
fibroblast growth factor, fibronectin, vitronectin, and cytokines such as
interleukins (e.g. IL-3
and IL-12).
Suitable stabilizing proteins include, but are not limited to: serum proteins,
i.e.,
3o albumin (especially recombinant and defatted), arnylins, atrial natriuretic
peptides,
endothelins and endothelin inhibitors, urokinase, streptokinase,
staphylokiase, vasoactive
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intestinal peptide, HDL, LDL, VLDL, etc.; agglutination (antihemophillia)
factors, i.e.,
Factor VIII, Factor IX and subtypes thereof, decorsin, serum thymic factor,
etc.; peptide
hormones, i.e., ACTH, FSH, LH, parathyroid hormone, thyroxin, insulin,
vasopressin,
bradykinin and bradykinin potentiators, HGH, CRF (corticotropin releasing
factor), oxytocin,
gastrins, LH-RIi, MSH (melanocyte stimulating hormone) and MSH releasing
factor;
parathyroid hormones and analogs; pituitary adenylate cyclase activating
polypeptide;
secretins; thyrotropin releasing hormone, etc.; structural proteins, i.e.,
collagens, amyloid
proteins, brain natriuretic peptides, elafin, fibronectin and fibronectin
fragments, laminin,
sarafotoxins, etc.; growth factors, i.e., nerve growth factor, platelet
derived growth factor,
l0 epidermal growth factor, vascular endothelial growth factor, tumor necrosis
factor, CINC- I
(cytokine-induced neutrophil chemoattractant), growth hormone releasing
factor, liver cell
growth factor, midkines, neurokinins, neuromedins, etc., metabolic
potentiators, i.e.,
erythropoietin, adrenomedullin and adrenomedullin antagonists, (o-agatoxin TK,
agelenin,
angiotensins, calcicludine, calciseptine, calcitonin and calcitonin
antagonists, calmodulin,
charybdotoxin, chlorotoxin, conotoxins, endorphins, neo-endorphins, glucagon
and variants,
guanylins, iberiotoxin, kaliotoxin, maxgatoxin, mast cell degranulating
peptide, neurotensins,
pancreastatins, PLTX-11, scylotoxin, ATPase inhibitors, somatostatins,
somatomedin,
uroguanylin, etc.; nuclear binding proteins, i.e., histones, spermine,
spermidine, nuclear
localization sequences, telomerase, etc.; enzymes, i.e., cholecystokinin,
cathepsins, etc.;
antivirals, i.e., IFN-oc, IFN-P, IFN-Z, virus replication inhibiting peptide,
etc.;
immunoglobulins, i.e., IgA, IgD, IgE, IgG, IgH and subtypes; and miscellaneous
proteins
such as apamin, bombesin, casomorphins, conantokins, defensin-1, dynorphins,
enkephalins,
galanins, magainin, nociceptin, osteocalcins, substance P, xenin, etc. While
not wishing to be
limited to the preceding examples, one of skill in the art will recognize that
the examples
given may be used individually or in combination.
The stabilizing protein may also serve as a targeting agent or binding ligand
to
direct the nanoparticles and drugs therein to a certain site. The preferred
protein is albumin,
in particular, human serum albumin and even more preferably recombinant
derived human
albumin. Another preferred protein is defatted albumin, either native or
recombinant. For
veterinary applications, the albumin is preferably from the patient's species.
The stabilizing
albumin is generally added to the nanoparticles at an effective stabilizing
concentration,
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generally in the range of about 0.001 % w/v to up to about 10% wlv, more
preferably in the
range of about 0.01% to about 5%, in the range of about 0.1 % to about 2.5%,
and most
preferably in the range of about 0.25% to about 1.5%. Note that more than one
protein may
be used to stabilize the nanoparticles. For example, the particles may be
formulated with
about 1.0% w/v albumin and about 0.1 % w/v EGF. In this case, the EGF serves
as a
targeting ligand to help the nanoparticle to bind to tissues with increased
expression of the
EGF receptor.
The protein may be naturally occurring, a protein fragment, e.g. a fragment of
the
gamma-carboxy terminus of fibrinogen, or chemically modified. For example,
albumin or
l0 other proteins may be modified with one or more hydrophilic or targeting
moieties. For
example, the protein may be modified by binding one or more PEG residues per
protein
molecule, typically between 1 and 100 PEG molecules per protein molecule, but
more
preferably between 1 and 10 PEG residues. For example, mono or bifunctional
PEG groups
may be coupled to the protein through linkages such as ethers or biodegradable
bonds such as
i5 esters, amides, carbamates, thioesters, disulfides, thiocarbamates,
phosphate esters and
phosphoamides. The resulting "PEGylated" protein enables the protein to
stabilize the
surface of the nanoparticle while the PEG groups help to protect the
nanoparticle surface
from nonspecific interaction with serum proteins. In this regard, the
"PEGylated" proteins
increase the serum half lives of the nanoparticles.
20 In addition to the proteins enumerated above, the polymers may be other
natural
polymers, such as: cellulose and dextran; semi-synthetic cellulose derivatives
such as
methylcellulose and carboxymethylcellulose; and synthetic polymers such as
polvinylalcohol
polyvinylpyrrolidone and copolymers containing PEG and a second polymer such
as
polypropylene glycol (PPG) (e.g. those available under the Pluronic
trademaxk). Synthetic
25 polymers such as the Pluronics, i.e. copolymers of PEG and PPG, may be
incorporated into
mixtures of stabilizing agents, e.g., with albumin. Preferred block copolymers
include, but
axe not limited to, polyethylene glycol-N-caxboxyanhydride of 6-
(benzyloxycarbonyl)-1-
lysine, polyethylene glycol-poly-1-lysine and polyethylene glycol-polyaspartic
acid. Methods
for synthesizing the above copolymers are detailed in Haxada and Kataoka
(1995)
30 Macromolecules 28:5294-5299. One of skill in the art will readily recognize
that the same
synthetic methods can be used to substitute polypropylene glycols for PEG to
make the PPG
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block copolymer analogs of the above.
Other suitable PEG copolymers may be synthesized from polymerizable aldehydes
that optionally contain additives and/or crosslinking elements capable of
copolymerization,
surfactants or surfactant mixtures, coupling agents, biomolecules or
macromolecules bound
by these coupling agents, as well as diagnostically or therapeutically
effective components.
The monomers encompassed herein include, but are not limited to, alpha/beta-
unsaturated aldehydes, e.g., acrolein, crotonaldehyde, propionaldehyde, alpha-
substituted
acrolein derivatives, e.g., alpha-methyl acrolein, alpha-chloroacrolein, alpha-
phenyl acrolein,
alpha-ethyl acrolein, alpha-isopropyl acrolein, alpha-n-butyl acrolein, alpha-
n-propyl acrolen;
l0 dialdehydes, e.g., glutaraldehyde, succinaldehyde or their derivatives or
their mixtures with
additives capable of copolymerization (comonomers), e.g., alpha-substituted
acroleins, beta-
substituted acroleins, ethyl cyanoacrylates, methyl cyanoacrylates, butyl
cyanoacrylates,
hexyl eyanoacrylate, methyl methacrylates, vinyl alcohols, acrylic acids,
methacrylic acids,
acrylic acid chlorides, methacrylic acid chlorides, acrylonitrile,
methacrylonitriles,
acrylarnides, substituted acrylamides, hydroxy methyl methacrylates, mesityl
oxide,
dimethylaminoethylmethacrylates 2-vinylpyridines and N-vinyl-2-pyrrolidinone.
Suitable coupling agents that may be employed in the synthesis of PEG
copolymers
include, but are not limited to: compounds containing amino groups (e.g.,
hydroxylamine,
butylamine, allylamine, ethanolamine, trishydroxymethylaminomethane, 3-amino-1-
propanesulfonic acid, 5-aminovaleric acid, 8-aminooetanoic acid, D-glucosamine
hydrochloride, aminogalactose, aminosorbitol, aminomannitol,
diethylaminoethylamine,
anilines, sulfonyl acid amide, choline, N-methylglucamine, piperazine, 1,6-
hexanediamine,
urea, hydrazine, glycine, alanine, lysine, serine, valine, leucine, peptides,
proteins, albumin,
human serum albumin, polylysine, gelatin, polyglycolamines, aminopolyalcohols,
dextran
sulfates with amino groups, N-aminopolyethylene glycol (HO-PEG-NHZ), N,N'-
diaminopolyethylene glycol (NHz PEG-NHZ), antibodies, irnmunoglobulins, etc.);
compounds containing acid groups, e.g., carboxylic acids such as acetic acid,
propionic acid,
butyric acid, valeric acid, caproic acid, caprylic acid, capric acid, lauric
acid, myristic acid,
palmitic acid, stearic acid, oleic acid, linolic acid, linolenic acid,
cyclohexane carboxylic acid,
phenylacetic acid, benzoylacetic acid, chlorobenzoic acid, bromobenzoic acid,
nitrobenzoic
acid, ortho-phthalic acid, meta-phthalic acid, para-phthalic acid, salicylic
acid,
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hydroxybenzoic acid, aminobenzoic acid, methoxybenzoic acid, PEG-linker-
glutaminic acid,
PEG-linker-DTPA, PEG-linker-EDTA, etc.; compounds containing hydroxy groups,
i.e.,
alcohols such as methanol, ethanol, propanol, butanol, pentanol, hexanol,
heptanol, octanol,
decanol, dodecanol, tetradecanol, hexadecanol, octadecanol, isopropyl alcohol,
isobutyl
alcohol, isopentyl alcohol, cyclopentanol, cyclohexanol, crotyl alcohol,
benzyl alcohol,
phenyl alcohol, diphenyl methanol, triphenyl methanol, cinnamyl alcohol,
ethylene glygol,
1,3-propanediol, glycerol, pentaerythritol and the like; polymerizable
substances, such as
alpha, beta-unsaturated aldehydes, e.g., acrolein, crotonaldehyde,
propionaldehyde, etc.;
alpha-substituted acrolein derivatives, e.g., alpha-methylacrolein, alpha-
chloroacrolein, alpha-
1o phenylacrolein, alpha-ethylacrolein, alpha-isopropylacrolein, alpha-n-
butylacrolein, alpha-n-
propylacrolein, etc.; and dialdehydes, e.g., glutaraldehyde, succinaldehyde or
their derivatives
or their mixtures with additives capable of copolymerization, such as alpha-
substituted
acroleins, beta-substituted acroleins, ethyl cyanoacrylates, methyl
cyanoacrylates, butyl
acrylates, hexyl cyanoacrylates, methylmethacrylates, vinyl alcohols, acrylic
acids,
methacrylic acids, acrylic acid chlorides, acrylonitrile, methacrylonitriles,
acrylamides,
substituted acrylamides, hydroxymethylmethacrylates, mesityl oxide,
dimethylaminoethylmethacrylates 2-vinylpyridines, vinylpyrrolidinone, etc.
Particularly preferred coupling agents include hydroxylamine,
trishydroxymethylaminomethane, 3-amino-1-propane sulfonic acid, D-
z0 glucosaminohydrochloride, aminomamlitol, urea, human serum albumin,
hydrazine, proteins,
polyglycolamines, aminopolyalcohols (e.g., HO-PEG-NHZ or NHz-PEG-NHZ), and
compounds containing acid groups such as PEG-linker-asparaginic acid, PEG-
linker-
glutaminic acid, PEG-linker-DTPA and PEG-linker-EDTA, wherein the molecular
weight of
the PEG is less than about 100 kD, preferably less than about 40 kD.
Z5 The amount of coupling agent is typically present in the range of about 1 %
wt. to
about 95% wt. of the polyaldehyde in the PEG copolymer. The coupling agents
can be
condensed by their amino group or on the formyl groups located on the surface
of
nanoparticles synthesized from polymerized aldehydes and optional surfactants.
Also, such
formyl groups may also bind those monomers listed above that are
polymerizable. However,
3o the acids and alcohols named above are typically coupled on the
nanoparticles only after
previous conventional conversion of the aldehyde function.
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Generally the stabilizing agent is added to the complex in aqueous media. The
complex and stabilizing agent are then subjected to a mechanical dispersal
process that helps
to break the complex into nanoparticles that are then stabilized by the
stabilizing agent.
Useful mechanical dispersal processes include shaking, agitation (e.g.,
vortexing), sonication,
extrusion under pressure, microfluidization, microemulsification and high
speed blending.
Compounds other than free phospholipids are also useful for reducing particle
size,
and can be used in addition to or in lieu of free phospholipids; these other
compounds
include, but are not limited to, cholic acids, cholic acid salts, saccharides
(such as sorbitol,
sucrose and trehalose), polyhydroxyalcohols (such as glycerol), and liquid
polyethylene
l0 glycols (i.e., PEG having a molecular weight less than about 1000 Daltons).
The
formulations of the invention can also contain pharmaceutically acceptable
auxiliary agents as
required in order to approximate physiological conditions; such auxiliary
agents include pH
adjusting and buffering agents, tonicity adjusting agents, and the like. Lipid-
protecting
agents that serve to minimize free radical and peroxidative damage upon
storage may also be
advantageous. Suitable lipid protective agents include alpha-tocopherol and
water-soluble,
iron-specific chelators such as deferoxamine and ethylenediaminetetraacetic
acid (EDTA).
Additionally, for lyophilized compositions that are to be hydrated prior to
use, it may be
desirable to include one or more cryoprotectants, or anti-flocculants in order
to facilitate
rehydration and formation of a substantially homogeneous suspension. For
compositions that
2o are to be stored in liquid form, it is preferred that one or more
conventional anti-bacterial
agents be included. Still other additives that may be incorporated into the
present
formulations include radioactive or fluorescent markers useful for imaging
purposes.
Radioactive markers include, for example, technitium and indium, while an
exemplary
fluorescent marker is fluorescein. The excipients can be included in an amount
up to about
50 wt.% of the formulation, but preferably represent less than about 10 wt.%
of the
formulation.
E. MANUFACTURE AND STORAGE
The formulations of the invention are manufactured using standard techniques
and
3o reagents known to those skilled in the art of pharmaceutical formulation
and drug delivery
and/or described in the pertinent texts and literature. See Remington: The
Science ahd
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Practice of Pharmacy, 19th Ed. (Easton, PA: Mack Publishing Co., 1995), which
discloses
conventional methods of preparing pharmaceutical compositions that may be used
as
described or modified to prepare pharmaceutical formulations of the invention.
In a preferred
embodiment, the hydrophilic polymer, drug, optional free phospholipid and all
other
components of the final composition are mixed together in an organic solvent
or solvent
system such as isopropanol, t-butanol, benzene/methanol, ethanol, or an
alternative suitable
solvent as will be apparent to those of skill in the art and then lyophilizing
the mixture. The
solvent may also be removed by subjecting the mixture to rotary evaporation to
yield a
powder or a solid matrix. When a solid matrix is obtained, the material may be
ground via
l0 ball milling or subjected to other mechanical shear stress to achieve a
finely ground powder
of nanoparticulate material. The resulting nanoparticles may be stabilized
with surfactants,
phospholipids, stabilizing agents including albumin, and other stabilizing
materials, as
discussed above. Another method of manufacturing the formulation is spray
drying. In this
method, a suitable organic solvent, ideally having a flash point sufficiently
above the drying
temperature. Formulations made using this method are in the form of a fluffy,
dry powder.
Alternatively, the components of the final product may be dissolved in a
supercritical fluid such as compressed carbon dioxide, and then ejected under
pressure and
shearing force to form dried particles of the drug-containing formulation. The
formulation is
preferably stored in lyophilized form, in which case, the lyophilized
composition is
rehydrated prior to use. Rehydration is carried out by mixing the lyophilized
composition
with an aqueous liquid (e.g., water, isotonic saline solution, phosphate
buffer, etc.) to provide
a total solute concentration in the range of about SO to 100 mg/ml and a drug
concentration in
the range of about 1 to 20 mg/ml, preferably about 5 to 15 mg/ml. The
formulation may,
however, be stored in the aqueous state, e.g., in pre-filled syringes or
vials. The formulation
may also be stored as a liquid in a physiologically acceptable organic solvent
such as ethanol,
propylene glycol or glycerol, to be diluted with water prior to injection into
a patient. The
lyophilized and rehydrated formulations may be stored at various temperatures
such as
freezing conditions (below about 0°C and as low as about -40°C
to -100°C), refrigerated
conditions generally between about 0°C and 15°C, room
temperature conditions generally
3o between about 15°C and 28°C, or at elevated temperatures as
high as about 40°C.
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The particle size of individual particles within the formulation will vary,
depending
upon the molecular weight and concentration of the hydrophilic polymer, the
amount of drug
as well as its solubility profile (i.e., its solubility in water and the
hydrophilic polymer), the
use of stabilizing agents, and the conditions used in manufacturing. That is,
as noted in the
preceding section, stabilizing agents and various excipients may be used to
facilitate
rehydration and provide a substantially homogeneous dispersion. Additionally,
mechanical
processing techniques can be used to adjust particle size to the appropriate
diameter for the
intended application; for example, after rehydration, the formulation can be
subjected to shear
forces with microfluidization, sonication, extrusion, or the like.
Formulations made with
l0 stabilizing agents can have a particle size on the order of about 20 nm to
100 nm. These
smaller particles, by virtue of their larger accessible surface-to-volume
ratio, tend to release
drug quite rapidly, while larger particles, e.g., over 10 ~,m in diameter,
will provide for far
more gradual, sustained release of drug. The preferred particle size herein is
in the range of
about 1 nm to 500-1000 ~,m in diameter. For intramuscular and subcutaneous
injection,
particle size should be in the range of about 1 nm to 500 ~,m, preferably in
the range of about
10 nm to 300 ~,m, and most preferably in the range of about 20 ~.m to 200 ~,m.
For
intravenous administration, as noted previously, particle size is optionally
in the range of
about 30 nm to 250 nm. For interstitial administration and fracture or wound
packing,
particle size can be up to 1000 ~,m, while for embolization, particle size
will generally be
2o between about 100 ~,m and 250 ~,m.
The formulation can be sterilized using heat, ionizing radiation or
filtration. For drugs
that are thermally stable, heat sterilization is preferable. Lower viscosity
formulations can be
filter sterilized, in which case the particle size should be under about 200
nm. Aseptic
manufacturing conditions may be employed as well, and lyophilization is also
helpful to
maintain sterility and ensure long shelf life. In addition, as noted in the
preceding section,
anti-bacterial agents may be included in aqueous formulations in order to
prevent bacterial
contamination.
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F. INCORPORATION OF AN ACOUSTICALLY ACTIVE GAS
In a further embodiment of the invention, the present formulations are made
with
small quantities of an acoustically active gas instilled therein. In order to
instill the selected
gas into the present formulations, a headspace of gas (preferably an insoluble
gas) is applied
atop the lyophilized composition in a closed container, which is then exposed
to mild
agitation during rehydration. Microquantities of gas will become entrapped in
the interstices
of the dispersion. The presence of the acoustically active gas is useful in
conjunction with
ultrasound imaging, as the gas-instilled dispersion produces an echogenic
contrast that allows
the drug to be tracked in the body. In addition, if a sufficient quantity of
gas is entrapped in
to the formulation, therapeutic ultrasound can allow the microstructure to
unfold at the locus
where the ultrasound is applied, releasing the active agent and thus enhancing
targeting
effectiveness. The acoustically active gas lowers the cavitation threshold,
i.e., the energy
required for cavitation with ultrasound. Preferably, the cavitation energy
used will be under
about 1.5 MegaPascals, and more preferably under about 1.0 MegaPascals. The
gas also
effects dB reflectivity, and a gas concentration of about 1 mg per mI of
particles will
generally have a reflectivity approximately 2 dB higher than that of pure
water.
In general, the amount of acoustically active gas that is imbibed by the
particles of
the formulation is approximately equal to the void space within the particles,
which can be
approximated by their density. For example, particles having a density of 0.10
will imbibe
2o about 90 vol.% gas. Lower density particles will imbibe a higher volume of
gas (i.e., 95
vol.% for particles having a density of 0.05), while higher density particles
will imbibe a
lower volume of gas (i.e., 85 vol.% for particles having a density of 0.15).
Gas may also
adhere to the surface of the particles, typically up to about two times the
volume of the
particles. Normally, the amount of acoustically active gas that is employed is
such that the
gas-instilled formulation will contain at least about 5 vol.% gas, preferably
about 10-15 vol.%
gas.
Typical acoustically active gases are chemically inert gases having 1 to 12
carbon
atoms, and particularly preferred acoustically active gases are
perfluorocarbons, including
saturated perfluorocarbons, unsaturated perfluorocarbons, and cyclic
perfluorocarbons. The
saturated perfluorocarbons, which are usually preferred, have the formula
C~FZn+z, where n is
from 1 to 12, preferably 2 to 10, more preferably 4 to 8, and most preferably
5. Examples
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of suitable saturated perfluorocarbons are the following: tetrafluoromethane;
hexafluoroethane; octafluoropropane; decafluorobutane; dodecafluoropentane;
perfluorohexane; and perfluoroheptane. Saturated cyclic perfluorocarbons,
which have the
formula C"FZn, where n is from 3 to 8, preferably 3 to 6, may also be
preferred, and include,
e.g., hexafluorocyclopropane, octafluorocyclobutane, and
decafluorocyclopentane. Other
gases that can be used include air, nitrogen, helium, argon, xenon and other
such gases.
Alternatively, a gaseous precursor can be used that is in the liquid state at
room
temperature and that is either (1) volatilized prior to introduction into the
headspace above the
lipid- and drug-containing dispersion, or (2) volatilized and instilled into a
microemulsion
l0 which is then introduced into the lipid- and drug-containing dispersion.
Suitable gaseous
precursors are described, for example, in U.S. Patent No. 5,922,304 to Unger,
and include,
without limitation, hexafluoro acetone, isopropyl acetylene, allene,
tetrafluoroallene, boron
trifluoride, isobutane, 1,2-butadiene, 2,3-butadiene, 1,3-butadiene, 1,2,3-
trichloro-2-fluoro-
1,3-butadiene, 2-methyl-1,3-butadiene, hexafluoro-1,3-butadiene, butadiyne, 1-
fluoro-butane,
2-methyl-butane, decafluorobutane, 1-butene, 2-butene, 2-methyl-1-butene, 3-
methyl-1-
butene, perfluoro-1-butene, perfluoro-2-butene, 4-phenyl-3-butene-2-one, 2-
methyl-1-butene-
3-yne, butyl nitrate, 1-butyne, 2-butyne, 2-chloro-1,1,1,4,4,4-
hexafluorobutyne, 3-methyl-1-
butyne, perfluoro-2-butyne, 2-bromobutyraldehyde, carbonyl sulfide,
crotononitrile,
cyclobutane, methylcyclobutane, octafluorocyclobutane, perfluorocyclobutene, 3-
chlorocyclopentene, octafluorocyclopentenecyclopropane, 1,2-
dimethylcyclopropane, 1,l-
dimethylcyclopropane, 1,2-dimethylcyclopropane, ethylcyclopropane,
methylcyclopropane,
diacetylene, 3-ethyl-3-methyl diaziridine, l,l,l-trifluorodiazoethane,
dimethylamine,
hexafluorodimethylamine, dimethylethylamine, bis(dimethylphosphine)amine,
perfluorohexane, 2,3-dimethyl-2-norbornane, perfluorodirnethylamine,
dimethyloxonium
chloride, 1,3-dioxolane-2-one, 4-methyl-1,1,1,2-tetrafluoroethane, 1,1,1-
trifluoroethane,
1,1,2,2-tetrafluoroethane, 1,1,2-trichloro-1,2,2-trifluoroethane, 1,1-
dichloroethane, 1,1-
dichloro-1,2,2,2-tetrafluoroethane, 1,2-difluoroethane, 1-chloro-1,1,2,2,2-
pentafluoroethane,
2-chloro-1,1-difluoroethane, l,l-dichloro-2-fluoroethane, 1-chloro-1,1,2,2-
tetrafluoroethane,
2-chloro-I,1-difluoroethane, chloroethane, chloropentafluoroethane,
dichlorotrifluoroethane,
fluoroethane, hexafluoroethane, nitropentafluoroethane,
nitrosopentafluoroethane,
perfluoroethylamine, ethyl vinyl ether, 1,1-dichloroethane, l,l-dichloro-1,2-
difluoroethane,
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1,2-difluoroethane, methane, trifluoromethanesulfonylchloride,
trifluoromethane-
sulfonylfluoride, bromodifluoronitrosomethane, bromofluoromethane, bromochloro-
fluoromethane, bromotrifluoromethane, chlorodifluoronitromethane,
chlorodinitromethane,
chlorofluoromethane, chlorotrifluoromethane, chlorodifluoromethane,
dibromodifluoromethane, dichlorodifluoromethane, dichlorofluoromethane,
difluoromethane,
difluoroiodomethane, disilanomethane, fluoromethane, iodomethane,
iodotrifluoromethane,
nitrotrifluoromethane, nitrosotrifluoromethane, tetrafluoromethane,
trichlorofluoromethane,
trifluoromethane, 2-methylbutane, methyl ether, methyl isopropyl ether,
methyllactate,
methylnitrite, methylsulfide, methyl vinyl ether, neon, neopentane, nitrogen
(N2), nitrous
oxide, 1,2,3-nonadecane-tricarboxylic acid-2-hydroxytrimethylester, 1-nonene-3-
yne, oxygen
(Oz), 1,4-pentadiene, n-pentane, perfluoropentane, 4-amino-4-methylpentan-2-
one, 1-pentene,
2-pentene (cis), 2-pentene (trans), 3-bromopent-1-ene, perfluoropent-1-ene,
tetrachlorophthalic acid, 2,3,6-trimethylpiperidine, propane, 1,1,1,2,2,3-
hexafluoropropane,
1,2-epoxypropane, 2,2-difluoropropane, 2-aminopropane, 2-chloropropane,
heptafluoro-1-
nitropropane, heptafluoro-1-nitrosopropane, perfluoropropane, propene,
hexafluoropropane,
1,1,1,2,3,3-hexafluoro-2,3 dichloropropane, 1-chloropropane, chloropropane
(trans), 2-
chloropropane, 3-fluoropropane, propyne, 3,3,3-trifluoropropyne, 3-
fluorostyrene, sulfur
hexafluoride, sulfur (di)-decafluoride (SZFIO), 2,4-diaminotoluene,
trifluoroacetonitrile,
trifluoromethyl peroxide, trifluoromethyl sulfide, tungsten hexafluoride,
vinyl acetylene,
2o vinyl ether, and xenon.
III. UTILITY:
The formulations of the invention are used to treat a mammalian individual,
generally a human patient, suffering from a condition, disease or disorder
that is responsive to
systemic administration of a particular drug. The formulations may be
administered orally,
parenterally, topically, transdermally, rectally, vaginally, by inhalation,
intra-ocularly, in an
implanted reservoir (i.e., in a sustained release depot for subcutaneous or
intramuscular
administration), or as a packing material for wounds and fractures. The term
"parenteral" as
used herein is intended to include subcutaneous, intravenous, intramuscular,
intra-arterial,
intrathecal and intraperitoneal injection, and the formulation may be injected
as either a bolus
or an infusion. Since the invention substantially increases the systemic
bioavailability of a
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drug having low aqueous solubility, dosage can be significantly reduced
relative to that used
in conjunction with conventional pharmaceutical compositions containing the
same active
agent. Analogously, a conventional dosage--or an increased dosage--can be used
to provide
substantially higher blood levels of a drug than previously obtained using
conventional
formulations. For paclitaxel, by way of example, a bolus dosage of at least
3.5 mg/kg and
even 7.0 mg/kg can be administered using the present formulations, while with
continuous
infusion, a dosage of at least 140 mg/kg can be administered and with using a
stabilized
formulation, a dosage of up to 200 mg/kg can be administered.
The formulations of the invention may be also be used so that a drug is
targeted to
l0 a particular cell type or tissue. In this embodiment, a targeting agent is
employed that is
covalently coupled to the hydrophilic polymer such as through a terminal
hydroxyl group of
polyethylene glycol. Suitable targeting agents are those that are generally
used with
liposomal formulations, e.g., peptides, peptide fragments, antibodies or
peptidomimetics,
although other ligands such as saccharides and folates can also be used.
Preferred targeting
agents are integrins such as the (33 integrins ("cytoadhesins"), with cyclized
oligopeptides
containing the Arg-Gly-Asp (RGD) motif particularly preferred.
The present formulations are also useful as packing materials for wounds and
fractures, and as coating materials for endoprostheses such as stems, grafts
and joint
prostheses. It is known that restenosis (narrowing of the blood vessels) may
occur after
angioplasty, placement of a stmt, and/or other coronary intervention
procedures, as a result of
fibroblast proliferation and smooth muscle hypertrophy. Thus, the formulations
of the
invention may be used as coating materials for endoprostheses to provide local
drug delivery
following coronary intervention.
It is to be understood that while the invention has been described in
conjunction
with the preferred specific embodiments thereof, that the foregoing
description as well as the
examples that follow are intended to illustrate and not limit the scope of the
invention. Other
aspects, advantages and modifications within the scope of the invention will
be apparent to
those skilled in the art to which the invention pertains.
All patents, patent applications, and publications mentioned herein are hereby
incorporated by reference in their entirety.
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EXPERIMENTAL
The following examples are put forth so as to provide those of ordinary skill
in the
art with a complete disclosure and description of how to prepare and use the
formulations
disclosed and claimed herein. Efforts have been made to ensure accuracy with
respect to
numbers (e.g., amounts, temperature, etc.) but some errors and deviations
should be
accounted for. Unless indicated otherwise, parts are parts by weight,
temperature is in °C and
pressure is at or near atmospheric.
Also, in these examples and throughout this specification, the abbreviations
employed have their generally accepted meanings, as follows:
g = gram
ml = milliliter
mmol = millimole
nm = nanometer
~,1= microliter
~m = micron
EXAMPLE 1
The solubility of various drugs was evaluated in both polyethylene glycol and
water. The drugs tested and their source, catalog number and lot number are
given in Table
1.
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TABLE 1
COMPOUND MANUFACTURER CATALOG # LOT #
trans- retinol (vitamin Sigma R-7632 69H5019
A)
oc-tocopherol (vitamin E) Sigma T-3251 6981224
dexamethasone Sigma D 1756 88H1266
dexamethasone acetate Sigma D 1881 115H1008
vancomycin . Sigma V 2002 118H0955
amikacin sulfate Sigma A 2324 37H602
etoposide Sigma E 1383 68H1001
quinine Sigma Q-1878 58H2505
verapamil hydrochloride Sigma V-4629 56H0925
5-fluorouracil Sigma F-6627 39H0901
poly-L-trytophan (MW 3900-4500)Sigma P-4647 84H5511
poly-L-trytophan (MW 9000-11500)Sigma P-0644 115F50011
cyclosporin A Calbiochem 239835 B24596
amphotericin B Sigma A 4888 68H4111
tamoxifen Sigma T-5648 28H1033
cetrorelix acetate Asta Ber x 544
A known amount of each compound (determined using a Metler Analytical Balance
Model
AG245) was placed in a 10 ml scintillation vial. Two or three ml of PEG 400
(Kodak,
Catalog # 1369941, Lot # 1156703112) were added to each vial using a
micropipettor (a
Gilson Pipetman, 1000 ~1). Each vial was gently swirled to determine whether
or not the
drug had dissolved. If dissolution was less than complete, the vial was
vortexed at a low
speed. If vortexing failed to effect dissolution, the vial was sonicated in a
water bath until the
l0 drug had dissolved. The time to dissolution was noted. Following
dissolution, the sample
was transferred to a cuvette and the absorption monitored using a LJV6Vis
spectrophotometer
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(Model Lambda 3B, Perkin Elmer). All samples with the exception of traps-
retinol and a-
tocopherol were monitored for absorption in the 200-400 nm range.
The masses of each drug and the wavelength at which maximum absorption was
measured are set forth in Table 2.
TABLE 2
COMPOUND MASS USED (MG) a. OF MAXIMUM
ABSORBANCE (NM)
traps- retinol (vitamin 4.80 360
A)
a-tocopherol (vitamin E) 11.25 280
dexamethasone 6.75 250
dexamethasone acetate 5.88 285
vancomycin 3.97 ~ N/A
amikacin sulfate 9.82 N/A
etoposide 5.85 232/280
quinine 6.17 280
verapamil hydrochloride 6.51 280
5-fluorouracil 5.68 280
poly-L-trytophan (MW 3900-4500)5.10 280
poly-L-trytophan (MW 9000-11500)4.40 280
cyclosporin A 5.00 280
amphotericin B 4.30 N/A
tamoxifen 7.95 280
cetrorelix acetate 5.48 N/A
The relative solubility in PEG 400 and water is indicated in Table 3, below.
In the table, "+"
l0 ' refers to a solubility in PEG 400 that is at least 1.5 times the
solubility in water, "++" refers to
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a solubility in PEG 400 that is at least ten times the solubility in water,
and "+++" refers to a
solubility in PEG 400 that is at least fifty times the solubility in water.
'TABLE 3
COMPOUND SOLUBILITY IN PEG COMPARED TO WATER
trans- retinol (vitamin A) ++
alpha tocopherol (vitamin ++
E)
dexamethasone +++
dexamethasone acetate ~ +++
vancomycin ---
amikacin sulfate ---
etoposide +
quinine ++
verapamil hydrochloride ---
5-fluorouracil +++
poly-L-trytophan (MW 3900-4500)+
poly-L-trytophan (MW 9000-11500)+++
cyclosporin A ++
amphotericin B +
tamoxifen +++
cetrorelix acetate +
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EXAMPLE 2
FORMULATION METHODOLOGY
100 mg of a PEG component (either a PEGylated phospholipid or branched PEG,
40 kD, Shearwater Polymers, Huntsville, AL) is dissolved in 10 ml t-butanol.
The solution is
heated over a 45-60°C hot water bath and subjected to sonication until
the solution clarifies.
The optional next component (free phospholipid or dispersing agent) is added
in an amount as
in Table 1 and sonication is applied again until the mixture clarifies. 10 mg
of paclitaxel
(Hawser Laboratories or Natural Pharmaceuticals) is then added, followed by
heating and
sonication as above. The mixture is flash frozen over liquid nitrogen and
lyophilized on an
l0 ice-water bath for 4 hours followed by room temperature overnight to remove
t-butanol. The
final lyophilisate may be optionally microfluidized at about 15,000 psi and
then lyophilized
again for storage. The dry powder so obtained may be rehydrated in 1.0 ml
saline.
EXAMPLE 3
15 ALTERNATIVE FORMULATION USING STAR-PEG (STAR-PEG 631, MW=154 KD, 1 S ARMS,
SHEARWATER POLYMERS, HUNTSVILLE, AL)
The protocol of Example 2 is duplicated using 100 mg of the star-PEG or
branched
PEG described above: No additional phospholipid is added in this case.
2o EXAMPLE 4
ETHANOL INJECTION IN WATER
1.6664 g of branched polyethylene glycol, MW 20,000, 4 branches (bPEG) is
first
dissolved in 20 ml of ethanol in a round bottom flask at approximately
35°C in a water bath
with a rotor stirrer for approximately 15 min or until the bPEG has dissolved.
This results in
25 a clear solution to which the 332.7 mg of paclitaxel is added and dissolved
under the same
conditions. This is then taken up into a syringe and injected into a cold
stirring solution of
638.6 mg of albumin and 1.6428 g of sucrose. This results in the instant
formation of
nanoparticles (less than 1 ~.m). The suspension is immediately lyophilized.
Sucrose (20%), human serum albumin (2%) with sucrose (5%), sodium salt of
30 taurocholic acid (5%) with sucrose (20%), and lipids (1,2 dioctanol-sn-
glycerol-3-
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phosphocholine, 1,2 dilauroyl-sn-glycerol-3-phosphocholine and 1,2 dipalmitoyl-
sn-glycerol-
3-phosphocholine) (2%) are some of the stabilizing agents that have been
tested using this
method.
EXAMPLE 5
ETHANOL IN METHYL T-BUTYL ETHER
Branched polyethylene glycol (1.6590 g) and paclitaxel (332.0 mg) were
dissolved
in ethanol as described in Example 4. The solution was then transferred to a
dripping funnel
and allowed to drip slowly into cold and stirring methyl t-butyl ether. This
resulted in the
1o instant formation of nanoparticles (less than 1 Vim).
EXAMPLE 6
CHOLIC SALTS AS STABILIZING AGENTS
a) 1.6579 g of branched polyethylene glycol, MW 20,000, 4 branches (bPEG) vvas
first
dissolved in 30 mL of t-butanol in a round bottom flask at approximately
55°C in a
water bath with a rotor stirrer for approximately 15 min or until the bPEG had
dissolved. This resulted in a clear solution to which the 329.5-mg of
paclitaxel was
added and dissolved under the same conditions. This solution was then freeze-
dried
and lyophilized. The white flaky powder that resulted was hydrated with a
solution of
631.3mg sodium taurocholate and 1.6360 g of sucrose. The suspension was
microfluidized for 15 min using the Model 1 l OS, Microfluidics International
Corp.,
Newton MA. This resulted in an almost clear (translucent) solution; the
particle size
was less than 50 nm.
b) The procedure described in part a) was applied using three other cholic
acid salts
(sodium cholate, sodium glycholate and sodium deoxycholate). The ratio of
polyethylene glycol to paclitaxel was kept the same (5:1) and the amounts of
the
cholic acid salts and sucrose used was 2% and 5% respectively. This is similar
to that
used in Example 4. The particle size after microfluidizing was less than 1
~,m.
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EXAMPLE 7
TWEEN~ HO (POLYOXYETHYLENE 2O SORBITAN MONOOLEATE)
Branched polyethylene glycol (1.6429 g) and paclitaxel (330.8 mg) were
prepared
as described in Example 4 above. After lyophilization the dry material was
hydrated with a
1% solution of Tween~ 80 (356.8 mg) and microfluidized. This resulted in
particles less than
1 ~,m.
EXAMPLE 8
IN VIVO EVALUATION -- MAXIMUM TOLERATED DOSE (MTD) STUDIES
(a) BMS TAXOL~:
Taxol° (Bristol Myers Squibb) was acquired from the University of
Arizona
Animal Care Center at 6 mg/ml. This solution was further diluted in saline to
reach various
concentrations needed in the experiment. Mice were injected with 500 ~,1 in a
slow bolus
over 1.5 minutes.
The dosages for the experimental groups were as follows: 40 mg/kg; 30 mg/kg;
20
mg/kg; 10 mg/kg; and control saline. These animals were injected once a day
for 8 days.
The 40 mg/kg group died immediately after receiving the first injection. The
30 mg/kg and
mg/kg groups survived a single injection on day one but all of the animals in
these groups
died after receiving the injection on day two.
The 10 mg/kg groups all survived the entire 8 days of dosing and showed a
delayed
(1-2 minutes) severe response, i.e., they fell on their side, could not move,
and had very
labored breathing and occasional gasping for 5-10 minutes after which they
slowly recovered.
The animals were weighed daily until the final day. On the final day of dosing
the animals
z5 were sacrificed by COZ asphyxiation. A blood sample was collected by heart
puncture and
split for cell count and liver function assays. The heart, liver, left kidney
and spleen were
collected and weighed from each animal. These tissues were placed in formalin
for
histological analysis.
The animal weights and tissue weights were analyzed statistically. There was
no
3o significant effect of Taxol~ at 10 mg/kg/day on body weight when compared
to the saline
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control. There was a significant increase in spleen weight in the 10 mg/kg/day
Taxol~ group.
The difference was between a mean spleen weight of 0.074 g for the saline
group to 0.094 g
for the 10 mg/kg/day Taxol~ group with a p value < 0.015.
(b) Branched PEG/Paclitaxel:
A formulation of the invention was prepared with 5.3126 g branched PEG 40,000
("PEG
40K"), 8 branches (Shearwater Polymers), 1.0022 g paclitaxel, 100 ml t-butanol
and 2.6919 g
sucrose.
The following procedure was carried out on each of the two flasks
independently:
l0 The PEG 40K was dissolved in 100 ml t-butanol with heating and sonication
at
approximately 55-60°C. The solution was heated and sonicated until a
clear solution resulted.
The paclitaxel was added and the mixture was sonicated at the aforementioned
temperature
until a clear solution resulted (approximately 20 minutes). Each flask was
then lyophilized
overnight.
Each lyophilisate was then hydrated with 100 ml saline containing the quantity
of
sucrose indicated above. The mixtures were then heated in a water bath to
52°C. The
contents of the two flasks were combined. Total theoretical amounts of
material combined:
10.6162 g branched PEG 40K; 2.0271 g paclitaxel; and 5.3847 g sucrose. This
combined
batch was then microfluidized for 15 minutes at 14,000 psi.
Approximately 160 ml of sample was collected. Two approximately 80 ml batches
were placed in a round bottom flask and lyophilized over 72 hours.
Paclitaxel content in the lyophilized material (dry powder) was determined by
HPLC analysis as 0.078 mg/mg. Daily stock vials were then prepared each
containing 768.5
mg of the aforementioned lyophilized formulation. This was dissolved in a
total volume of 6
rnl of water, making a 10 mg/ml paclitaxel solution.
Seven groups of 5 mice were given increasing dosages of diluted paclitaxel
solution. The groups received either saline, BMS Taxol~ 10 mg/kg, or the
branched PEG
40K/paclitaxel solution as prepared above. Dosages were 20 mg/kg, 40 mg/kg, 60
mg/kg,
100 mg/kg and 140 mg/kg paclitaxel. The paclitaxel formulations (BMS Taxol~ or
PEG
40K/paclitaxel) were further diluted in saline to reach concentrations needed
in the
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experiment. Mice were injected with 500 ~,l in a slow bolus over 1.5 minutes.
The mice
were injected once a day for 8 days, a Monday through Thursday schedule.
The saline group had no response to injection on day 1 or 2. All mice in the
BMS
Taxol° group showed a delayed (I-2 minutes) severe response, i.e., they
fell on their side,
could not move, and had very labored breathing and occasional gasping for 5-10
minutes after
which they slowly recovered on both day l and 2. All mice receiving 20, 40,
and 60 mg/kg
paclitaxel using the branched PEG 40K/paclitaxel formulation prepared above
showed no
response to the injections on day 1 or 2. 100 mg/kg paclitaxel resulted in
slight distress, i.e.,
movements were slowed for 1-2 minutes but no breathing problems were observed
on day 1
or day 2. The 140 mg/kg group showed moderate distress immediately after
injection, i.e.,
they did not move and their breathing was slightly labored. Within this group
one mouse
died on day l, one-hour post injection, and one mouse died two hours post
injection. On day
2 one mouse died immediately after injection.
In summary, over three days, the average dose tolerance was found to be at
least
1 OX in terms of toxicity tolerance for the branched PEG/paclitaxel
formulation of the
invention compared to Taxol°. By the end of two weeks all of the
animals receiving 100 mg
and 140 mg/kg/day of the branched PEG/paclitaxel formulation had died. All of
the animals
receiving 60 mg/kg/day and below survived, however. Compared to Taxol°,
the results of
this i~ vivo study support a ten-fold improvement in acute safety for the
branched
PEG/paclitaxel formulation of the invention and a six-fold improvement for
sitbacute
administration.
EXAMPLE 9
PREPARATION OF PACLITAXEL "MICROBUBBLES"
The formulation described in Example 2 is suspended in a 3 ml glass vial
volume
in a 1.6 ml saline solution, paclitaxel concentration 10 mg per~ml, and the
vial is sealed with a
headspace of perfluorobutane gas. The vial is agitated at 4,500 rpm using a
Capmix (ESPE,
Morris, IL) dental amalgamator for 60 seconds. This results in microbubbles
bearing
paclitaxel.
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EXAMPLE 10
TREATMENT USING THE PACLITAXEL MICROBUBBLES
A patient with prostate cancer receives an IV injection of the microbubbles
described in Example 5, dose of paclitaxel = 30 mg. An endorectal ultrasound
probe is placed
in the patient's rectum and ultrasound, l .OMHz, 1.0 Watt/cm2, is applied
across the patient's
rectum for 30 minutes. The ultrasound probe is adjusted so that the energy is
concentrated on
the prostate gland. Ultrasound energy is adjusted so that the microbubbles are
ruptured as
they circulate within the patient's prostate gland. Increased delivery of
paclitaxel to the
cancer within the prostate is achieved. The patient is then treated with
radiation therapy with
l0 a fraction of 300 Rads as part of a multi-dose treatment regiment. The
paclitaxel acts as a
radiation sensitizer and the ultrasound-mediated delivery enhances cellular
uptake of the drug
by the cancer.
EXAMPLE 11
~ TREATMENT OF A BREAST CANCER PATIENT
A patient with breast cancer may develop an allergic reaction to Taxol~ due to
the
surfactants in the formulation. Still the patient will need the drug to
control her disease and
require pre-medication to avoid allergic reaction as well as sustained, slow
infusion of the
drug. The new formulation described in Example 3 is made available. Because of
the absence
of surfactants, the paclitaxel can be administered as an IV bolus injection
without
premedication.
EXAMPLE 12
COATED GRAFTS
z5 Paclitaxel and branched PEG are dissolved in t-butanol at a drug
concentration of
10 mg per ml and a weight ratio of branched PEG to the drug of approximately
5:1. This
material is atomized and sprayed onto a Gortex~ graft for coronary bypass. The
graft is dried
as the drug/branched PEG adsorbs on the surface of the graft. The resultant
graft is implanted
in a patient who needs a coronary artery bypass graft. Compared to grafts
without this
pretreatment there is a much higher rate of graft restenosis. The PEG adsorbed
on the surface
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retains the paclitaxel as a depot, releasing it over the proper time course to
prevent
fibrointimal hyperplasia.
EXAMPLE 13
SUSTAINED RELEASE DEPOT OF PACLITAXEL
Paclitaxel is dissolved in t-butanol at a concentration of 20 mg per ml with
100 mg
per ml of branched PEG. The material is lyophilized and reconstituted at a
concentration of
25 mg per ml paclitaxel in physiological buffered saline (PBS). The material
appears as a
viscous suspension of particles up to about an average size of 100 to 200
microns. This
l0 material is applied as a paste to the peritoneal surfaces of a patient with
ovarian cancer
following laparotomy and debulking for recurrent tumor. The formulation acts
as a sustained
release depot within the peritoneal cavity helping to control the patient's
cancer.
EXAMPLE 14
SUSTAINED RELEASE DEPOT DELIVERY OF A PEPTIDE
The peptide leuprolide acetate is dissolved in t-butanol at a concentration of
20 mg
per ml with 100 mg per ml of branched PEG. This is reconstituted with PBS at a
drug
concentration of 50 mg per ml. Two cc of this material is injected
subcutaneously into a
patient with prostate cancer. The sustained release of leuprolide acts to
decrease hormonal
stimulation and help control his cancer.
EXAMPLE 15
DERIVATIZATION OF POLYETHYLENE GLYCOL WITH PHOSPHATE MOIETIES
In a 1 liter round bottom flask is added 5 grams (125 micromoles, 1 equiv.) of
~-armed,
branched polyethylene glycol (co or terminally hydroxylated PEG, Shearwater
Polymers,
Huntsville Alabama) and 17.4 microliters of triethylamine (131.5 micromoles,
1.05 equiv.,
Aldrich, Milwaukee, Wisc.) in 250 ml dimethylformamide (DMF, Mallinckrodt, St.
Louis,
Mo.). The solution was cooled to 0 ° C in an ice bath. To this solution
was added by
dropwise addition, 20.2 mg (131.5 micromoles, 1.05 equiv.) of phosphorous
oxychloride
(Mallinckrodt, St. Louis, Mo.) in 5 ml of DMF. The mixture was allowed to
equilibrate to
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room temperature followed by stirring for an additional 8 hours. The solution
was then
quenched with 10 ml of water and the pH adjusted to neutrality.
The reaction mixture was then concentrated in vacuo followed by
resuspension/dissolution in 100 ml of water. The product was dialyzed in a I
000 Molecular
weight cutoff (MWCO) dialysis bag against deionized water. The solution from
the bag was
recovered, frozen, and lyophilized; yielding a white product containing mono-
phosphorylated
branched PEG.
If desired, the reaction may be repeated in the identical manner using twice,
three
times, four times, five times, etc., the stoichiometric amount of phosphorous
oxychloride
(POCl3) to yield di-, tri-, tetra-, penta-, hexa-, hepta-, and per-
phosphorylated branched PEGs.
Note that the same procedure can be applied to any terminally hydroxylated
branched PEG.
EXAMPLE 16
CATHETER COATING
I5 A balloon dilatation catheter (Boston Scientific, Quincy, MA, model no. 13-
188) is
coated with a formulation of the invention, as follows: Branched PEG,
MW=40,000
(Shearwater Polymers) is dissolved with paclitaxel (Natural Pharmaceuticals,
Cambridge,
MA) in t-butanol at a PEG concentration of 50 mg per ml and a paclitaxel
concentration of 10
mg per ml. This material is atomized and deposited on the surfaces of the stmt
within a
drying chamber. The balloon catheter is dried. The paclitaxel formulation
appears as a white
powder material coating the surfaces of the balloon. The catheter is then used
for an
angioplasty. The balloon is inflated at the site of vessel narrowing. The
paclitaxel-containing
coating impregnates the vessel wall as the balloon is inflated under high
pressure. The local
effects of the paclitaxel diminish fibroblast proliferation.
EXAMPLE 17
STENT COATING
A Wallstent~ (Boston Scientific, Quincy, MA, model no. 42054) metallic stmt is
coated at described above. The coated stmt is covered by a hazy white
clathrate. The stmt is
advanced into the iliac artery of a patient with stenotic narrowing due to
advanced
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atherosclerosis. The stent is positioned and deployed at the site of stenosis
using a Unistep
Plus Delivery System (Boston Scientific). The paclitaxel aids in prevention of
restenosis and
the formulation releases the drug over a delayed period of time for maximal
therapeutic
benefit.
EXAMPLE 18
ALTERNATIVE STENT COATING
Another Wallstent~ (same model) is treated with a gold plating process using
electrochemistry to deposit a thin film of gold on the surface of the stmt. A
different
l0 branched PEG is prepared by substituting the terminal hydroxyl moieties of
the PEG with
thiol groups. The branched PEG is mixed with paclitaxel as described above in
an organic
solvent. The mixture is atomized and sprayed on the stmt as described above in
Example 12.
Compared to the coating used in Example 12, the thiolated PEG used in this
coating
provides for a slower release rate, delivering the paclitaxel over a longer
period of time.
EXAMPLE 19
The procedure of Example 14 is repeated with a poly(alkylene oxide) comprising
50% ethylene oxide monomers and 50% propylene oxide monomers. The
incorporation of
propylene oxide groups prolongs the release of the paclitaxel, providing
therapy for a longer
period of time.
EXAMPLE 20
PREPARATION OF NANOPARTICLES WITH BRANCHED PEG STABILIZED WITH ALBUMIN
Samples were prepared according to the procedure of Example I except that
after
the first lyophilization the samples were hydrated with solutions of different
concentrations of
human serum albumin. Microfluidizing then followed for 40 minutes. This was
done to
ensure that the suspension was homogenous and that the particle size was
consistent. 1:5
bPEG 20I~ 4a was used as the determinant on how the variation of albumin
affects the
formulation.
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TABLE 4
AMOUNT OF ALBUMIN AS W/W% PARTICLE DIAMETER
0.5 % 0.07-0.3 E.~,IvI
1% 0.03-0.2~NI
2% . 0.02-0.2 ~,NI
EXAMPLE 21
PREPARATION OF NANOPARTICLES WITH LINEAR PEG STABILIZED IN ALBUMIN
The procedure of Example 2 was used. The ratio of paclitaxel to PEG was
maintained at 1:5 for PEG 35K, 20K & l OK. The difference between linear and
branched
PEG was not apparent without the use of human serum albumin. When the
different
to formulations were suspended in an albumin solution, the difference in
particle size was
apparent and in the linear PEG the toxicity was greater.
TABLE 5
FORMULATIONS MADE USING LINEARSIZE
PEG W/HSA (Z-5%~
PEG 1 OK > 1 ~,M
PEG 20K 0.05-0.7~1vI
PEG 35K 0.05-0.7~IvI
BRANCHED PEG 20K & 40K 0.02-0.2~IvI
Is
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EXAMPLE 22
TABLE OF STABILIZING AGENTS AND RESULTS
The formulation used was of 1:S paclitaxel to bPEG 20I~ 4a, prepaxed in the
same
way as described in Example 1. The stabilizing agents tabulated below were
added to the
formulation in the same manner as described for albumin in Example 16.
T~hlo ~
COATING AGENT RESULT (PARTICLE DIAMETER)
HUMAN SERUM ALBUMIN 2O-300NM
POLYVINYL ALCOHOL SO-SOONM
2O% SUCROSE <1 ~I,M BUT RE-AGGREGATE AFTER
TIME
POLYVINYL PYRROLIDONE 2OONM-1 ~,M
METHYLCELLULOSE <1~LM
CARBOXYMETHYL CELLULOSE <1 ~,M
ALGINIC ACID <1 ~,M
PEG 400 <1 ~,M
DEXTRIN <1 ~,M
to EXAMPLE 23
NANOPARTICLES USING PHOSPHORYLATED PEG
The procedure of Example I was repeated (ratio of 1:S paclitaxel: PEG) using a
2%
albumin solution in a phosphate buffer (pH7.24) to hydrate the dry complex of
the PEG and
paclitaxel. This suspension was microfluidized for 4S minutes. No crystals
were observed.
Laxge particles resulted which appeared to aggregate.
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EXAMPLE 24
EXAMPLES USING POLOXAMERS (PLURONICS) AS A STABILIZER FOR NANOPARTICLES
The procedure and conditions used were as described in Example T with the
exception that Pluronic L64 (25%w/v) was dissolved into the t-butanol after
the PEG. The
paclitaxel was dissolved at a 1:5 ratio with PEG and the solution lyophilized.
The resultant
white, flaky powder was hydrated with an equivalent amount of a saline
solution. The
suspension was then placed in a flask with a rotor stirrer and immersed in a
hot water bath set
at 40 ° to 43 ° C. This was stirred for 45 min. No crystals were
observed, but the suspension
appeared to be more fluid than the PEG/paclitaxel suspension. If this
formulation had been
l0 microfluidized or subjected to conditions where the particle size could
have been altered, this
would have resulted in a stable suspension of smaller particles (<1 ~.m). This
is due to the
surfactant nature of poloxamers.
EXAMPLE 25
THE COUPLING OF AN ACTIVATED PEG TO ALBUMIN
The PEG is initially activated using trichloro-s-triazine (TsT), which is a
symmetrical heterocyclic compound containing three reactive acyl-like
chlorines. PEG
activation is necessary to form derivatives that are amine reactive where
proteins linkages, in
this case albumin, can develop. The activated PEG is slowly added to a
solution of albumin
in a 0.1 M sodium borate buffer at a concentration of 2-10 mg/ml. Note that at
least a five-
fold molar excess of the activated PEG should be present. This reaction takes
1 hr. at 4°C
and the excess PEG can be removed by dialysis or gel filtration using a column
of Sephacryl
S-300 (Hermanson G.T, (1996) "Bioconjugate Techniques" Academic Press, San
Diego).
2s EXAMPLE 26
IN Y IVO MAXIMUM TOLERATED DOSE OF THE PEG/PACLITAXEL FORMULATION
A maximum tolerated dose (MTD) study was designed to test the safety of the
stabilized paclitaxel formulation described in Example 16. These experiments
were carried
out in non-tumor bearing nude mice of between 6 and ~ weeks and an approximate
body
weight of 25 g. The animals were warmed under a heat lamp for 15 minutes and
then placed
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into a mouse restraint. An injection dose was then administered through the
tail vein at a
volume not exceeding 20 ml/kg. The maximum tolerated dose was defined to be
the highest
dose where the animals did not lose more than 10% body weight for two weeks
after a single
dose of drug. The mice were caged in groups of 5 for the holding period and
were provided
with food and water ad libitum. Animals completing the study were euthanized
by COZ
asphyxiation after the final weighing. A control study was carried out using
Bristol-Myers'
Squibb's TAXOL~ in this strain of mice.
A group of three nude mice were tested, each receiving different doses of the
formulation: 150, 200, and 250 mg/kg. Their body weight was monitored
following the
l0 injection and results are present in the figure below. In all tested mice,
body weight change
was approximately proportional to the dose given. The body weight decreased
less than 10%
of the initial weight, except for the mouse that received 250 mglkg, (that
mouse died on day
4). In the control study, the MTD of BMS TAXOL~ was determined as 20 mg/kg.
The
single dose MTD for the formulation is 200 mglkg and exceeds ten times that of
BMS
TAXOL°. (See Table 7 below.)
TABLE 7
FORMULATION MAXIMUM TOLERATED DOSE
MGlKG
PEG/Paclitaxel Complex 200
TAXOL~ 20
EXAMPLE 27
IN Y IVO EFFICACY OF THE PEG/PACLITAXEL FORMULATION
Efficacy experiments were carried out in athymic, nude mice of same age,
between
6 and 8 weeks, with an approximate body weight of 25 g. The mice were
implanted with two
LS 180 human colon adenocarcinoma tumors per animal, with one tumor being
implanted in
each flank. Following tumor cell implantation, the animals were held until
tumors were
measurable. They then received the test article at doses that were based on
MTD. The
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animals were warmed under a heat lamp for 15 minutes and then placed into a
mouse
restraint. An injection dose was then administered through the tail vein at a
volume not
exceeding 20 ml/kg. Animals were provided with food and water ad libitum.
Tumors were
measured using a digital caliper. The weight of the tumor was calculated using
the
approximate equation of weight = (length x width2)/2, where length and width
is expressed in
mm and weight in mg. Animals were euthanized by COZ administration when the
estimated
tumor weight exceeded 1 g.
The efficacy of the formulation was monitored following single administration
at
2/3rd of MTD (maximum tolerated dose, 135 mg/kg) and full MTD (200 mg/kg), and
compared to the efficacy of BMS TAXOL° administered at its MTD, i.e.,
20 mg/kg.
Efficacy at 2/3ras MTD. A significant tumor growth inhibition was seen after a
single
administration of the product of Example 16, i.e., at 135 mg/kg. Tumor
inhibition was
determined by calculating the minimum value of the T/C ratio defined as (mean
tumor weight
in treated group/mean tumor weight in control group) x 100%. According to NCl
definitions,
T/C ratios less than 42% indicate an active agent and T/C ratios below 10%
indicate a highly
active agent. The result of the 2/3'as MTD testing is represented in Figure 7.
A minimum T/C
ratio of 8.5% was observed on day 10. Therefore, we conclude that the
formulation of PEG
stabilized paclitaxel at 2/3'as MTD is a highly active agent.
z0
Efficacy at Full MTD. Efficacy experiments were then carried out in the same
model at full
MTD, i.e., 200 mg/kg. Following the single administration, all tumors
regressed throughout
the duration of experiment. Tumor inhibition as defined above is not
applicable here.
Instead, tumor regression was calculated using the formula (1 - mean tumor
weight/initial
~5 mean tumor weight) x 100%. A maximum tumor regression of 82% was seen on
day 8. The
body weight of the animals undergoing efficacy experiment was monitored and is
shown in
Figure 8. The maximum weight loss occurred eight days after injection. The
animals fully
recovered the initial weight within two weeks after injection. The same
pattern was observed
following the first and second injections; this suggests that the toxic
effects are reversible.
