U.S. patent application number 10/191377 was filed with the patent office on 2003-05-29 for biodegradable ph/thermosensitive hydrogels for sustained delivery of biologically active agents.
This patent application is currently assigned to Amgen Inc.. Invention is credited to Dai, Weiguo, Shah, Subodh.
Application Number | 20030099709 10/191377 |
Document ID | / |
Family ID | 22826693 |
Filed Date | 2003-05-29 |
United States Patent
Application |
20030099709 |
Kind Code |
A1 |
Shah, Subodh ; et
al. |
May 29, 2003 |
Biodegradable pH/thermosensitive hydrogels for sustained delivery
of biologically active agents
Abstract
The present invention relates generally to the development of
pharmaceutical compositions which provide for sustained release of
biologically active polypeptides. More specifically, the invention
relates to the use of pH/thermosensitive, biodegradable hydrogels,
consisting of a A-B di block or A-B-A tri block copolymer of
poly(d,l- or l-lactic acid) (PLA) or poly(lactide-co-glycolide)
(PLGA) (block A) and polyethylene glycol (PEG) (block B), with
ionizable functional groups on one or both ends of the polymer
chains, for the sustained delivery of biologically active
agents.
Inventors: |
Shah, Subodh; (Newbury Park,
CA) ; Dai, Weiguo; (Winnetka, CA) |
Correspondence
Address: |
AMGEN INCORPORATED
MAIL STOP 27-4-A
ONE AMGEN CENTER DRIVE
THOUSAND OAKS
CA
91320-1799
US
|
Assignee: |
Amgen Inc.
|
Family ID: |
22826693 |
Appl. No.: |
10/191377 |
Filed: |
July 8, 2002 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10191377 |
Jul 8, 2002 |
|
|
|
09221178 |
Dec 23, 1998 |
|
|
|
6451346 |
|
|
|
|
09221178 |
Dec 23, 1998 |
|
|
|
09221187 |
Dec 23, 1998 |
|
|
|
6311266 |
|
|
|
|
Current U.S.
Class: |
424/469 ;
712/E9.049 |
Current CPC
Class: |
A61K 9/06 20130101; A61K
9/0024 20130101; Y10S 514/964 20130101; A61K 47/34 20130101; Y10S
514/965 20130101; Y10S 514/944 20130101 |
Class at
Publication: |
424/469 |
International
Class: |
A61K 009/26 |
Claims
What is claimed is:
1. A pharmaceutical composition for the sustained administration of
an effective amount of a biologically active agent, or a
derivative, analog, fusion, conjugate, or chemically modified form
thereof, comprising an injectable biodegradable polymeric matrix
into which said biologically active agent has been incorporated,
said polymeric matrix having reverse thermal gelation properties
and pH-responsive gelation/de-gelation properties.
2. The composition of claim 1, wherein said polymeric matrix is a
biodegradable block copolymer comprising: (a) 20% to 80% by weight
of a hydrophobic A polymer block and; (b) 20% to 80% by weight of a
hydrophilic B polymer block comprising a polyethylene glycol having
an average molecular weight of between 500-10,000; wherein said
copolymers have ionizable functional groups on one or both ends of
the polymer chains.
3. The composition of claim 2, wherein said ionizable functional
groups have a pK.sub.a in the range of 3-8.
4. The composition of claim 2, wherein said hydrophobic A polymer
block is a poly(.alpha.-hydroxy acid) having an average molecular
weight of between 1000-20,000.
5. The composition of claim 4, wherein said poly(.alpha.-hydroxy
acid) is selected from the group consisting of poly(lactide)s (d,l-
or l-forms), poly(glycolide)s, polyanhydrides, polyorthoesters,
polyetheresters, polycaprolactone, polyesteramides, polycarbonate,
polycyanoacrylate, polyurethanes, polyacrylate, blends and
copolymers thereof.
6. The composition of claim 5, wherein said poly(.alpha.-hydroxy
acid) is poly lactide-co-glycolide (PLGA).
7. The composition of claim 6, wherein said block copolymer is a
tri block copolymer having a configuration selected from the group
consisting of A-B-A or B-A-B block segments.
8. The composition of claim 7, wherein said hydrophobic A polymer
block comprises 74% by weight of said block copolymer and said
hydrophilic B polymer block comprises 26% by weight of said block
copolymer.
9. The composition of claim 8 further comprising an excipient which
will vary the lower critical solution temperature and increase the
rate of gelation of said block copolymer.
10. The composition of claim 1, wherein said biologically active
agent is a protein selected from the group consisting of interferon
consensus, interleukins, erythropoietins, granulocyte-colony
stimulating factor (GCSF), stem cell factor (SCF), leptin (OB
protein), interferons (alpha, beta, gamma), tumor necrosis factor
(TNF), tumor necrosis factor-binding protein (TNF-bp),
interleukin-1 receptor antagonist (IL-1ra), brain derived
neurotrophic factor (BDNF), glial derived neurotrophic factor
(GDNF), neurotrophic factor 3 (NT3), fibroblast growth factors
(FGF), neurotrophic growth factor (NGF), bone growth factors such
as osteoprotegerin (OPG), granulocyte macrophage colony stimulating
factor (GM-CSF), megakaryocyte derived growth factor (MGDF),
keratinocyte growth factor (KGF), thrombopoietin, platelet-derived
growth factor (PGDF), novel erythropoiesis stimulating protein
(NESP), tissue plasminogen activator (TPA), urokinase,
streptokinase and kallikrein.
11. The composition of claim 1, wherein said biologically active
agent is a small molecule.
12. A method for the parenteral administration of a biologically
active agent, or a derivative, analog, fusion, conjugate, or
chemically modified form thereof, in a biodegradable polymeric
matrix to a warm blooded animal with the resultant sustained
release of said agent concomitant with biodegradation and clearance
from injection site of said polymeric matrix, which comprises: (a)
providing an injectable liquid polymeric matrix having reverse
thermal gelation properties and pH-responsive gelation/degelation
properties, and into which a biologically active agent has been
incorporated; (b) maintaining said liquid polymeric matrix at a
temperature below the lower critical solution temperature of said
polymeric matrix; and (c) injecting said liquid parenterally into
said animal, thus forming a gel depot of said agent and polymeric
matrix as the temperature of said liquid is raised in the body of
said animal above the lower critical solution temperature of the
polymeric matrix.
13. The method of claim 12, wherein said polymeric matrix is a
biodegradable block copolymer comprising: (a) 20% to 80% by weight
of a hydrophobic A polymer block and; (b) 20% to 80% by weight of a
hydrophilic B polymer block comprising a polyethylene glycol having
an average molecular weight of between 500-10,000; wherein said
copolymers have ionizable functional groups on one or both ends of
the polymer chains.
14. The method of claim 13, wherein said hydrophobic A polymer
block is a poly(.alpha.-hydroxy acid) having an average molecular
weight of between 1000-20,000.
15. The method of claim 14, wherein said poly(.alpha.-hydroxy acid)
is poly lactide-co-glycolide (PLGA).
16. The method of claim 15, wherein said block copolymer is a tri
block copolymer having a configuration selected from the group
consisting of A-B-A or B-A-B block segments.
17. The method of claim 16, wherein said hydrophobic A polymer
block comprises 74% by weight of said block copolymer and said
hydrophilic B polymer block comprises 26% by weight of said block
copolymer.
18. The method of claim 17 further comprising an excipient which
will vary the lower critical solution temperature and increase the
rate of gelation of said block copolymer.
19. The method of claim 12, wherein said biologically active agent
is a protein selected from the group consisting of interferon
consensus, interleukins, erythropoietins, granulocyte-colony
stimulating factor (GCSF), stem cell factor (SCF), leptin (OB
protein), interferons (alpha, beta, gamma), tumor necrosis factor
(TNF), tumor necrosis factor-binding protein (TNF-bp),
interleukin-1 receptor antagonist (IL-1ra), brain derived
neurotrophic factor (BDNF), glial derived neurotrophic factor
(GDNF), neurotrophic factor 3 (NT3), fibroblast growth factors
(FGF), neurotrophic growth factor (NGF), bone growth factors such
as osteoprotegerin (OPG), granulocyte macrophage colony stimulating
factor (GM-CSF), megakaryocyte derived growth factor (MGDF),
keratinocyte growth factor (KGF), thrombopoietin, platelet-derived
growth factor (PGDF), novel erythropoiesis stimulating protein
(NESP), tissue plasminogen activator (TPA), urokinase,
streptokinase and kallikrein.
20. The method of claim 12, wherein said biologically active agent
is a small molecule.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to the use of biodegradable,
pH/thermosensitive hydrogels, consisting of a A-B-A tri block
copolymer of poly(d,l- or l-lactic acid) (PLA) or
poly(lactide-co-glycolide) (PLGA) (block A) and polyethylene glycol
(PEG) (block B), with ionizable functional groups on one or both
ends of the polymer chains, for the sustained delivery of
biologically active agents.
BACKGROUND OF THE INVENTION
[0002] Due to recent advances in genetic and cell engineering
technologies, proteins known to exhibit various pharmacological
actions in vivo are capable of production in large amounts for
pharmaceutical applications. Such proteins include erythropoietin
(EPO), novel erythropoiesis stimulating protein (NESP), granulocyte
colony-stimulating factor (G-CSF), interferons (alpha, beta, gamma,
consensus), tumor necrosis factor binding protein (TNFbp),
interleukin-1 receptor antagonist (IL-1ra), brain-derived
neurotrophic factor (BDNF), kerantinocyte growth factor (KGF), stem
cell factor (SCF), megakaryocyte growth differentiation factor
(MGDF), osteoprotegerin (OPG), glial cell line derived neurotrophic
factor (GDNF) and obesity protein (OB protein). OB protein may also
be referred to herein as leptin.
[0003] Because proteins such as leptin generally have short in vivo
half-lives and negligible oral bioavailability, they are typically
administered by frequent injection, thus posing a significant
physical burden on the patient (e.g., injection site reactions are
particularly problematic with many leptin formulations) and
associated administrative costs. As such, there is currently a
great deal of interest in developing and evaluating
sustained-release formulations. Effective sustained-release
formulations can provide a means of controlling blood levels of the
active ingredient, and also provide greater efficacy, safety,
patient convenience and patient compliance. Unfortunately, the
instability of most proteins (e.g. denaturation and loss of
bioactivity upon exposure to heat, organic solvents, etc.) has
greatly limited the development and evaluation of sustained-release
formulations.
[0004] Biodegradable polymer matrices have thus been evaluated as
sustained-release delivery systems. Attempts to develop
sustained-release formulations have included the use of a variety
of biodegradable and non-biodegradable polymer (e.g.
poly(lactide-co-glycolide)) microparticles containing the active
ingredient (see e.g., Wise et al., Contraception, 8:227-234 (1973);
and Hutchinson et al., Biochem. Soc. Trans., 13:520-523 (1985)),
and a variety of techniques are known by which active agents, e.g.
proteins, can be incorporated into polymeric microspheres (see
e.g., U.S. Pat. No. 4,675,189 and references cited therein).
[0005] Utilization of the inherent biodegradability of these
materials to control the release of the active agent and provide a
more consistent sustained level of medication provides improvements
in the sustained release of active agents. Unfortunately, some of
the sustained release devices utilizing microparticles still suffer
from such things as: active agent aggregation formation; high
initial bursts of active agent with minimal release thereafter; and
incomplete release of active agent.
[0006] Other drug-loaded polymeric devices have also been
investigated for long term, therapeutic treatment of various
diseases, again with much attention being directed to polymers
derived from alpha hydroxycarboxylic acids, especially lactic acid
in both its racemic and optically active form, and glycolic acid,
and copolymers thereof. These polymers are commercially available
and have been utilized in FDA-approved systems, e.g., the Lupron
Depot.TM., which consists of injectable microcapsules which release
leuprolide acetate for about 30 days for the treatment of prostate
cancer.
[0007] Various problems identified with the use of such polymers
include: inability of certain macromolecules to diffuse out through
the matrix; deterioration and decomposition of the drug (e.g.,
denaturation caused by the use of organic solvents); irritation to
the organism (e.g. side effects due to use of organic solvents);
low biodegradability (such as that which occurs with
polycondensation of a polymer with a multifunctional alcohol or
multifunctional carboxylic acid, i.e., ointments); and slow rates
of degradation.
[0008] The use of polymers which exhibit reverse thermal gelation
have also been reported. For example, Okada et al., Japanese Patent
Application 2-78629 (1990) describe biodegradable block copolymers
synthesized by transesterification of poly(lactic acid) (PLA) or
poly(lactic acid)/glycolic acid (PLA/GA) and poly(ethylene glycol)
(PEG). PEGs with molecular weights ranging from 200 to 2000, and
PLA/GA with molecular weights ranging from 400 to 5000 were
utilized. The resultant product was miscible with water and formed
a hydrogel. The Okada et al. reference fails to provide any
demonstration of sustained delivery of drugs using the
hydrogels.
[0009] Cha et al., U.S. Pat. No. 5,702,717 describe systems for
parenteral delivery of a drug comprising an injectable
biodegradable block copolymeric drug delivery liquid having reverse
thermal gelation properties, i.e., ability to form semi-solid gel,
emulsions or suspension at certain temperatures. Specifically,
these thermosensitive gels exist as a mobile viscous liquid at low
temperatures, but form a rigid semisolid gel at higher
temperatures. Thus, it is possible to use these polymers to design
a formulation which is liquid at room temperature or at lower
temperatures, but gels once injected, thus producing a depot of
drug at the injection site. The systems described by Cha et al.
utilize a hydrophobic A polymer block comprising a member selected
from the group consisting of poly(.alpha.-hydroxy acids) and
poly(ethylene carbonates) and a hydrophilic B polymer block
comprising a PEG. The Cha et al. system requires that less than 50%
by weight hydrophobic A polymer block be utilized and greater than
50% by weight hydrophilic B polymer block be utilized.
Interestingly, however, it appears that several of the disclosed
hydrogels might not be commercially useful in that the lower
critical solution temperature (LCST) for many of the gels is
greater than 37.degree. C. Although Cha et al. propose use of their
hydrogels for controlled release of drugs, no such demonstration is
provided.
[0010] Churchill et al., U.S. Pat. No. 4,526,938, describe a
continuous release composition comprising a biodegradable
(PLGA/PEG) block copolymer admixed with a drug which is
continuously released from the block copolymer. The example
described in Churchill et al. uses 50%/50% weight percentage
copolymer. Churchill et al. do not discuss whether the compositions
exhibit reverse thermal gelation properties, nor teach aqueous
solutions of drug-containing block copolymers that are soluble at
the time of injection and that undergo gelation as they reach body
temperature. Rather, Churchill et al. teach administration of a
block copolymer in solid form.
[0011] Martini et al., J. Chem. Soc., 90(13):1961-1966 (1994)
describe low molecular weight ABA type tri block copolymers which
utilize hydrophobic poly(.epsilon.-caprolactone) (PCL) and PEG.
Unfortunately, in vitro degradation rates for these copolymers was
very slow, thus calling into question their ability as
sustained-release systems.
[0012] Stratton et al., PCT/US97/13479 (WO 98/02142) Jan. 22, 1998,
describe pharmaceutical compositions comprising a polymeric matrix
having thermal gelation properties, for the delivery of proteins.
The class of block copolymers described are generically referred to
as polyoxyethylene-polyoxypropylene condensates (also known as
Pluronics). Unfortunately, only high molecular weight Pluronics at
higher concentrations (25-40 wt. %) exhibit thermoreversible
gelation, and the very nature of gelation caused by formation of
densely packed liquid crystalline phases in concentrated Pluronic
solutions limits the applicability of Pluronics in drug
delivery.
[0013] Kim et al., J. Appl. Polym. Sci., 45:1711 (1992) describe
various pH-sensitive hydrogels and the use of such hydrogels to
fabricate a glucose-sensitive insulin release device.
[0014] Chen and Hoffman, Nature, 373:49-52 (1995) describe a new
generation of `intelligent` copolymers of thermogelling surfactants
and pH-responsive bioadhesive polymers containing ionizable
carboxylic groups, that obtain pH and temperature sensitivity. The
polymers are prepared by grafting a temperature-sensitive polymer
(PNIPAAm) onto a pH-sensitive polymer (PAAc) backbone, and have
been shown to possess bioadhesive properties due to the backbone
polymer. It is necessary to obtain a graft (or block) copolymer
because it was found that random copolymers of the temperature- and
pH- sensitive monomers lose their temperature-sensitivity at body
temperatures when the levels of the pH-sensitive component are high
enough to obtain a sufficiently bioadhesive material. Drawbacks to
the copolymers described by Chen and Hoffman are the potentially
poor biocompatibility and non-biodegradability of PNIPAAm polymers,
and the fact that drugs contained within some NIPAAm-containing
hydrogels are known to be effectively squeezed out of the hydrogel
as the hydrogel collapses, leading to a burst of drug each time the
gel collapses, which is not ideal for sustained drug delivery.
[0015] Lee et al., J. Appl. Polym. Sci., 62:301-311 (1996) report
on the preparation and swelling properties of pH- and
temperature-dependent poly(vinyl alcohol) (PVA)/poly(acrylic acid)
(PAAc) interpenetrating polymer networks (IPN) hydrogels by a
unique freezing-thawing method. It was reported that the hydrogels
showed both positive and negative swelling behaviors depending on
PAAc content. It is postulated that the hydrogels could be strong
candidates as drug delivery materials, but there is no
demonstration of such use.
[0016] It is the object of the present invention to provide
biodegradable, pH/termosensitive hydrogels for the sustained
delivery of drugs. The hydrogels of the present invention utilize
copolymer compositions containing ionizable functional groups which
provide for instant gelation with trapping of all the biologically
active agent within the gel, i.e., no burst, and, importantly,
which upon injection, possess improved rates of degradation,
de-gelation and clearance of the depot from the injection site,
making this class of hydrogels more commercially practical than
those previously described.
SUMMARY OF THE INVENTION
[0017] In one embodiment, the present invention provides
pharmaceutical compositions comprising an effective amount of a
biologically active agent incorporated into a polymeric matrix,
said polymeric matrix comprising a di block or tri block copolymer
which is thermosensitive, exhibits pH-responsive
gelation/de-gelation, and is capable of providing for the
sustained-release of the biologically active agent.
[0018] In another embodiment, the present invention provides a
method for the parenteral administration of a biologically active
agent in a biodegradable polymeric matrix to a warm blooded animal,
wherein a gel depot is formed within the body of said animal and
the biologically active agent is released from the depot at a
controlled rate concomitant with biodegradation of the polymeric
matrix.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] FIG. 1 is a schematic which depicts the pH/thermosensitive
nature of the hydrogels of the present invention. T=temperature,
and the darkened hydrogel depicts the hydrogel in a gelled form,
while the clear hydrogel depicts the hydrogel in solution form.
[0020] FIG. 2 depicts the two methods by which the A-B-A tri block
copolymers of the present invention can be prepared.
[0021] FIG. 3 depicts the in vitro release characteristics of
leptin released from various hydrogels. The -.diamond-solid.-
depicts the release from a 100% hydroxy-terminated PLGA-PEG-PLGA
hydrogel; -.box-solid.- depicts the release from a 80%
hydroxy-terminated+20% carboxy-terminated PLGA-PEG-PLGA hydrogel
(weight ratio); and -.diamond-solid.- depicts the release from a
50% hydroxy-terminated+50% carboxy-terminated PLGA-PEG-PLGA
hydrogel (weight ratio). % protein released is plotted vs. time
(days).
[0022] FIG. 4 depicts the in vivo bioactivity of various
leptin-containing hydrogel formulations. The -.quadrature.-
represents the buffer control (0.1 ml of 10 mM acetate buffer, pH
4.0, (day 0 only)); -.circle-solid.- represents the leptin control
(0.1 ml of 20 mg/ml (100 mg/kg) leptin formulated in 10 mM acetate
buffer, pH 4.0 (day 0 only)); -.diamond-solid.- represents 0.1 ml
of a 95% hydroxy-terminated+5% carboxy-terminated PLGA-PEG-PLGA
hydrogel (weight ratio) consisting of 20 mg/ml (100 mg/kg) leptin,
in 10 mM acetate, pH 4.0 (day 0 only); and -.box-solid.- represents
0.1 ml of a 70% hydroxy-terminated+30% carboxy-terminated
PLGA-PEG-PLGA hydrogel (weight ratio) consisting of 20 mg/ml (100
mg/kg) leptin, in 10 mM acetate, pH 4.0 (day 0 only). % body weight
change (from the day 0 body weight) is plotted vs. time (days).
DETAILED DESCRIPTION OF THE INVENTION
[0023] As used herein, the following terms shall have the following
meaning:
[0024] "Reverse thermal gelation temperature" is defined as meaning
the temperature below which a copolymer is soluble in water and
above which the block copolymer solution forms a semi-solid, i.e.
gels, emulsions, dispersions and suspensions.
[0025] "LCST", or lower critical solution temperature, is defined
as meaning the temperature at which a block copolymer undergoes
reverse thermal gelation (solution to gel to solution). For
purposes of the present invention, the term "LSCT" can be used
interchangeably with "reverse thermal gelation temperature".
[0026] "Depot" is defined as meaning a drug delivery liquid which,
following injection into a warm blooded animal, has formed a gel
upon having the temperature raised to or above the LCST.
[0027] "Biodegradable" is defined as meaning that the block
copolymer will erode or degrade in vivo to form smaller non-toxic
components.
[0028] "Parenteral administration" is defined as meaning any route
of administration other than the alimentary canal, including, for
example, subcutaneous and intramuscular.
[0029] For purposes of the present invention, the terms
thermosensitive, thermoreversible, and thermoresponsive can be used
interchangeably.
[0030] The present invention involves utilization of block
copolymers having biodegradable hydrophobic ("A") block segments
and hydrophilic ("B") block segments. The block copolymers are di
block, e.g., A-B, or tri block copolymers, e.g., A-B-A or B-A-B,
type block copolymers.
[0031] Biodegradable hydrophobic A block segments contemplated for
use include poly(.alpha.-hydroxy acid) members derived from or
selected from the group consisting of homopolymers and copolymers
of poly(lactide)s (d,l- or l-forms), poly(glycolide)s,
polyanhydrides, polyesters, polyorthoesters, polyetheresters,
polycaprolactone, polyesteramides, polycarbonate,
polycyanoacrylate, polyurethanes, polyacrylate, blends and
copolymers thereof.
[0032] The term "PLGA" as used herein is intended to refer to a
polymer of lactic acid alone, a polymer of glycolic acid alone, a
mixture of such polymers, a copolymer of glycolic acid and lactic
acid, a mixture of such copolymers, or a mixture of such polymers
and copolymers. Preferably, the biodegradable A block polymer will
be poly lactide-co-glycolide (PLGA). The PLGA may be non-ionic,
e.g., hydroxy-terminated, or may be ionic, e.g.,
carboxy-terminated. As relates to the ionic polymers, the ionizable
functional groups may be on either one or both ends of the polymer
chain, and terminal ionizable groups contemplated for use include
any ionizable group having a pK.sub.a 3-8, e.g., carboxylic acids,
amines, sulfonic acids, ammonium salts.
[0033] The range of molecular weights contemplated for the A block
polymers to be used in the present processes can be readily
determined by a person skilled in the art based upon such factors
the desired polymer degradation rate. Typically, the range of
molecular weight for the A block will be 1000 to 20,000
Daltons.
[0034] Hydrophilic B block segments contemplated for use include
polyethylene glycols having average molecular weights of between
about 500 and 10,000. These hydrophilic segments may also contain
ionizable groups, if for example, B-A-B type copolymers are
used.
[0035] The copolymer compositions for the tri block copolymers of
the present invention are specially regulated to assure retention
of the desired water-solubility and gelling properties, i.e., the
ratios must be such that the block copolymers possess water
solubility at temperatures below the LCST, and such that there is
instant gelation under physiological conditions (i.e. pH 7.0 and
37.degree. C.) so as to minimize the initial burst of drug. In the
hydrogels of the present invention the hydrophobic A block makes up
20% to 80% by weight of the copolymer and the hydrophilic B block
makes up 80% to 20% of the copolymer.
[0036] The concentration at which the block copolymers of the
present invention remain soluble below the LCST is typically up to
about 60% by weight, with 10%-30% preferred. The concentration
utilized will depend upon the copolymer composition actually used,
as well as whether or not a gel or emulsion is desired.
[0037] The pH/thermosensitive hydrogels of the present invention
comprise ionic block copolymers such that the resultant hydrogels
exhibit pH-responsive gelation/de-gelation in addition to the
reverse thermal gelation properties (see FIG. 1). The hydrogels may
comprise non-ionic block copolymers mixed or "blended" with ionic
block copolymers and the weight ratios of ionic block copolymer to
non-ionic block copolymer in the blends can be adjusted such that
the resultant hydrogels possess the desirable rate of degradation,
de-gelation and rate of clearance from the injection site. Because
this new class of hydrogels provide for an improved rate of
clearance of the hydrogel from the injection site, they are more
commercially practicable than those hydrogels and compositions
previously described in that they.
[0038] The biodegradable, pH/thermosensitive block copolymers of
the present invention can be prepared in a two-step procedure which
utilizes thermal condensation. In step 1, thermosensitive,
hydroxy-terminated A-B-A block copolymers of PLGA/PLA (block A) and
PEG (block B) are synthesized by mixing either homopolymer of poly
lactide (PLA) or copolymer of poly lactide-co-gycolide (PLGA) with
polyethylene glycol (PEG) and allowing di-hydroxy PEG to react with
PLGA or PLA at 160.degree. C. under reduced pressure. Different
weight ratios of PLGA and PEG were used for thermal condensation to
obtain a series of block copolymers with desirable copolymer
composition and block lengths. Copolymer composition and relative
block lengths were confirmed by .sup.1H-NMR spectroscopy. In step
2, the thermosensitive, hydroxy-terminated A-B-A block copolymers
are further reacted with, e.g., succinic anhydride, to obtain A-B-A
block copolymers with succinic acid groups at one or both ends of
the polymer chain, thus providing hydrogels which exhibit
thermosensitive/pH-responsive gelation. This two-step procedure is
graphically depicted in FIG. 2, Scheme 1.
[0039] The biodegradable, ionic block copolymers of the present
invention can also be synthesized by single step condensation of
PLGA with activated PEG. This procedure is graphically depicted in
FIG. 2, Scheme 2.
[0040] Alternatively, the thermosensitive, non-ionic block
copolymers could be synthesized in a melt process which involves
ring opening polymerization of A block using B block as the
initiator. In a typical experiment, the A-B-A tri block copolymer
is prepared by stannous octoate catalyzed ring-opening
polymerization of d,l-dilactide (or PLGA) using
.alpha.,.omega.-dihydroxy-terminated PEG as the initiator. The mole
ratio of B block to d,l-dilactide (or PLGA) is used to control the
lengths of the A blocks, and provide a series of polymers with
increasing A block contents and hydrophobicities. The relative A
and B block lengths can be confirmed by .sup.1H-NMR
spectroscopy.
[0041] The process used to mix the copolymers with a biologically
active agent and/or other materials involves dissolving the A-B-A
tri block copolymers in an aqueous solution, followed by addition
of the biologically active agent (in solution, suspension or
powder), followed by thorough mixing to assure a homogeneous
distribution of the biologically active agent throughout the
copolymer. Alternatively, the process can involve dissolving the
A-B-A tri block copolymer in a biologically active agent-containing
solution. In either case, 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 the compositions of the present
invention, the biologically active agent will generally have a
concentration in the range of 0 to 200 mg/mL.
[0042] Buffers contemplated for use in the preparation of the
biologically active agent-containing hydrogels are buffers which
are all 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.
[0043] It is also envisioned that other excipients, e.g., various
sugars (glucose, sucrose), salts (NaCl, ZnCl) or surfactants, may
be included in the biologically active agent-containing hydrogels
of the present invention in order to alter the LCST or rate of
gelation of the gels. The ability to alter the rate of gelation
and/or LCST is important and an otherwise non-useful hydrogel may
be made useful by addition of such excipients.
[0044] As used herein, biologically active agents refers to
recombinant or naturally occurring proteins, whether human or
animal, useful for prophylactic, therapeutic or diagnostic
application. The biologically active agent can be natural,
synthetic, semi-synthetic or derivatives thereof. In addition,
biologically active agents of the present invention can be
perceptible. A wide range of biologically active agents are
contemplated. These include but are not limited to hormones,
cytokines, hematopoietic factors, growth factors, antiobesity
factors, trophic factors, anti-inflammatory factors, small
molecules and enzymes (see also U.S. Pat. No. 4,695,463 for
additional examples of useful biologically active agents). One
skilled in the art will readily be able to adapt a desired
biologically active agent to the compositions of present
invention.
[0045] Proteins contemplated for use would 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
including drawings), interleukins (see, U.S. Pat. No. 5,075,222,
hereby incorporated by reference including drawings),
erythropoietins (see, U.S. Pat. Nos. 4,703,008, 5,441,868,
5,618,698 5,547,933, and 5,621,080 hereby incorporated by reference
including drawings), granulocyte-colony stimulating factors (see,
U.S. Pat. Nos. 4,810,643, 4,999,291, 5,581,476, 5,582,823, and PCT
Publication No. 94/17185, hereby incorporated by reference
including drawings), stem cell factor (PCT Publication Nos.
91/05795, 92/17505 and 95/17206, hereby incorporated by reference
including drawings), and leptin (OB protein) (see PCT publication
Nos. 96/40912, 96/05309, 97/00128, 97/01010 and 97/06816 hereby
incorporated by reference including figures).
[0046] The type of leptin used for the present preparations may be
selected from those described in PCT International Publication
Number WO 96/05309, as cited above and herein incorporated by
reference in its entirety. FIG. 3 of that publication (as cited
therein SEQ ID NO: 4) depicts the full deduced amino acid sequence
derived for human leptin (referred to as the human "OB" protein).
The amino acids are numbered from 1 to 167. A signal sequence
cleavage site is located after amino acid 21 (Ala) so that the
mature protein extends from amino acid 22 (Val) to amino acid 167
(Cys). For the present disclosure, a different numbering is used
herein, where the amino acid position 1 is the valine residue which
is at the beginning of the mature protein. The amino acid sequence
for mature, recombinant methionyl human leptin is presented herein
as SEQ ID NO: 1, where the first amino acid of the mature protein
is valine (at position 1) and a methionyl residue is located at
position -1 (not included in the sequence below).
1 V P I Q K V Q D D T K T L I K T I V SEQ ID NO:1 T R I N D I S H T
Q S V S S K Q K V T G L D F I P G L H P I L T L S K M D Q T L A V Y
Q Q I L T S M P S R N V I Q I S N D L E N L R D L L H V L A F S K S
C H L P W A S G L E T L D S L G G V L E A S G Y S T E V V A L S R L
Q G S L Q D M L W Q L D L S P G C
[0047] However, as with any of the present leptin moieties, the
methionyl residue at position -1 may be absent.
[0048] Alternatively, one may use a natural variant of human
leptin, which has 145 amino acids and, as compared to rmetHu-leptin
of SEQ ID NO: 1, has a glutamine absent at position 28.
[0049] Generally, the leptin moiety for human pharmaceutical use
herein will be capable of therapeutic use in humans (see also,
animal leptins, below). Thus, one may empirically test activity to
determine which leptin moieties may be used. As set forth in WO
96/05309, leptin protein in its native form, or fragments (such as
enzyme cleavage products) or other truncated forms and analogs may
all retain biological activity. Any of such forms may be used as a
leptin moiety for the present preparations, although such altered
forms should be tested to determine desired characteristics. See
also, PCT International Publication Numbers WO 96/40912, WO
97/06816, 97/18833, WO 97/38014, WO 98/08512 and WO 98/28427,
herein incorporated by reference in their entireties.
[0050] One may prepare an analog of recombinant human leptin by
altering amino acid residues in the recombinant human sequence,
such as substituting the amino acids which diverge from the murine
sequence. Murine leptin is substantially homologous to human
leptin, particularly as a mature protein and, further, particularly
at the N-terminus. Because the recombinant human protein has
biological activity in mice, such an analog would likely be active
in humans. For example, in the amino acid sequence of native human
leptin as presented in SEQ ID NO: 1, one may substitute with
another amino acid one or more of the amino acids at positions 32,
35, 50, 64, 68, 71, 74, 77, 89, 97, 100, 101, 105, 106, 107, 108,
111, 118, 136, 138, 142 and 145. One may select the amino acid at
the corresponding position of the murine protein (see Zhang et al.,
1994, supra) or another amino acid.
[0051] One may further prepare "consensus" molecules based on the
rat OB protein sequence. Murakami et al., Biochem. Biophys. Res.
Comm., 209:944-52 (1995) herein incorporated by reference. Rat OB
protein differs from human OB protein at the following positions
(using the numbering of SEQ ID NO: 1): 4, 32, 33, 35, 50, 68, 71,
74, 77, 78, 89, 97, 100, 101, 102, 105, 106, 107, 108, 111, 118,
136, 138 and 145. One may substitute with another amino acid one or
more of the amino acids at these divergent positions. The positions
underlined are those in which the murine OB protein as well as the
rat OB protein are divergent from the human OB protein and, thus,
are particularly suitable for alteration. At one or more of the
positions, one may substitute an amino acid from the corresponding
rat OB protein, or another amino acid.
[0052] The positions from both rat and murine OB protein which
diverge from the mature human OB protein are: 4, 32, 33, 35, 50,
64, 68, 71, 74, 77, 78, 89, 97, 100, 101, 102, 105, 106, 107, 108,
111, 118, 136, 138, 142 and 145. An OB protein according to SEQ ID
NO: 1 having one or more of the above amino acids replaced with
another amino acid, such as the amino acid found in the
corresponding rat or murine sequence, may also be effective.
[0053] In addition, the amino acids found in rhesus monkey OB
protein which diverge from the mature human OB protein are (with
identities noted in parentheses in one letter amino acid
abbreviation): 8 (S), 35 (R), 48 (V), 53 (Q), 60 (I), 66 (I), 67
(N), 68 (L), 89 (L), 100 (L), 108 (E), 112 (D) and 118 (L). Since
the recombinant human OB protein is active in cynomolgus monkeys, a
human OB protein according to SEQ ID NO: 1 having one or more of
the rhesus monkey divergent amino acids replaced with another amino
acid, such as the amino acids in parentheses, may be effective. It
should be noted that certain rhesus divergent amino acids are also
those found in the above murine and rat species (positions 35, 68,
89, 100, 108 and 118). Thus, one may prepare a
murine/rat/rhesus/human consensus molecule (using the numbering of
SEQ ID NO: 1) having one or more of the amino acids replaced by
another amino acid at positions: 4, 8, 32, 33, 35, 48, 50, 53, 60,
64, 66, 67, 68, 71, 74, 77, 78, 89, 97, 100, 102, 105, 106, 107,
108, 111, 112, 118, 136, 138, 142 and 145. The positions underlined
are those in which all three species are divergent from human OB
protein. A particularly preferred human leptin analog is one
wherein the amino acids at position 100 (Trp) or 138 (Trp), and
more preferably, both positions are substituted with another amino
acid, preferably Gln.
[0054] Other analogs may be prepared by deleting a part of the
protein amino acid sequence. For example, the mature protein lacks
a leader sequence (-22 to -1). One may prepare the following
truncated forms of human OB protein molecules (using the numbering
of SEQ ID NO: 1):
[0055] (i) amino acids 98-146;
[0056] (ii) amino acids 1-99 and (connected to) 112-146;
[0057] (iii) amino acids 1-99 and (connected to) 112-146 having one
or more of amino acids 100-111 sequentially placed between amino
acids 99 and 112.
[0058] In addition, the truncated forms may also have altered one
or more of the amino acids which are divergent (in the murine, rat
or rhesus OB protein) from human OB protein. Furthermore, any
alterations may be in the form of altered amino acids, such as
peptidomimetics or D-amino acids.
[0059] It is desirable to have such protein containing
sustained-release compositions as such compositions could serve to
enhance the effectiveness of either exogenously administered or
endogenous protein, or could be used, for example, to reduce or
eliminate the need for exogenous protein administration.
[0060] Moreover, because the materials utilized in the present
invention are biocompatible and biodegradable, use of the protein
compositions of the present invention help prevent adverse
injection site reactions sometimes associated with injections of
various proteins such as leptin.
[0061] In addition, biologically active agents can also include
insulin, gastrin, prolactin, adrenocorticotropic hormone (ACTH),
thyroid stimulating hormone (TSH), luteinizing hormone (LH),
follicle stimulating hormone (FSH), human chorionic gonadotropin
(HCG), motilin, interferons (alpha, beta, gamma), tumor necrosis
factor (TNF), tumor necrosis factor-binding protein (TNF-bp),
interleukin-1 receptor antagonist (IL-1ra), brain derived
neurotrophic factor (BDNF), glial derived neurotrophic factor
(GDNF), neurotrophic factor 3 (NT3), fibroblast growth factors
(FGF), neurotrophic growth factor (NGF), insulin-like growth
factors (IGFs), macrophage colony stimulating factor (M-CSF),
granulocyte macrophage colony stimulating factor (GM-CSF),
megakaryocyte derived growth factor (MGDF), novel erythropoiesis
stimulating protein, keratinocyte growth factor (KGF),
thrombopoietin, platelet-derived growth factor (PGDF), colony
simulating growth factors (CSFs), bone morphogenetic protein (BMP),
superoxide dismutase (SOD), tissue plasminogen activator (TPA),
urokinase, streptokinase and kallikrein. The term proteins, as used
herein, includes peptides, polypeptides, consensus molecules,
analogs, derivatives or combinations thereof.
[0062] Also included are those polypeptides with amino acid
substitutions which are "conservative" according to acidity,
charge, hydrophobicity, polarity, size or any other characteristic
known to those skilled in the art. See generally, Creighton,
Proteins, W. H. Freeman and Company, N.Y., (1984) 498 pp. plus
index, passim. One may make changes in selected amino acids so long
as such changes preserve the overall folding or activity of the
protein. Small amino terminal extensions, such as an amino-terminal
methionine residue, a small linker peptide of up to about 20-25
residues, or a small extension that facilitates purification, such
as a poly-histidine tract, an antigenic epitope or a binding
domain, may also be present. See, in general, Ford et al., Protein
Expression and Purification 2:95-107 (1991), which is herein
incorporated by reference. Polypeptides or analogs thereof may also
contain one or more amino acid analogs, such as
peptidomimetics.
[0063] In general, comprehended by the invention are pharmaceutical
compositions comprising effective amounts of chemically modified
protein, or derivative products, together with pharmaceutically
acceptable diluents, preservatives, solubilizers, emulsifiers,
adjuvants and/or carriers needed for administration. (See PCT
97/01331 hereby incorporated by reference.) The optimal
pharmaceutical formulation for a desired biologically active agent
will be determined by one skilled in the art depending upon the
route of administration and desired dosage. Exemplary
pharmaceutical compositions are disclosed in Remington's
Pharmaceutical Sciences (Mack Publishing Co., 18th Ed., Easton,
Pa., pgs. 1435-1712 (1990)).
[0064] The pharmaceutical compositions of the present invention are
administered as a liquid via intramuscular or subcutaneous route
and undergo a phase change wherein a gel is formed within the body,
since the body temperature will be above the gelation temperature
of the material. The release rates and duration for the particular
biologically active agents will be a function of, inter alia,
hydrogel density and the molecular weight of the agent.
[0065] Therapeutic uses of the compositions of the present
invention depend on the biologically active agent used. One skilled
in the art will readily be able to adapt a desired biologically
active agent to the present invention for its intended therapeutic
uses. Therapeutic uses for such agents are set forth in greater
detail in the following publications hereby incorporated by
reference including drawings. Therapeutic uses include but are not
limited to uses for proteins like interferons (see, U.S. Pat. Nos.
5,372,808, 5,541,293, hereby incorporated by reference including
drawings), interleukins (see, U.S. Pat. No. 5,075,222, hereby
incorporated by reference including drawings), erythropoietins
(see, U.S. Pat. Nos. 4,703,008, 5,441,868, 5,618,698 5,547,933, and
5,621,080 hereby incorporated by reference including drawings),
granulocyte-colony stimulating factors (see, U.S. Pat. Nos.
4,999,291, 5,581,476, 5,582,823, 4,810,643 and PCT Publication No.
94/17185, hereby incorporated by reference including drawings),
stem cell factor (PCT Publication Nos. 91/05795, 92/17505 and
95/17206, hereby incorporated by reference including drawings),
novel erythropoiesis stimulating protein (PCT Publication No.
94/09257, hereby incorporated by reference including drawings), and
the OB protein (see PCT publication Nos. 96/40912, 96/05309,
97/00128, 97/01010 and 97/06816 hereby incorporated by reference
including figures). In addition, the present compositions may also
be used for manufacture of one or more medicaments for treatment or
amelioration of the conditions the biologically active agent is
intended to treat.
[0066] In the sustained-release compositions of the present
invention, an effective amount of active ingredient will be
utilized. As used herein, sustained release refers to the gradual
release of active ingredient from the polymer matrix, over an
extended period of time. 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.
Typically, a sustained release of the active agent will be for a
period of days to weeks, depending upon the desired therapeutic
effect.
[0067] The following examples are offered to more fully illustrate
the invention, but are not to be construed as limiting the scope
thereof.
[0068] Materials
[0069] Low molecular weight (Mn 2000-6000) PLGA (poly lactic
acid-co-glycolic acid) and PLA (poly lactic acid) were synthesized
by direct thermal condensation of glycolic acid and lactic acid at
180.degree. C. under reduced pressure. High molecular weight PLGAs
were obtained from B.I. Chemicals. Polyethylene glycols (PEG) were
obtained from Fluka Chemicals. Leptin, zinc-leptin, G-CSF,
Fc-Leptin, and Fc-OPG were obtained from Amgen Inc. All other
chemicals are from sources well known in the art.
EXAMPLE 1
[0070] This example describes synthesis of a hydroxy-terminated
A-B-A (PLGA-PEG-PLGA), tri block copolymer by thermal condensation
(FIG. 2, Scheme 1a).
[0071] 30 grams PLGA (75%/25% LA/GA ratio) (Mn 3740, MW 7050) and
10.7 grams polyethylene glycol (MW 1000) were placed into a
three-neck round bottom flask equipped with a thermometer, a
nitrogen gas inlet, and a distillation condenser connected to a
vacuum pump. After addition of the polymers, the temperature of the
reaction mixture was raised slowly to 160.degree. C. under nitrogen
purging. The condensation reaction was further carried out at
160.degree. C. for 14 hours under 500 millitorr pressure and with
continuous bubbling of dry nitrogen gas. At the end of the
condensation reaction, the reaction mixture was cooled, dissolved
in methylene chloride and precipitated with an excess of cold
isopropanol.
[0072] The isolated polymer was dried at 40.degree. 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 by .sup.1H-NMR.
[0073] The PLGA-PEG-PLGA tri block copolymer dissolved either in
100 mM sodium acetate, pH 6.0, or 100 mM sodium phosphate, pH 7.0,
exhibited a unique thermoreversible property (solution below room
temperature and gel above room temperature, sol-gel-sol) with lower
critical solution temperature (LCST) at about 30.degree. C. to
35.degree. C.
EXAMPLE 2
[0074] This example describes modification of hydroxy-terminated
PLGA-PEG-PLGA tri block copolymer to carboxylic acid-terminated
PLGA-PEG-PLGA tri block copolymer (FIG. 2, Scheme 1b).
[0075] To a hydroxy-terminated PLGA-PEG-PLGA copolymer (30 grams)
described in Example 1, 120 ml of anhydrous 1,4-dioxane was added
under continuous nitrogen purging. After complete dissolution of
the polymer, 8.57 grams of succinic anhydride (Sigma) in
1,4-dioxane was added, followed by addition of 1.9 grams
triethylamine (Aldrich) and 2.3 grams of 4-dimethylaminopyridine
(Aldrich). 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 methylene chloride (40 ml) and precipitating from ether.
The isolated polymer was dried at 40.degree. C. under vacuum for 48
hours. The dried acid-terminated block copolymer (21 grams) was
dissolved in 400 ml of 100 mM sodium phosphate buffer (pH 7.4), and
filtered through 0.45 .mu.m filter. The polymer solution was then
placed in a dialysis membrane (2,000 Molecular Weight cut-off)
(Spectrum) and dialyzed against deionized water at 4.degree. C.
After dialysis, the polymer solution was lyophilized and the dried
polymer was stored at -20.degree. C. under a nitrogen
environment.
[0076] 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 .sup.1H-NMR.
[0077] The carboxy-terminated PLGA-PEG-PLGA tri block copolymer
dissolved in 100 mM sodium acetate, pH 4.8 exhibited similar
thermoreversible gelation as described in Example 1 (solution below
room temperature and gel above room temperature, sol-gel-sol) with
lower critical solution temperature (LCST) of about 30.degree. C.
to 35.degree. C. The carboxy-terminated PLGA-PEG-PLGA tri block
hydrogel also demonstrated complete de-gelation as the pH of the
hydrogel gradually increased from acidic to neutral under
physiological conditions.
EXAMPLE 3
[0078] This example describes synthesis of carboxylic
acid-terminated PLGA-PEG-PLGA tri block copolymers using different
weight ratios of PLGA to PEG.
[0079] The synthesis procedures described in Examples 1 and 2 were
utilized to prepare carboxy-terminated PLGA-PEG-PLGA tri block
copolymers with various PLGA to PEG ratios (See Table 1 below). All
the tri block copolymers listed below showed thermoreversible
gelation (sol-gel-sol) with LCST in the range of 25.degree.
C.-35.degree. C.
2TABLE 1 PLGA PLGA (MW) (LA/GA PLGA/PEG Polymer PEG (MW) (Mn) molar
ratio) (w/w) 1 1000 3550 75/25 64/36 2 1000 3550 75/25 66/34 3 1000
3550 75/25 70/30 4 1000 4200 75/25 72/28 5 1000 3500 75/25 74/26 6
1000 3500 75/25 76/24 7 1000 3158 100/0 72/28 8 1000 3557 56/44
72/28
EXAMPLE 4
[0080] This example describes synthesis of carboxylic
acid-terminated PLGA-PEG-PLGA tri block copolymer by condensation
of PLGA with activated PEG. (FIG. 1, Scheme 2).
[0081] Under a nitrogen stream, 1 gram PEG-bis-isocynate
(NCO-PEG-NCO, MW 980 from Shearwater Polymers, Inc.), 2.3 grams
PLGA (Mn 1652, polydispersity 1.4) and 1.1 grams dibutyltin
dilaurate (Aldrich) were added to a 100 ml flask with 30 ml
anhydrous methylene chloride. The reaction mixture was stirred at
room temperature for 24 hours under nitrogen environment. The
reaction was followed by IR spectrophotometer. After completion of
the reaction a crude polymer was isolated from the solution by
precipitation using excess of diethyl ether/petroleum ether (50/50
(v/v)). The isolated polymer was dried at 40.degree. C. under
vacuum for 48 hours. The dried acid terminated block copolymer (2
grams) was dissolved in 38 ml of 100 mM sodium phosphate buffer (pH
7.4), and filtered through a 0.45 .mu.m filter. The polymer
solution was placed in a dialysis membrane (2,000 Molecular Weight
cut-off) (Spectrum) and dialyzed against deionized water at
4.degree. C. After dialysis, the polymer solution was lyophilized
and the dried polymer was stored at -20.degree. C. under nitrogen
environment.
[0082] 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 .sup.1H-NMR. The tri block copolymer synthesized
by this method exhibited similar pH/thermoreversible gelation as
described in above examples.
EXAMPLE 5
[0083] The following example demonstrates pH dependent gelation of
the carboxy-terminated PLGA-PEG-PLGA tri block copolymer
solution.
[0084] The carboxy-terminated PLGA-PEG-PLGA tri block copolymer
described in Example 2 was dissolved in 50 mM sodium acetate or
sodium phosphate buffers to obtain 30% (by weight) polymer solution
with final pH in the range of 4.0-8.0. One milliliter polymer
solution, formulated in different pH buffers, was placed in a glass
vial at 37.degree. C. and the gelation was monitored visually as a
function of time. The results are summarized in Table 2. As
depicted in Table 2, the carboxy-terminated tri block copolymer
solution showed pH dependent gelation with no sol-gel property at
any time above pH 6.5. All the tri block copolymers listed in Table
1 (Example 3) showed similar pH sensitive gelation at 37.degree.
C.
3TABLE 2 Initial hydrogel pH Gel formation at 37.degree. C. 4 quick
gel 4.5 quick gel 5.0 quick gel 5.5 quick gel 6.0 slow gelation 6.5
highly viscous solution 7.0 no gel any time 7.4 no gel any time 8.0
no gel any time
EXAMPLE 6
[0085] The following example demonstrates pH dependent de-gelation
(gel to solution) of the carboxy-terminated PLGA-PEG-PLGA
hydrogel.
[0086] The carboxy-terminated PLGA-PEG-PLGA tri block copolymer
described in Example 2 was dissolved in 50 mM sodium acetate buffer
to obtain 30% (by weight) polymer solution with final pH 4.5. One
milliliter polymer solution was placed into dialysis cassettes (MW
cutoff 10,000) (Pierce). The cassettes were then placed in a
37.degree. C. incubator to ensure gelation of the hydrogel inside
the dialysis cassettes. Upon complete gelation, each cassette was
placed in a beaker containing 500 ml buffer with various pHs
ranging from 4.0 to 7.4 and incubated at 37.degree. C. The
consistency of the gel at different pHs was monitored as a function
of time over one week period. The observations are summarized in
Table 3. As depicted in Table 3, the hydrogel from the cassette
degelled into a solution, due to increase in a pH of the hydrogel
during buffer exchange with external buffer when the pH of the
external medium was 6.5 or higher. The gel remains intact and firm
at all pHs below 6.0. The experiment suggests that the invented
acid-terminated PLGA-PEG-PLGA tri block copolymer hydrogel is
pH-responsive to a change in surrounding pH. All the block
copolymers listed in Table 1 (Example 3) showed similar degelation
at closer to neutral pH.
4TABLE 3 External Buffer Hydrogel morphology pH 4.0 gel over one
week pH 5.0 gel over one week pH 5.5 gel over one week pH 6.0 soft
gel in 2-3 days and solution within one week pH 7.4 solution in 1-2
hours
EXAMPLE 7
[0087] This example demonstrates manipulation of the rate of
de-gelation of the hydrogel by blending carboxy-terminated PLGA/PEG
block copolymers with hydroxy-terminated PLGA/PEG block
copolymers.
[0088] 30% (by weight) solutions of hydroxy-terminated
PLGA-PEG-PLGA tri block copolymer described in Example 1 (Polymer
A) and carboxylic acid-terminated PLGA-PEG-PLGA tri block copolymer
described in Example 2 (Polymer B) were prepared separately by
dissolving the polymers in 50 mM sodium acetate buffer. The final
pHs of both the solutions were adjusted to 4.5 using dilute
solutions of either hydrochloric acid or sodium hydroxide. The two
polymer solutions were mixed together with different proportions to
obtain solutions of polymer blends with various weight ratios of
polymer A to polymer B.
[0089] One ml of each polymer blend solutions was placed in an
individual dialysis cassette (MW cutoff 10,000) (Pierce) and the
cassettes were then placed in a 37.degree. C. incubator to ensure
gelation of the hydrogel inside the dialysis cassettes. Upon
complete gelation the cassettes were placed in a beaker containing
500 ml sodium phosphate buffer, pH 7.4, incubated at 37.degree. C.
The consistency of the hydrogel and rate of de-gelation (conversion
of the hydrogel into a solution) was monitored as a function of
time over one week period. The observations are summarized in Table
4. As depicted in Table 4 the rate of de-gelation of the hydrogel,
under physiological conditions, was increased with increasing the
amount of carboxylic acid terminated block copolymer in the
blend.
5 TABLE 4 Sample (by weight %) Degelation rate 100% A Firm gel over
one week 80% A + 20% B Soft gel after 4 days Viscous solution after
1 week 50% A + 50% B Solution within 6-10 hours 100% B Solution
within 1 hour A: Hydroxy-terminated PLGA-PEG-PLGA copolymers B:
Carboxy-terminated PLGA-PEG-PLGA copolymers
EXAMPLE 8
[0090] This example demonstrates clearance of the hydrogel depot
from the injection site of normal mice.
[0091] The hydrogel solutions with different weight ratios of
hydroxy-terminated and carboxy-terminated PLGA-PEG-PLGA tri block
copolymers were prepared as described in example 7. Mice were
injected subcutaneously with 100 .mu.l of the hydrogel blend
solutions. At desirable time points 2 mice from each group were
sacrificed by carbon dioxide asphyxiation. A small incision was
made near the site of injection and the skin was peeled back
carefully so as not to disturb the hydrogel depot. After exposing
the injection site, surrounding tissues were carefully dissected
away to allow clear observation of the surrounding tissue and the
hydrogel depot. The gross visual observation was recorded and the
injection sites were photographed using polaroid camera. The
observations are summarized in Table 5. As depicted in Table 5, the
rate of disappearance of the hydrogel depot from the injection site
was gradually increased with increasing the amount
carboxy-terminated tri block copolymer in the hydrogel blend.
6 TABLE 5 Sample (by weight %) Clearance from injection site 100% A
4-6 weeks 90% A + 10% B 2-3 weeks 80% A + 20% B 3 days 70% A + 30%
B 1 day 100% B 1-2 hours A: Hydroxy-terminated PLGA-PEG-PLGA
copolymers B: Carboxy-terminated PLGA-PEG-PLGA copolymers
EXAMPLE 9
[0092] This example describes the preparation of a leptin/hydrogel
formulation and the methods used to determine the in vitro release
kinetics, and in vivo bioactivity of the leptin/hydrogel
formulation.
[0093] Preparation of Leptin/Hydrogel Formulation
[0094] The hydroxy-terminated PLGA-PEG-PLGA tri block copolymer
described in Example 1 and carboxy-terminated PLGA-PEG-PLGA block
copolymer described in Example 2 were dissolved separately in 50 mM
sodium acetate buffer, pH 6.0. The two polymer solutions were mixed
with different proportions to obtain blends with various ratios of
carboxy-terminated to hydroxy-terminated copolymers. Leptin
solution (formulated in 10 mM acetate, pH 4.0) was slowly added to
the hydrogel solution and the mixture was gently swirled on an
orbital shaker at 5.degree. C. to assure a homogeneous mixing of
leptin throughout the hydrogel solution. The final concentration of
the copolymer was 28% (by weight) with pH 4.5. The leptin
concentration in leptin/hydrogel formulations was 20 mg/ml. The
final leptin/hydrogel formulation was filtered through 0.2 .mu.m
filter and stored either as a solution at 5.degree. C. or stored as
a frozen mass at -20.degree. C.
[0095] In Vitro Release Study
[0096] The in vitro release of leptin from the leptin/hydrogel
formulation was carried out in 20 mM sodium phosphate, pH 7.4, at
37.degree. C. One gram of leptin/hydrogel solution formulation was
placed in a glass vial at 37.degree. C. Upon gelation of the
leptin/hydrogel formulation, 1 ml of 20 mM phosphate, pH 7.4,
buffer was added directly above and in contact with the gel. The
amount of leptin released in the top buffer phase was determined by
UV spectrophotometer at 280 nm as well as by SEC-HPLC at 220 nm. To
maintain a perfect sink condition the aqueous receptor phase above
the gel was completely removed at definite time intervals and
replaced by fresh buffer. The % leptin released over time is
depicted in FIG. 2. The integrity of the leptin released from the
hydrogel formulation was confirmed by gel & HPLC.
[0097] In Vivo Bioactivity
[0098] The in vivo bioactivity of leptin/hydrogel formulations were
evaluated in normal mice.
[0099] Mice were injected subcutaneously (s.c.) with either: a) 0.1
ml of 10 mM acetate buffer, pH 4.0, (n=5, day 0 only); (b) 0.1 ml
of 20 mg/ml leptin formulated in 10 mM acetate buffer, pH 4.0 (n=5,
100 mg/kg, day 0 only); (c) 0.1 ml of a leptin/hydrogel blend
solution (95% hydroxy-terminated+5% carboxy-terminated polymer)
(w/w)) formulation consisting of 20 mg/ml leptin, in 10 mM acetate,
pH 4.0 (n=5, 100 mg/kg, day 0 only); (d) 0.1 ml of a
leptin/hydrogel blend solution (70% hydroxy-terminated+30%
carboxy-terminated polymer) (w/w)) formulation consisting of 20
mg/ml leptin, in 10 mM acetate, pH 4.0 (n=5, 100 mg/kg ,day 0
only).
[0100] % body weight change (from the day 0 body weight) was
determined by weighing the animals daily until the body weight of
the animals injected with sample (b), (c) and (d) reached the body
weights of the animals injected with buffer control (sample (a)).
Importantly, a single s.c. injection of 100 mg/kg leptin/hydrogel
formulations (samples (c), (d)) showed sustained weight loss in
normal mice over a 5 day period (FIG. 3).
EXAMPLE 10
[0101] This example describes the incorporation of Fc-leptin into
the hydrogel and the results of in vitro release studies using the
formulation.
[0102] Fc-leptin solution (formulated in 10 mM phosphate, 2.7%
arginine, 0.01% Tween-20, pH 6.0) was added to the copolymer
hydrogel blend solution (formulated in 50 mM acetate, pH 6.0) as
described in Example 7. The final concentration of the copolymer in
the Fc-leptin/hydrogel formulation was 10-30% (w/w) and the
Fc-leptin concentration was in the range of 20 mg/ml. The in vitro
release of Fc-leptin from the hydrogel was carried out in 20 mM
sodium phosphate buffer, pH 7.4, at 37.degree. C. as described in
Example 9. The Fc-leptin/hydrogel formulation showed sustained
release of Fc-leptin over a 7-10 day period of time.
EXAMPLE 11
[0103] This example describes the incorporation of BDNF into the
hydrogel and the results of in vitro release studies using the
formulation.
[0104] BDNF solution (formulated in 10 mM sodium phosphate, 150 mM
sodium chloride, pH 7.0) was added to the copolymer hydrogel blend
solution (formulated in 50 mM acetate, pH 6.0) as described in
Example 7. The final concentration of the copolymer in the
BDNF/hydrogel formulation was 20-30% (w/w) and the BDNF
concentration was in the range of 20 mg/ml. The in vitro release of
BDNF from the hydrogel was carried out in 20 mM sodium phosphate
buffer, pH 7.4, at 37.degree. C. as described in Example 9. The
release of BDNF could be maintained over a 6-9 day period of
time.
[0105] The present invention has been described in terms of
particular embodiments found or proposed to comprise preferred
modes for the practice of the invention. It will be appreciated by
those of ordinary skill in the art that, in light of the present
disclosure, numerous modifications and changes can be made in the
particular embodiments exemplified without departing from the
intended scope of the invention.
Sequence CWU 1
1
1 1 146 PRT Human Leptin 1 Val Pro Ile Gln Lys Val Gln Asp Asp Thr
Lys Thr Leu Ile Lys Thr 1 5 10 15 Ile Val Thr Arg Ile Asn Asp Ile
Ser His Thr Gln Ser Val Ser Ser 20 25 30 Lys Gln Lys Val Thr Gly
Leu Asp Phe Ile Pro Gly Leu His Pro Ile 35 40 45 Leu Thr Leu Ser
Lys Met Asp Gln Thr Leu Ala Val Tyr Gln Gln Ile 50 55 60 Leu Thr
Ser Met Pro Ser Arg Asn Val Ile Gln Ile Ser Asn Asp Leu 65 70 75 80
Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala Phe Ser Lys Ser Cys 85
90 95 His Leu Pro Trp Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly
Gly 100 105 110 Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala
Leu Ser Arg 115 120 125 Leu Gln Gly Ser Leu Gln Asp Met Leu Trp Gln
Leu Asp Leu Ser Pro 130 135 140 Gly Cys
* * * * *