Note: Descriptions are shown in the official language in which they were submitted.
CA 02719855 2010-11-02
B&P File No. 10723-332
TITLE: TEMPERATURE SENSITIVE HYDROGEL AND BLOCK COPOLYMERS
FIELD
The present disclosure relates to temperature sensitive hydrogel and
block copolymers, processes for the production thereof, and pharmaceutical
conjugates using the copolymers.
BACKGROUND OF THE DISCLOSURE
Recently, the advance of biomaterials for myocardial tissue engineering
includes in situ polymer gels such as injectable fibrin glue, matrigel,
collegen,
alginate gels and self-assembling peptide. These in situ polymer scaffolds
(injectable extracellular matrix, iECM), by themselves or in combination with
cells
or biological molecules, have proven to more precisely control the myocardial
microenvironment, which enhances cell transplant survival, reduces infarct
expansion, and induces neovasculature formation in ischemic myocardium as
well as the sustained release for delivery of growth factors and cells.
SUMMARY OF THE DISCLOSURE
The present disclosure relates to biodegradable, body temperature-
sensitive hydrogel and block copolymers, which optionally act as a delivery
matrix for the delivery of therapeutic compounds. The disclosure also includes
a
process for the preparation of such hydrogel and block copolymers.
Accordingly, the present disclosure includes a block copolymer comprising
an A block and a B block, wherein the block copolymer has the formula.
A-B;
A-B-A; or
B-A-B;
the A block is a poly(b-valerolactone), poly(s-caprolactone), poly(lactide),
poly(a-
hydroxy acid), poly(glycolide), polyanhydride, polyester, polyorthoester,
polyetherester, polyesteramide, polycarbonate, polycyanoacrylate,
polyurethane,
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CA 02719855 2010-11-02
polyacrylate, or a co-polymer thereof, all of which are optionally
substituted;
wherein the B block is an optionally substituted polyethylene glycol or
optionally
substituted polypropylene glycol;
wherein the A block has a number average molecular weight between 500 and
30,000 and the B block has a number average molecular weight between 500
and 10,000;
wherein the optional substituents are selected from halo, OH, (C1_6)-alkyl and
fluoro-substituted (C1_6)-alkyl; and
wherein the block copolymer forms a hydrogel at a temperature of above about
30 C.
In another embodiment, the A block has a number average molecular
weight between 500 and 10,000, or 1,000 and 5,000, or 1,000 and 3,000, or
about 1,750. In another embodiment, the B block has a number average
molecular weight of between 1,000 and 8,000, optionally between 1,500 and
8,000, or 1,500 and 5,000.
In another embodiment, the block copolymer has the formula: A-B-A.
In a further embodiment, the A block comprises a poly(8-valerolactone),
poly(s-caprolactone), poly(lactide), poly(a-hydroxy acid), poly(glycolide) or
a
copolymer thereof, optionally poly(8-valerolactone) or poly(E-caprolactone) or
copolymers thereof, or poly(8-valerolactone).
In an embodiment of the disclosure, the B block comprises polyethylene
glycol.
In another embodiment of the disclosure, the block copolymer comprises
A B O A
O O 1' H
H O O
W x y
O
wherein the integers w, x and y represent the number of repeating units to
obtain
a block copolymer wherein the A block has a number average molecular weight
between 500 and 30,000 and the B block has a number average molecular
weight between 500 and 10,000.
The present disclosure also includes a process for the preparation of a
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block copolymer comprising at least one A block and at least one B block
having
the formula A-B, A-B-A, B-A-B, the process comprising reacting
(i) an optionally substituted polyethylene glycol or polypropylene oxide
comprising the B block;
with,
(ii) monomeric units of the A block, the monomeric units comprising 8-
valerolactone, E-caprolactone, lactide, an a-hydroxy acid, glycolic acid, an
anhydride, an ester, an orthoester, an etherester, an esteramide, a
carbonate, a cyanoacrylate, a urethane, an acrylate, or a mixture thereof,
all of which are optionally substituted, wherein the optional substituents
are selected from halo, OH, P_6)-alkyl and fluoro-substituted (C1_6)-alkyl;
in the presence of an acid catalyst having a pKa of less than -12, and wherein
the process is optionally performed at a temperature between -10 C and 35 C.
In another embodiment, the number average molecular weight of the A
block is controlled by the molar ratio of the monomeric units of the A block
to the
B block during the process.
In another embodiment of the process, the A block has a number average
molecular weight between 500 and 30,000 and the B block has a number
average molecular weight between 500 and 10,000. In another embodiment,
the A block has a number average molecular weight between 500 and 10,000, or
1,000 and 5,000, or 1,000 and 3,000, or about 1,750. In another embodiment,
the B block has a number average molecular weight of between 1,000 and 8,000,
optionally between 1,500 and 8,000, or 1,500 and 5,000.
In another embodiment of the process, the block copolymer has the
formula: A-B-A.
In another embodiment of the process, the monomeric units of the A block
comprise b-valerolactone, E-caprolactone, lactide, an a-hydroxy acid,
glycolide or
a copolymer thereof, optionally S-valerolactone or s-caprolactone or a
copolymer
thereof, or 8-valerolactone.
In an embodiment of the process, the B block comprises polyethylene
glycol.
In a further embodiment of the process, the acid catalyst is a sulfonic acid,
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optionally trifluoromethanesulfonic acid or fluorosulfonic acid. In an
embodiment,
the sulfonic acid is trifluoromethanesulfonic acid.
In another embodiment of the disclosure, there is also included a block
copolymer comprising at least one A block and at least one B block, wherein
the
block copolymer has the formula: A-B; A-B-A; or B-A-B; in which the block
copolymer is produced by the process of the disclosure. In another embodiment,
the block copolymer produced by the process comprises
A B O A
H O O H
O O
W x y
O
wherein the integers w, x and y represent the number of repeating units to
obtain
a block copolymer wherein the A block has a number average molecular weight
between 500 and 30,000 and the B block has a number average molecular
weight between 500 and 10,000.
The present disclosure also includes a pharmaceutical conjugate
comprising a block copolymer as defined in the disclosure and a therapeutic
compound. In another embodiment, the therapeutic compound is a biologic,
optionally stem cell factor (SCF) or vascular endothelial growth factor
(VEGF).
The present disclosure also includes a method for the treatment of cardiac
abnormality and/or vascular abnormality in a patient in need thereof
comprising
administering a therapeutically effective amount of a pharmaceutical conjugate
as defined in the disclosure to the site of the cardiovascular defect. In an
embodiment, the cardiac abnormality is myocardial infarction and the vascular
abnormality is a vascular aneurysm.
Other features and advantages of the present disclosure will become
apparent from the following detailed description. It should be understood,
however, that the detailed description and the specific examples while
indicating
preferred embodiments of the disclosure are given by way of illustration only,
since various changes and modifications within the spirit and scope of the
disclosure will become apparent to those skilled in the art from this detailed
description.
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BRIEF DESCRIPTION OF THE DRAWINGS
The invention will now be described in greater detail with reference to the
following drawings in which:
Figure 1 is a photomicrograph illustrating the distribution of rat bone marrow
stromal cells in a block copolymer gelled matrix in an embodiment of the
disclosure;
Figure 2 is an 1H NMR spectrum of a block copolymer in an embodiment of the
present disclosure;
Figure 3 illustrates the characterization of a vascular endothelial growth
factor
conjugated to a block copolymer in an embodiment of the disclosure;
Figure 4 is a graph illustrating the gelling temperature versus the
concentration of
a block polymer in an embodiment of the disclosure;
Figure 5 shows a photograph of the gelling of a block copolymer in an
embodiment of the disclosure;
Figure 6 shows photographs heart slices after myocardial infarction;
Figure 7 is a graph illustrating the scar area (%) after myocardial
infarction; and
Figure 8 is a graph illustrating the ejection fraction after myocardial
infarction.
DETAILED DESCRIPTION
(I) DEFINITIONS
The term "C1_6alkyl" as used herein means straight and/or branched chain,
saturated alkyl radicals containing from one to six carbon atoms and includes
methyl, ethyl, propyl, isopropyl, n-butyl, s-butyl, isobutyl, t-butyl and the
like.
The term "halo" as used herein means halogen and includes chloro, fluoro,
bromo and iodo.
The term "fluoro-substituted C1_6alkyl" as used herein that at least one
(including all) of the hydrogens on the referenced group is replaced with
fluorine.
The term "block copolymer" as used herein refers to a polymer built of
linearly linked polymeric units, prepared by the polymerization of a plurality
of
different monomer units in each block. The block copolymer is of the formula A-
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B, A-B-A, or B-A-B in which A and B represent different polymeric blocks built
from repeating monomeric subunits. For example, a polyethylene glycol polymer
represents one example of a B block polymer built from repeating ethylene
glycol
monomeric units, while poly(b-valerolactone) represents one example of an A
block polymer built from repeating S-valerolactone monomeric units (through
cationic lactone ring-opening polymerization), and as such the block copolymer
for this example would be PVL-PEG.
The terms "polyethylene glycol" or "polypropylene glycol" as used herein
means a polymer built from repeating ethylene glycol or propylene glycol
monomeric units, respectively. Polyethylene glycol is formed of repeating
units
comprising
O H
H n 0~
while polypropylene glycol is formed of repeating units comprising
O H
H n 01--,'
The term "hydrogel" as used herein refers to a block copolymer of the
present disclosure and forms, to various degrees, a jelly-like or gelled
product
when heated to a particular temperature, for example body temperature (37 C),
or a temperature higher than 30 C. The block copolymer is accordingly a liquid
at room temperature and soluble in water, but upon reaching a particular
temperature, forms a hydrogel when mixed with water such that water is a
dispersion medium forming the hydrogel.
The term "reverse thermal gelation temperature" as used herein is defined
as meaning the temperature below which a block copolymer of the disclosure is
soluble in water and above which the block copolymer solution forms a semi-
solid, for example, a gel, emulsion, dispersion or suspension.
In understanding the scope of the present disclosure, the term
"comprising" and its derivatives, as used herein, are intended to be open
ended
terms that specify the presence of the stated features, elements, components,
groups, integers, and/or steps, but do not exclude the presence of other
unstated
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features, elements, components, groups, integers and/or steps. The foregoing
also applies to words having similar meanings such as the terms, "including",
"having" and their derivatives. Finally, terms of degree such as
"substantially",
"about" and "approximately" as used herein mean a reasonable amount of
deviation of the modified term such that the end result is not significantly
changed. These terms of degree should be construed as including a deviation of
at least 5% of the modified term if this deviation would not negate the
meaning
of the word it modifies.
(II) BLOCK COPOLYMERS OF THE DISCLOSURE
The present disclosure relates to block copolymers comprising at least
one A block which comprises hydrophobic, biodegradable, and non-swellable
domains and at least one B block which comprises hydrophilic and swellable
soft
domains, and have the formula A-B, A-B-A or B-A-B. In one embodiment, the
block copolymer is a di-block copolymer or a triblock copolymer. In one
embodiment, the block copolymers are thermoplastic and biodegradable
hydrogel copolymers which are liquid and dissolve in water or buffer solution
at
room temperature and form a hydrogel at certain temperatures, optionally at a
temperature above 30 C. The block copolymers of the disclosure are
biodegradable such that the copolymer erodes or degrades in vivo to form
smaller non-toxic compounds.
In another embodiment, the block copolymers are thermo-sensitive
hydrogels comprise amphiphilic block copolymers (comprising hydrophilic and
hydrophobic blocks), wherein the hydrogels exhibit temperature-responsive
gelation/de-gelation in addition to the reverse thermal gelation properties.
In one
embodiment, the block copolymer of the disclosure forms a gel by aggregation
in
a solution when heated to a certain temperature, and also disassociates (de-
aggregates) in solution when removed from that certain temperature
environment. In another embodiment, the block copolymers of the disclosure,
when dissolved in solution, possess reverse thermal gelation properties in
that
the solution forms a gel when heated to a certain temperature, whereas
typically,
polymers usually lose viscosity upon heating.
In one embodiment, the block copolymers are easily administered to
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patients in need of treatment (when the block copolymers are conjugated to a
therapeutic compound) such as syringe or catheter injection, and also are
easily
processed using infusion methods or solvent casting methods as there is no
chemical crosslinkage of the block copolymers. In one embodiment, the gelling
of a solution of a block copolymer of the disclosure is a physical aggregation
which results in the gelling of the solution, and does not involve chemical
changes to the polymer (for example, chemical crosslinking).
In another embodiment, the copolymers are easily degraded into small
and nontoxic molecules by simple intra-molecular ester hydrolysis or enzyme
hydrolysis in order to be easily excreted through the kidney. In another
embodiment, the formation of the hydrogel is reversible by heating. In one
embodiment, a solution of a block copolymer of the present disclosure forms a
hydrogel at a temperature above about 30 C, or about 35 C, or about 37 C. In
another embodiment, a solution of a block copolymer of the disclosure returns
to
a solution (liquid state) when cooled to less than about 37 C, or less than
about
35 C, or less than about 30 C. In another embodiment, a solution of a block
copolymer of the disclosure becomes a solution when heated to a temperature of
greater than about 45 C, or greater than about 40 C, or greater than about 39
C.
Accordingly, in one embodiment the present disclosure includes a block
copolymer comprising at least A block and at least one B block, wherein the
block copolymer has the formula:
A-B;
A-B-A; or
B-A-B;
the A block is a poly(b-valerolactone), poly(E-caprolactone), poly(lactide),
poly(a-
hydroxy acid), poly(glycolide), polyanhydride, polyester, polyorthoester,
polyetherester, polyesteramide, polycarbonate, polycyanoacrylate,
polyurethane,
polyacrylate, or a co-polymer thereof, all of which are optionally
substituted;
wherein the B block is an optionally substituted polyethylene glycol or
optionally
substituted polypropylene glycol;
wherein the optional substituents are selected from halo, OH, (C1_6)-alkyl and
fluoro-substituted (C1.6)-alkyl; and
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wherein the block copolymer forms a hydrogel at a temperature of above about
30 C.
In one embodiment, it will be understood by a person skilled in the art that
the determination of the desired polymer degradation rate, the reverse thermal
gelation temperature etc., is based upon the molecular weight of the A block
polymers.
In another embodiment, to increase the rate at which the a block
copolymer of the disclosure is solubilized in an aqueous solution (such as
water
or a phosphate buffer), the solution is heated to a temperature of greater
than
about 40 C, or 50 C, optionally 60 C, and then cooled to a temperature below
room temperature, or below about 20 C, optionally below 10 C.
In another embodiment, the present disclosure includes a block copolymer
comprising at least A block and at least one B block, wherein the block
copolymer has the formula:
A-B;
A-B-A; or
B-A-B;
the A block is a poly(b-valerolactone), poly(c-caprolactone), poly(lactide),
poly(a-
hydroxy acid), poly(glycolide), polyanhydride, polyester, polyorthoester,
polyetherester, polyesteramide, polycarbonate, polycyanoacrylate,
polyurethane,
polyacrylate, or a co-polymer thereof, all of which are optionally
substituted;
wherein the B block is an optionally substituted polyethylene glycol or
optionally
substituted polypropylene glycol;
wherein the A block has a number average molecular weight between 500 and
30,000 and the B block has a number average molecular weight between 500
and 10,000;
wherein the optional substituents are selected from halo, OH, (C1.6)-alkyl and
fluoro-substituted (C1_6)-alkyl; and
wherein the block copolymer forms a hydrogel at a temperature of above about
30 C.
In another embodiment, the A block has a number average molecular
weight between 500 and 10,000, or 1,000 and 5,000, or 1,000 and 3,000, or
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about 1,750. In another embodiment, the B block has a number average
molecular weight of between 1,000 and 8,000, optionally between 1,500 and
8,000, or 1,500 and 5,000.
In another embodiment, the block copolymer has the formula: A-B-A.
In a further embodiment, the A block comprises a poly(b-valerolactone),
poly(E-caprolactone), poly(lactide), poly(a-hydroxy acid), poly(glycolide) or
a
copolymer thereof, optionally poly(b-valerolactone) or poly(E-caprolactone) or
copolymers thereof, or poly(b-valerolactone). In another embodiment, the A
block
comprises a copolymer of poly(b-valerolactone) and glycolic acid.
In an embodiment of the disclosure, the B block comprises polyethylene
glycol.
In another embodiment, the hydrophilic B block hydrophilic segments may
also contain ionizable groups, if for example, B-A-B type copolymers are used.
In another embodiment of the disclosure, the block copolymer comprises
A B O A
O O H
H O O
W H_
Y
0
wherein the integers w, x and y represent the number of repeating units to
obtain
a block copolymer wherein the A block has a number average molecular weight
between 500 and 30,000 and the B block has a number average molecular
weight between 500 and 10,000.
In one embodiment, the block copolymers of the present disclosure
possess water-solubility and gelling properties, such that the copolymers
possess water solubility at temperatures below the gelling temperature and
that
there is rapid gelation under physiological conditions (for example, a
temperature
of about 37 C). Accordingly, in one embodiment, when the polymers are
conjugated to, or contain therapeutic compounds, and administered to a
patient,
the rapid gelling minimizes the initial burst of the therapeutic compound,
such as
a cell or cytokines. In one embodiment, the temperature at which the block
copolymers gel (or the reverse thermal gelation temperature) is controlled by
the
molecular weights of the A block and the B block in the block copolymer.
CA 02719855 2010-11-02
In one embodiment of the disclosure, the hydrophobic A block comprises
about 20% to 80% by weight of the copolymer, optionally 30% to 70%, or about
50%, and the hydrophilic B block makes up 80% to 20% by weight of the
copolymer, optionally, 70% to 30%, or about 50%.
In another embodiment, the concentration at which the block copolymers
of the present disclosure are soluble (in aqueous solution (water, buffer
etc.))
below the gelling temperature is up to about 60% by weight, optionally about
10% to about 40%.
(III) PROCESS OF THE DISCLOSURE
The present disclosure also includes processes for the preparation of
block copolymers comprising at least one A block and at least one B block,
having the formula A-B, A-B-A or B-A-B. In one embodiment, a hydrophilic B
block polymer, such as polyethylene glycol is used as a cationic macro-
initiator
for the polymerization reaction with the monomeric subunits of the A block
polymer, in which the cationic macro-initiator begins the cationic
polymerization
with biodegradable monomeric units of the B block. Accordingly, the block
copolymers of the disclosure comprise biodegradable linkages, which are
hydrolyzed in vivo and excreted through the kidney. In one embodiment, the
process of the disclosure using a hydrophilic B block polymer, such as
polyethylene glycol, increases the processability of higher molecular weight B
block polymers, such as polyethylene glycol.
The present disclosure also includes a process for the preparation of a
block copolymer comprising at least one A block and at least one B block
having
the formula A-B, A-B-A, B-A-B, the process comprising reacting
(i) an optionally substituted polyethylene glycol or polypropylene glycol
comprising the B block;
with,
(ii) monomeric units of the A block, the monomeric units comprising b-
valerolactone, s-caprolactone, lactide, an a-hydroxy acid, glycolic acid, an
anhydride, an ester, an orthoester, an etherester, an esteramide, a
carbonate, a cyanoacrylate, a urethane, an acrylate, or a mixture thereof,
all of which are optionally substituted, wherein the optional substituents
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are selected from halo, OH, (C1_6)-alkyl and fluoro-substituted (C1.6)-alkyl;
in the presence of an acid catalyst having a pKa of less than -12, and wherein
the process is performed at a temperature between -10 C and 35 C.
In another embodiment, the number average molecular weight of the A
block is controlled by the molar ratio of the monomeric units of the A block
to the
B block during the process.
In another embodiment of the process, the A block has a number average
molecular weight between 500 and 30,000 and the B block has a number
average molecular weight between 500 and 10,000. In another embodiment,
the A block has a number average molecular weight between 500 and 10,000, or
1,000 and 5,000, or 1,000 and 3,000, or about 1,750. In another embodiment,
the B block has a number average molecular weight of between 1,000 and 8,000,
optionally between 1,500 and 8,000, or 1,500 and 5,000.
In another embodiment of the process, the block copolymer has the
formula: A-B-A.
In another embodiment of the process, the monomeric units of the A block
comprise b-valerolactone, E-caprolactone, Iactide, an a-hydroxy acid,
glycolide or
a copolymer thereof, optionally S-valerolactone or E-caprolactone or a
copolymer
thereof, or 8-valerolactone. In another embodiment, the A block comprises a
copolymer of poly(8-valerolactone) and glycolic acid
In an embodiment of the process, the B block comprises polyethylene
glycol.
In a further embodiment of the process, the acid catalyst is a sulfonic acid,
optionally trifluoromethanesulfonic acid or fluorosulfonic acid. In an
embodiment,
the sulfonic acid is trifluoromethanesulfonic acid.
In another embodiment of the disclosure, there is also included a block
copolymer comprising at least one A block and at least one B block, wherein
the
block copolymer has the formula: A-B; A-B-A; or B-A-B; in which the block
copolymer is produced by the process of the disclosure. In another embodiment,
the block copolymer produced by the process comprises
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A B O A
~0"'- O H
H O O
W H,
y
O
wherein the integers w, x and y represent the number of repeating units to
obtain
a block copolymer wherein the A block has a number average molecular weight
between 500 and 30,000 and the B block has a number average molecular
weight between 500 and 10,000.
In one embodiment, the mole ratio of B block to the monomeric units of
the A block controls the lengths of the A blocks, and can provide a series of
polymers with increasing A block contents and hydrophobicities.
In one embodiment, the process of the disclosure follows a scheme as
shown in Scheme 1, in which for example, polyethylene glycol is the B block
polymer, and S-valerolactone is the monomeric unit forming the A block, to
form a
PVL-PEG-PVL triblock copolymer:
Scheme 1
O
H o + o
Ox H
Trifluoromethanesulfonic acid
Dichloromethane
A B O A
H wO\+~O H of H
X y
O
(IV) USES
The present disclosure also includes a pharmaceutical conjugate
comprising a block copolymer as defined in the disclosure and a therapeutic
compound. In another embodiment, the therapeutic compound is a biologic,
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optionally stem cell factor (SCF) or vascular endothelial growth factor
(VEGF).
The present disclosure also includes a method for the treatment of cardiac
abnormality and/or vascular abnormality in a patient in need thereof
comprising
administering a therapeutically effective amount of a pharmaceutical conjugate
as defined in the disclosure to the site of the cardiovascular defect. In an
embodiment, the cardiac abnormality is myocardial infarction and the vascular
abnormality is a vascular aneurysm.
In another embodiment, the block copolymers of the present disclosure
are drug delivery matrices which slowly release pharmaceutical agents as the
block copolymer biodegrades after administration.
In one embodiment, the block copolymers are carboxy-derivatized to allow
for the conjugation of a therapeutic compound, such as a biologic, such as
VEGF. For example, in one embodiment, the block copolymer is derivatized
using a compound which adds a carbvoxy group to the ends of the polymers,
such as succinic anhydride, to obtain block copolymers with succinic acid
groups
at one or both ends of the polymer chain, which can be conjugated to cytokine
such as VEGF. For example, in one embodiment, as shown in Scheme 2, the
triblock copolymer as shown in Scheme 1 is further derivatized:
0
O H
H O O
W
x y
O
Succinic Anhydride
HOOC-CH2-C H2-PVL-PEG-CH2-CH2-000H
VEGF
VEGF(O)C-CHZ-CH2-PVL-PEG-CH2-CHZ-C(O)-VEGF
In one embodiment, the process used to mix the copolymers with a
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biologically active agent and/or other materials involves dissolving the block
copolymers in an aqueous solution, followed by addition of the biologically
active
agent (in solution, suspension or powder such as VEGF and bone marrow cells),
followed by thorough mixing to assure a homogeneous distribution of the
biologically active agent throughout the copolymer. For example, Figure 1
shows
a photomicrograph of illustrating the distribution of rat bone marrow stromal
cells
in a block copolymer gelled matrix. In another embodiment, the process
involves
dissolving the block copolymer in a biologically active agent-containing
solution.
In both embodiments, the process is conducted at a temperature lower than the
gelation temperature of the copolymer and the material is implanted into the
body
as a solution which then gels into a depot in the body. In one embodiment, the
biologically active agent will generally have a concentration in the range of
0 to
100 mg/mL or 100 to 10 million cells.
In another embodiment, buffers for use in the preparation of the
biologically active agent-containing hydrogels are buffers which are well
known
by those of ordinary skill in the art and include sodium acetate, Tris, sodium
phosphate, MOPS, PIPES, MES and potassium phosphate, in the range of 25
mM to 500 mM and in the pH range of 4.0 to 8.5.
In another embodiment, other excipients, e.g., various sugars (glucose,
sucrose), salts (NaCl, ZnCI) or surfactants, are included in the biologically
active
agent-containing hydrogels of the present disclosure.
In one embodiment, proteins contemplated for use include but are not
limited to interferon consensus (see, U.S. Pat. Nos. 5,372,808, 5,541,293
4,897,471, and 4,695,623 hereby incorporated by reference), stem cell factor
(PCT Publication Nos. 91/05795, 92/17505 and 95/17206, hereby incorporated
by reference) and rat VEGF. In addition, biologically active agents can also
include fibroblast growth factors (FGF), insulin and Vascular endothelial
growth
factor (VEGF). The term proteins, as used herein, includes peptides,
polypeptides, consensus molecules, analogs, derivatives or combinations
thereof. In addition, in one embodiment, the block copolymers are useful for
the
treatment of damaged/diseased organs (or organ failure). In another
embodiment, the block copolymers are useful for drug delivery in oncology via
CA 02719855 2010-11-02
injection of the block copolymers (as conjugates and/or drug delivery devices)
directly into a tumor mass or the use of the polymers in conjunction with
photodynamic or temperature sensitive therapies into solid tumor masses.
In another embodiment, sustained-release compositions comprising an
effective amount of pharmaceutical ingredient, such as a biologic, will be
utilized.
As used herein, sustained release refers to the gradual release of ingredients
from the polymer matrix, over an extended period of time. In one embodiment,
the sustained release can be continuous or discontinuous, linear or non
linear,
and this can be accomplished using one or more polymer compositions, drug
loadings, selection of excipients, or other modifications. The sustained
release
will result in biologically effective serum levels of the active agent
(typically above
endogenous levels) for a period of time longer than that observed with direct
administration of the active agent. In another embodiment, a sustained release
of
the active agent will be for a period of days to weeks, depending upon the
desired therapeutic effect.
The following non-limiting examples are illustrative of the present
invention:
EXAMPLES
Example 1-Hydroxy- Terminated A-B-A Block Copolymer (polyvalerolactone)-
(polyethylene glycol)-(polyvalerolactone) (PVL-PEG-PVL)
0.5 g polyethylene glycol (PEG) (0.33 mmol) and 1.2 g valerolactone (VL, 12
mmol) were dissolved in 5 mL dichloromethane. Trifluorimethanesulfonic acid
catalyst, 61 uL (0.67 mmol) was added to the mixture at 0 C. The reaction was
maintained for 3 hours and terminated by the addition of 0.2 g of NaHCO3, and
then the mixture was filtered. The copolymer was collected after precipitation
in
hexane and dried in the oven.
The molecular weight of the poly-VL (PVL) block was calculated from 1H nuclear
magnetic resonance, with the known molecular weight of the PEG precursor
used as reference and CHC13 as the internal standard. The isolated polymer was
dried at 40 C under vacuum for 48 hours. The molecular weight of the block
copolymer was determined by gel permeation chromatography (GPC) using
polystyrene standards. The copolymer composition and relative block lengths
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were determined by 1H-NMR (as shown in Figure 2). The PVL-PEG-PVL tri
block copolymer was dissolved in 100 mM sodium phosphate, pH 7.4, and
exhibited the thermo-reversible property (solution below room temperature and
gel above room temperature).
Example 2-Hydroxy- Terminated A-B-A Block Copolymer (polyvalerolactone)-
(polyethylene glycol)-(polyvalerolactone) (PVL-PEG-PVL)
This example describes synthesis of a hydroxy-terminated A-B-A (PVL-PEG-
PVL), tri block copolymer by cationic polymerization method using a
polyethylene
glycol macro-initiator having a molecular weight of (Mn =5,000).
0.5 g polyethylene glycol (PEG) (0.132 mmol) and 1.2 g valerolactone (VL, 12
mmol) were dissolved in 5 mL dichloromethane. Trifluorimethanesulfonic acid
catalyst, 24 L (0.27 mmol) was added to the mixture at 0 C. The reaction was
maintained for 3 hours and terminated by the addition of 0.1 g of NaHCO3, and
then the mixture was filtered. The copolymer was collected after precipitation
in
hexane and dried in the oven.
The molecular weight of the poly-VL (PVL) block was calculated from 'H nuclear
magnetic resonance, with the known molecular weight of the PEG precursor
used as reference and CHCI3 as the internal standard.
The isolated polymer was dried at 40 C under vacuum for 48 hours. The
molecular weight of the block copolymer was determined by gel permeation
chromatography (GPC) using polystyrene standards. The copolymer composition
and relative block lengths were determined. The PVL-PEG-PVL tri block
copolymer dissolved either in 100 mM sodium phosphate, pH 7.4, exhibited the
thermo-reversible property (solution below room temperature and gel above
room temperature, or about 30 C).
Example 3-Hydroxy-Terminated A-B-A Block Copolymer (polyvalerolactone)-
(polyethylene glycol)-(polyvalerolactone) (PVL-PEG-PVL)
This example describes synthesis of a hydroxy-terminated A-B-A (PVL-PEG-
PVL), tri block copolymer by cationic polymerization method using a
polyethylene
glycol macro-initiator having a molecular weight of (Mn =8,000).
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0.5 g polyethylene glycol (PEG) (0.0625 mmol) and 1.2 g valerolactone (VL, 12
mmol) were dissolved in 5 mL dichloromethane. Trifluorimethanesulfonic acid
catalyst, 16 yL (0.17 mmol) was added to the mixture at 0 C. The reaction was
maintained for 3 hours and terminated by the addition of 0.2 g of NaHCO3, and
then the mixture was filtered. The copolymer was collected after precipitation
in
hexane and dried in the oven.
The molecular weight of the polyvalerolactone (PVL) block was calculated from
1H nuclear magnetic resonance, with the known molecular weight of the PEG
precursor used as reference and CHCI3 as the internal standard.
Example 4-Carboxy- Terminated A-B-A Block Copolymer (polyvalerolactone)-
(polyethylene glycol)-(polyvalerolactone) (PVL-PEG-PVL)
This example describes modification of hydroxy-terminated PVL-PEG-PVL tri
block copolymer to carboxylic acid-terminated PVL-PEG-PVL tri -block
copolymer.
To a hydroxy-terminated PVL-PEG-PVL copolymer (1.0 grams) as described in
Example 1, 10 ml of anhydrous 1,4-dioxane was added under continuous
nitrogen purging. After complete dissolution of the polymer, 1.0 grams of
succinic
anhydride in 1,4-dioxane was added, followed by addition of 0.2 grams
triethylamine and 0.1 grams of 4-dimethylaminopyridine. The reaction mixture
was stirred at room temperature for 24 hours under nitrogen atmosphere. The
conversion of terminal hydroxyl groups to carboxylic acid groups was followed
by
IR spectroscopy. After completion of the reaction the crude block polymer was
isolated by precipitation using ether. The crude acid-terminated polymer was
further purified by dissolving the polymer in methhylene chloride (40 ml) and
precipitating from ether. The isolated polymer was dried at 40 C under vacuum
for 48 hours.
The dried acid-terminated block copolymer (0.8 grams) was dissolved in 10 ml
of
100 mM sodium phosphate buffer (pH 7.4), and filtered through 0.45 m filter.
The polymer solution was then placed in a dialysis membrane (2,000 Molecular
Weight cut-off) and dialyzed against deionized water at 4 C. After dialysis,
the
polymer solution was lyophilized and the dried polymer was stored at -20 C.
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under a nitrogen environment.
The molecular weight of the tri block copolymer was determined by gel
permeation chromatography (GPC) using polystyrene standards. The copolymer
composition and relative block lengths were determined by 'H-NMR.
Example 5---Carboxy-Terminated A-B-A Block Copolymer (polyvalerolactone)-
(polyethylene glycol)-(polyvalerolactone) (PVL-PEG-PVL)
The carboxyl-terminated block copolymer was synthesized by reacting the
hydroxyl-terminated triblock copolymer with succicnic anhydride.
Synthesis of NHS-terminated block copolymer: In a round-bottom flask equipped
with a magnetic stir bar and a rubber septum, attached to a nitrogen line and
a
bubbler, the following materials were added: 0.5 g of dicarboxy- terminated
block
copolymer (0.128 mmol), 0.0396 g of N,N-dicyclohexylcarbodiimide (1.5x excess,
0.192 mmol), 0.0221 g of N-hydroxysuccinimide (1.5 excess, 0.192 mmol), and 5
mL of dichloromethane. The reaction was maintained for 24 h at room
temperature. The reaction mixture was then filtered, and precipitated in cold
diethyl ether. This reaction produced 0.31 g for a yield of 62 %.
Example 6-VEGF-Conjugated with Carboxy-Terminated A-B-A Block
Copolymer (polyvalerolactone)-(polyethylene glycol)-(polyvalerolactone) (PVL-
PEG-PVL)
This example describes synthesis of a VEGF conjugate from a carboxylic acid-
terminated PVL-PEG-PVL tri block copolymer. 10 mg NHS-PVL-PEG-PVL-NHS
(from Example 5) was added to a solution of 100 ng VEGF in 0.5 mL phosphate
buffered saline (PBS; pH = 7.4, equiv. 200 ng/mL). The reaction was maintained
for 24 h at room temperature. To remove the uncoupled VEGF, the reaction
mixture was dialyzed against water/PBS buffer using Spectra/Por 2 dialysis
membrane tubing with a molecular weight cut-off of 12-14 kDa for 48 h. The
reaction product (VEGF conjugate) was analyzed with sodium dodecyl sulfate-
polyacrylamide gel electrophoresis and the VEGF proteins were stained with
Coomassie Brilliant Blue, as shown in Figure 3. In Figure 3, "M" refers to a
protein ladder marker, Lane 1 refers to VEGF protein as a positive control,
Lane
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2 refers to the hydrogel alone as negative control and Lane 3 refers to the
hydrogel conjugated with VEGF.
Example 7-pH Dependent Gelation/De-Gelation of A-B-A Block Copolymer
(polyvalerolactone)-(polyethylene glycol)-(polyvalerolactone) (PVL-PEG-PVL)
(a) Gelation
The following example demonstrates temperature dependent gelation of a PVL-
PEG-PVL tri block copolymer solution.
The tri-block copolymer of PVL-PEG-PVL as described in Example 1 was
dissolved in sodium phosphate buffers to obtain 20% (by weight) polymer
solution with final pH in the range of 7.0-8.0 One milliliter polymer
solution,
formulated in different pH buffers, was placed in a glass vial at 37 C and the
gelation was monitored visually as a function of time.
(b) De-Gelation
The following example demonstrates temperature dependent de-gelation (gel to
solution) of the PVL-PEG-PVL hydrogel.
The above gel (from Example 7b) was heated to 60 C at which point it became a
liquid solution. Then, it was cooled down to room temperature and became a
solution again.
Example 8-Temperature-Sensitive A-B-A Block Copolymer (polyvalerolactone)-
(polyethylene glycol)-(polyvalerolactone) (PVL-PEG-PVL)
The temperature-sensitive hydrogel was prepared as follows: 0.2 g PVL-PEG-
PVL was added to 1.0 mL PBS. The mixture was heated to 60 C and stirred until
the polymer was completely dissolved, and then cooled to 10 C. A clear polymer
solution formed. The gelling temperature was determined by increasing the
temperature by 5 C per min until a gel formed. A VEGF-conjugated hydrogel
(HG-VEGF) was prepared by adding VEGF conjugates to PVL-PEG-PVL solution
at 10 C. The results are summarized in Figures 4 and 5, in which in Figure 4
the
graph illustrates the temperature dependent gelation at a pH above 7.4. Figure
5
illustrates the gelling process in which a solution of the block copolymer is
heated
from room temperature to about 37 C (10 minutes) to form a hydrogel.
CA 02719855 2010-11-02
Example 9-Degradation of A-B-A Block Copolymer (polyvalerolactone)-
(polyethylene glycol)-(polyvalerolactone) (PVL-PEG-PVL)
This example describes the hydrogel degradation in vitro and in vivo.
PVL-PEG-PVL (10, 20 or 30 pL) was added onto a cell culture dish formed a gel
at 37 C. The nodule diameter did not change for 4 weeks, indicating the gel
was
stable in vitro. The hydrogel injected subcutaneously (10, 20, or 30 L) into
a rat,
which immediately absorbed water and the nodule size initially increased at
all 3
tested concentrations. However, nodule size decreased beyond 7 days after
implantation and the nodules were completely degraded after 42 days indicating
biodegradation of the hydrogel copolymer.
Example 10-Comparison studies of hydrogel (PVL-PEG-PVL) and VEGF
conjugate of hydrogel
Myocardial infarction were created from a total of 44 Sprague Dawley rats
(body
weight = 200-250 g) and were used for the studies. 100 uL of PBS, HG, HG
mixed with VEGF (40 ng/rat) (HG+VEGF), or HG-VEGF (40 ng VEGF/rat) was
injected into 4 sites around the infarct area with a 28-gauge insulin syringe
(25
uL/injection), and the incision was closed. All animals received post-
operative
care. Function was evaluated at 35 days after injection with a pressure volume
catheter. The heart function was improved.
After the pressure/volume analysis was complete, hearts were rapidly excised
and fixed in 10% formaldehyde. Morphometry analysis were performed. It was
determined that hydrogel prevented ventricular dilation.
Discussion
As shown in Figure 6, representative heart slices obtained at 35 days after
myocardial infarction with injection of PBS, HG (hydrogel copolymer), HG + VGF
(mixture of hydrogel and VEGF) and HG-VEGF (conjugate of VEGF and
hydrogel), wherein the arrows indicate the location of the infarct in
individual
slices, and illustrates that HG, HG+VEGF or HG-VEGF helps to prevent scar
expansion. As shown in Figure 7, the left ventricular scar area after
myocardial
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infarction is lower when treated with the HG, HG+VEGF or HG-VEGF. Finally,
Figure 8 illustrates that the ejection fraction of the heart was greater when
treated
with HG, HG+VEGF or HG-VEGF.
It was determined that the block copolymer hydrogel of the present disclosure
provides a temporary scaffold to attenuate adverse cardiac remodeling and
helps
to prevent scar expansion. The block copolymer also provides a platform for
the
sustained release of a therapeutic compound (VEGF). In this Example, VEGF
further attenuated adverse cardiac remodeling, stimulates angiogenesis and
prevents heart failure. The sustained release of VEGF stimulates new blood
vessel formation and when VEGF is conjugated with block copolymer, they act
synergistically to impede scar expansion, maintain LV structure and preserve
LV
function.
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