CA2335852A1 - Thermosensitive biodegradable hydrogels for sustained delivery of biologically active agents - Google Patents
Thermosensitive biodegradable hydrogels for sustained delivery of biologically active agents Download PDFInfo
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- CA2335852A1 CA2335852A1 CA002335852A CA2335852A CA2335852A1 CA 2335852 A1 CA2335852 A1 CA 2335852A1 CA 002335852 A CA002335852 A CA 002335852A CA 2335852 A CA2335852 A CA 2335852A CA 2335852 A1 CA2335852 A1 CA 2335852A1
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
- A61K9/0024—Solid, semi-solid or solidifying implants, which are implanted or injected in body tissue
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
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 thermosensitive, biodegradable hydrogels, consisting of a block copolymer of poly(d, l- or l-lactic acid) (PLA) or poly(lactide-co-glycolide) (PLGA) and polyethylene glycol (PEG), for the sustained delivery of biologically active agents.
Description
THERMOSENSITIVE BIODEGRADABLE HYDROGELS FOR SUSTAINED
DELIVERY OF BIOLOGICALLY ACTIVE AGENTS
FIELD OF THE INVENTION
The present invention relates to the use of thermosensitive, biodegradable hydrogels, consisting of a block copolymer of poly(d,l- or 1-lactic acid)(PLA) or poly(lactide-co-glycolide)(PLGA) and polyethylene glycol (PEG), for the sustained delivery of biologically active agents.
BACKGROUND OF THE INVENTIQN
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), granulocyte colony-stimulating factor (G-CSF), interferons (alpha, beta, gamma, consensus), tumor necrosis factor binding protein (TNFbp), interleukin-1 receptor antagonist (IL-lra), 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.
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.
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. Patent No.
4,675,189 and references cited therein).
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.
DELIVERY OF BIOLOGICALLY ACTIVE AGENTS
FIELD OF THE INVENTION
The present invention relates to the use of thermosensitive, biodegradable hydrogels, consisting of a block copolymer of poly(d,l- or 1-lactic acid)(PLA) or poly(lactide-co-glycolide)(PLGA) and polyethylene glycol (PEG), for the sustained delivery of biologically active agents.
BACKGROUND OF THE INVENTIQN
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), granulocyte colony-stimulating factor (G-CSF), interferons (alpha, beta, gamma, consensus), tumor necrosis factor binding protein (TNFbp), interleukin-1 receptor antagonist (IL-lra), 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.
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.
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. Patent No.
4,675,189 and references cited therein).
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.
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 DepotT"", which consists of injectable microcapsules which release leuprolide acetate for about 30 days for the treatment of prostate cancer.
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.
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 polyethylene 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.
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.
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 polyethylene 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.
Cha et al., U.S. Patent No. 5,702,717 (Dec. 30, 1997) 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 temperature and below, 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 palymer block comprising a member selected from the group consisting of poly(a-hydroxy acids) and polyethylene 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°C. Although Cha et al. propose use of their hydrogels for controlled release of drugs, no such demonstration is provided.
Martini et al., J. Chem. Soc., 90(13):1961-1966 (1994) describe low molecular weight ABA type triblock copolymers which utilize hydrophobic poly(s-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.
Stratton et al., PCT/US97/13479 (WO 98/02142) January 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, systems utilizing Pluronics suffer from the fact that they are toxic to body organs and are nonbiodegradable. Moreover, only high molecular weight Pluronics at higher concentrations (25-40 wt. o) exhibit thermoreversible gelation.
It is thus the object of the present invention to provide thermosensitive, biodegradable hydrogels for the sustained delivery of drugs. The hydrogels of the present invention utilize copolymer compositions which provide for instant gelation, and which possess the necessary rate of degradation to make use of the hydrogels commercially practical.
CRY OF THE INVENTION
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 block copolymer which is biodegradable, exhibits thermal gelation behavior, and is capable of providing for the sustained-release of the biologically active agent.
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
Figure 1 depicts the two methods by which the A-B-A block copolymers of the present invention can be prepared.
Figure 2 depicts the in vitro release characteristics of leptin released from a hydrogel (PLGA/PEG (74~/26o w/w)). ~ protein released is plotted vs. time (days).
Figure 3 is a photograph of an SDS-PAGE gel characterizing samples of leptin released from a hydrogel on various days. Lane 1 is a leptin standard;
Lane 2 and 15 contain molecular weight markers; and Lanes 3-14 represent leptin samples at day 1-12, respectively.
Figure 4 depicts the in vivo bioactivity for various leptin-containing hydrogel (PLGA/PEG (74~5/26~
w/w)) formulations. The -*- depicts a 20mM acetate, pH 4.8, buffer control, 1001 on day 0; -~- depicts a hydrogel (74~/26~) control, 1001 on day 0; -depicts leptin (20 mg/mL), 100 mg/kg, 1001 on day 0;
-~- depicts leptin (2 mg/mL), 10 mg/kg, 1001 daily; -1- depicts a leptin-containing hydrogel (PLGA/PEG
(74°s/26~ w/w)), 20 mg/mL leptin, 100 mg/kg, 1001 on day 0; and -~- depicts a leptin-containing hydrogel (PLGA/PEG (74~/26~ w/w)), 20 mg/mL leptin, 200 mg/kg, 200~t1 on day 0. ~ body weight change (from the day 0 body weight) is plotted vs. time (days).
Figure 5 depicts the pharmacokinetics for a leptin-containing hydrogel (PLGA/PEG (74%/26~ w/w)) (-~-) and leptin solution (-~-). Serum leptin concentration (ng/mL) is plotted vs. time (hours).
Figure 6 depicts the in vitro release characteristics for GCSE from a GCSF-containing hydrogel (PLGA/PEG (74~/26~ w/w)) (-~-) and Fc-OPG from a Fc-OPG-containing hydrogel (PLGA/PEG (74~/26~ w/w)) (-~-). % protein released is plotted vs. time (days).
Figure 7 is a photograph of an SDS-PAGE gel characterizing samples of GCSF released from a hydrogel on various days. Lane 1 is a leptin standard; Lane 2 and 15 contain molecular weight markers; and Lanes 3-14 represent leptin samples at day 1-12, respectively.
Figure 8 depicts the in vivo bioactivity for a Zn:leptin-containing hydrogel (PLGA/PEG (74~/26~
w/w)) formulation. -*- depicts a 20mM acetate, pH 4.8, buffer control, 100~t1 on day 0; -~- depicts Zn:leptin, 100 mg/kg, 1001 on day 0; and -1- depicts a Zn:leptin-containing hydrogel (PLGA/PEG (74~/26~ w/w)), 20 mg/mL
leptin, 100 mg/kg, 100~t1 on day 0. ~ body weight 30. change (from the day 0 body weight) is plotted vs. time ( days ) .
1?ETAILED DESCRIPTION OF THE INVENTION
_g_ As used herein, the following terms shall have the following meaning:
"Reverse thermal gelation" is defined as meaning the temperature below which a copolymer is soluble in water and above which the block copolymer forms a semi-solid, i.e. gels, emulsions, dispersions and suspensions.
"LCST", or lower critical solution temperature, is defined as meaning the temperature at which a biodegradable block copolymer undergoes reverse thermal gelation. For purposes of the present invention, the term "LSCT" can be used interchangeably with "reverse thermal gelation temperature".
"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.
"Biodegradable" is defined as meaning that the block copolymer will erode or degrade in vivo to form smaller non-toxic components.
"Parenteral administration" is defined as meaning any route of administration other than the alimentary canal, including, for example, subcutaneous and intramuscular.
The present invention involves utilization of block copolymers having hydrophobic ("A") block segments and hydrophilic ("B") block segments. The block copolymers are triblock copolymers, e.g., ABA or BAB type block copolymers, which possess reverse thermal gelation properties and are biodegradable and biocompatible. Importantly, triblock copolymers of the present invention provide instant gelation and possess the necessary rate of degradation to be commercially useful.
_g_ Biodegradable hydrophobic A block segments contemplated for use include poly(a-hydroxy acid) members derived from or selected from the group consisting of homopolymers and copolymers of poly(lactide)s (d, l- or 1- forms), poly(glycolide)s, polyanhydrides, polyorthoesters, polyetheresters, polycaprolactone, polyesteramides, polycarbonate, polycyanoacrylate, polyurethanes, polyacrylate, blends and copolymers thereof.
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), and the PLGA composition will be such that the necessary rate of gelation and rate of degradation are obtained.
The range of molecular weights contemplated for the 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.
Hydrophilic B block segments contemplated for use include polyethylene glycols having average molecular weights of between about 500 and 10,000.
The copolymer compositions for the 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°C) so as to minimize the initial burst of drug. In the hydrogels of the present invention the hydrophobic A block makes up 55~ to 90~
by weight of the copolymer and the hydrophilic B block makes up 10~ to 45~ of the copolymer.
The concentration at which the block copolymers of the present invention remain soluble below the LCBT are generally up to about 60~ by weight, with 10~-30o preferred. The concentration utilized will depend upon the copolymer composition actually used, as well as whether or not a gel or emulsion is desired.
The thermosensitive block copolymers of the present invention can be prepared by thermal condensation. In a typical experiment, 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°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 1H-NMR
spectroscopy.
Alternatively, the 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 ABA triblock copolymer is prepared by stannous octoate catalyzed ring-opening polymerization of d,l-dilactide (or PLGA) using a,w-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 hydrophobicites. The relative A and B
block lengths can be confirmed by 1H-NMR spectroscopy.
The process used to mix the copolymers with a biologically active agent and/or other materials involves dissolving the ABA block capolymers 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 mixing of the biologically active agent throughout the copolymer. Alternatively, the process can involve the dissolving of the ABA 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 or solidifies 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.
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 25mM to 500mM and in the pH range of 4.0 to 8.5.
It is also envisioned that other excipients, e.g., various sugars, salts, 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. Examples of such sugars include glucose or sucrose in the range of 5~ to 20~. Examples of such salts include sodium chloride or zinc chloride in the range of 0.5% to 100.
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. Patent 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.
Proteins contemplated for use would include but are not limited to interferon consensus (fee, U.S.
Patent Nos. 5,372,808, 5,541,293 4,897,471, and 4,695,623 hereby incorporated by reference including drawings), interleukins (see, U.S. Patent No.
5,075,222, hereby incorporated by reference including drawings), erythropoietins (see, U.S. Patent 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 ( ee, U.S. Patent 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).
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.
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 i.v. injections of various proteins such as leptin.
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), bone growth factors such as osteoprotegerin (OPG), insulin-like growth factors (IGFs), macrophage colony stimulating factor (M-CSF), granulocyte macrophage colony stimulating factor (GM-CSF), megakaryocyte derived growth factor (MGDF), 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.
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.
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)).
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 re:Lease 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.
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. Patent Nos.
5,372,808, 5,541,293, hereby incorporated by reference including drawings), interleukins (see, U.S. Patent No.
5,075,222, hereby incorporated by reference including drawings), erythropoietins (see, U.S. Patent 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. Patent 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), 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.
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 a week or more, preferably up to one month.
The following examples are offered to more fully illustrate the invention, but are not to be construed as limiting the scope thereof.
Materials 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°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, GCSF, Fc-Leptin, and Fc-OPG
were obtained from Amgen Inc. All other chemicals are from sources well known in the art.
Example 1 This example describes synthesis of a PLGA/PEG, A-B-A (PLGA-PEG-PLGA), block copolymer by thermal condensation. The thermal condensation method is generally depicted in Figure 1.
30 g PLGA (75~/25~ LA/GA ratio) (Mn 3740, MW 7050) and 10.7 g 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°C under nitrogen purging. The condensation reaction was further carried out at 160°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.
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 by 1H-NMR.
The PLGA/PEG block copolymer dissolved either in 100mM sodium acetate, pH 6.0, or 100mM 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°C to 35°C.
Example 2 This example describes the synthesis of PLGA/PEG, A-B-A (PLGA-PEG-PLGA), block copolymers using PLGA with different lactic acid to g:Lycolic acid ratios.
The synthesis and characterization procedures described in Example 1 were utilized to prepare PLGA/PEG block copolymers using PLGA with different LA
to GA ratios (see Table 1 below). The block copolymers listed below showed thermoreversibility (Sol-gel-sol) with LCST at about 30°C to 35°C.
2 5 PLGA (LA/GA ratio) pEG 1000 PLGA/PEG PLGA/PEG Molar ratio wt (a) wt (a) w wr;n calcu a d by HI~iR
PLA (100%) 458 17.558 72/28 1.56 1.48 (Mn 3480, Mw 6590) 3 0 PLGA (75/25%) 308 11.748 72/28 1.65 1.56 (Mn 3740, MW 7090) PLGA (50/50%) 308 10.718 74/26 1.8 1.78 35 (~ 3480/MW 6590) PLGA (56/44%) 408 15.608 72/28 1.71 1.66 (Mn 3480/Mw 6590) Exam~l a 3 This example describes the synthesis of PLGA/PEG, A-B-A (PLGA-PEG-PLGA), block copolymers using different weight ratios of PLGA and PEG.
The synthesis and characterization procedure described in Example 1 were utilized to prepare PLGA/PEG block copolymers with various PLGA to PEG
ratios (see Table 2 below). All of the block copolymers listed below showed thermoreversibility (sol-gel-sol) with LCST in the range of 25°C - 35°C.
Table 2 PLGA (75/25)PEG 1000 PLGA/PEG PLGA/PEG Molar ratio 1 Mri.3790.MWwt () w atio cal-ylatedby NMR
308 9.478 76/24 2.03 408 14.288 74/26 1.79 1.70 408 14.908 73/27 I.72 1.65 308 11.848 72/28 1.65 1.56 2 308 12.638 70/30 1.53 1.50 308 14.218 68/32 1.36 1.32 308 15.488 66/34 1.24 1.17 308 16.708 64/36 1.15 1.08 308 18.408 62/38 1.05 Exams This example describes the preparation of a leptin/hydrogel formulation and the methods used to determine the in vitro release kinetics, in vivo release kinetics, and pharmacokinetics of the leptin/hydrogel.
Preparation of leptin/hydrogel formulation The PLGA/PEG block copolymer described in Example 1 was dissolved in 50mM sodium acetate, pH 6Ø
Leptin solution (formulated in 20mM acetate, pH 4.8) was slowly added to the hydrogel solution and the mixture was gently swirled on an orbital shaker at 5°C
to assure a homogeneous mixing of leptin throughout the hydrogel solution. The final concentration of the copolymer in the final leptin/hydrogel formulation was 10-50 (w/w) and the leptin concentration was in the range of 0-100 mg/ml. The final leptin/hydrogel formulation was filtered through 0.2~x filter and stored either as a solution at 5°C or stored as a frozen mass at -20°C.
Alternatively, the leptin/hydrogel formulation was prepared by dissolving the PLGA/PEG
block copolymer in a leptin solution. The leptin solution concentration was varied to obtain desirable copolymer as well as the desired protein concentration in final formulation.
In vitro Release Study The in vitro release of leptin from the leptin/hydrogel was carried~out in 20mM sodium phosphate, 5~ sorbitol, pH 7.4, at 37°C. 1 ml of leptin/hydrogel solution formulation was placed in a glass vial at 37°C. Upon gelation of the leptin/hydrogel formulation, 1 ml of 20mM phosphate, 5~ sorbitol, 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 280nm as well as by SEC-HPLC at 220nm. 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 Figure 2. The integrity of the leptin released from the hydrogel formulation was confirmed by HPLC (data not shown) and gel electrophoresis (SDS-PAGE)(see Figure 3).
In vivo bioactivity The in vivo bioactivity of leptin/hydrogel formulations were evaluated in normal mice.
Mice were injected subcutaneously (s. c.) with either:
a) 0.1 ml of 20mM acetate buffer, pH 4.8, (n=5, day 0 only); (b) 0.1 ml of 20 mg/ml leptin formulated in 20mM
acetate buffer, pH 4.8 (n=5, 100 mg/kg, day 0 only);
(c) 0.1 ml of 2 mg/ml leptin formulated in 20mM acetate buffer, pH 4.8 (n=5, 10 mg/kg, daily) ; (d) 0.1 ml of a leptin/hydrogel (74/26 (PLGA/PEG)(w/w)) formulation consisting of 20 mg/ml leptin, in 20mM acetate, pH 4.8 (n=5, 100 mg/kg, day 0 only) ; (e) 0.2 ml of a leptin/hydrogel (74/26 (PLGA/PEG)(w/w)) formulation consisting of 20 mg/ml leptin, in 20mM acetate, pH 4.8 (n=5, 200 mg/kg ,day 0 only); or (f) 0.1 ml of a hydrogel (74/26 (PLGA/PEG)(w/w)) control, formulated in 50mM acetate, pH 6.0 (n=5, day 0 only).
~ 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), (d) and (e) 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 formulation (sample (d)) showed sustained weight loss in normal mice over a 10 day period. The duration of sustained weight loss effect was further extended up to 14 days when the dose was increased to 200 mg/kg (sample (e)). It was also observed that a single injection of 100 mg/kg or 200 mg/kg leptin/hydrogel at day '0' was more efficacious up to 14 days than daily injections of 10 mg/kg leptin without hydrogel. These results are depicted in Figure 4.
Pharmacokinetics Study A pharmacokinetics study was carried out in male rats. After a single s.c. injection of either:
1) 100 mg/kg dose of leptin (20 mg/ml) formulated in 20mM acetate buffer, pH 4.8); or 2) a leptin/hydrogel (74/260 (PLGA/PEG)(w/w)) formulation consisting of 20 mg/ml leptin, in 20mM acetate, pH 4.8, blood samples were collected at various time intervals and analyzed for leptin by ELISA assay. As shown in Figure 5, serum concentrations of leptin were detectable for up to 168 hours for animals injected with the :leptin/hydrogel formulation.
This example describes the incorporation of G-CSF into the hydrogel and the results of in vitro release studies using the formulation.
GCSF solution (formulated in lOmM acetate, 5~ sucrose, pH 4.0) was added to the copolymer hydrogel solution (formulated in 20mM acetate, pH 6.0) as described in Example 4. The final concentration of the copolymer in the GCSF/hydrogel formulation was 10-50~
(w/w) and the GCSF concentration was in the range of 1-20 mg/ml. The in vitro release of GCSE from the hydrogel was carried out in 20mM sodium phosphate buffer, pH 7.4, at 37°C as described in Example 4.
The o GCSF released over time is shown in Figure 6. As depicted in Figure 6, nearly 1000 of the GCSF is released over a 9-10 day period of time. The integrity of the GCSF released from the hydrogel formulation was confirmed by HPLC (data not shown) and gel electrophoresis (SDS-PAGE)(see Figure 7).
Example 6 This example describes the incorporation of an Fc-OPG protein into the hydrogel and the results of in vitro release studies using the Fc-OPG/hydrogel formulation.
The Fc-OPG/hydrogel formulation was prepared as described in Example 4 by adding Fc-OPG solution (formulated in lOmM sodium acetate, 5~ sorbitol, 0.02 mg/ml tween 20, pH 5.0) to the copolymer solution (formulated in 50mM acetate, pH 6.0). The in vitro release of Fc-OPG from the hydrogel was carried out in 20mM sodium phosphate buffer, pH 7.4, at 37°C as described in Example 4. The ~ Fc-OPG released over time is shown in Figure 6. As depicted in Figure 6, nearly 100 of the Fc-OPG is released over a 8-9 day period of time.
Example 7 This example describes incorporation of the Zn:leptin suspension into PLGA/PEG hydrogel and the results of in vivo release kinetics of the leptin from the Zn:leptin/hydrogel.
The PLGA/PEG block polymers described in the examples above were hydrated in 100mM Tris, pH 8.0 buffer. The final pH of the hydrogel solution was maintained between 6.5 - 7.0 and then a zinc chloride Wp 00/00222 PCT/US99/14206 solution was added to the hydrogel to obtain a O.lmM ZnClz concentration in the final hydrogel solution. To this hydrogel solution, a Zn:leptin suspension was added as described in Example 4. The final Zn:leptin concentration in the hydrogel described in this example was 20 mg/ml. The in vivo bioactivity of a Zn:leptin/hydrogel (74/26 (PLGA/PEG)(w/w)) formulation was carried out as described in Example 4. The results of the in vivo bioactivity studies are depicted in Figure 8.
Example 8 This example describes the incorporation of Zn:GCSF into the PLGA/PEG hydrogel and the results of in vitro release studies using the formulation.
The PLGA/PEG block copolymer described in the examples above was hydrated in 100mM PIPES, pH 7.5 buffer. The final pH of the hydrogel solution was maintained between 6.5 - 7.0 and then a zinc chloride solution was added to the hydrogel to obtain a 0.lmM ZnClz concentration in the final hydrogel solution. To this hydrogel solution, a Zn:GCSF suspension was added as described in Example 4. The in vitro release of GCSE
from the hydrogel was carried out in 20mM sodium phosphate buffer, pH 7.4, at 37°C, as described in Example 4. It was demonstrated that sustained release of GCSE could be obtained from these hydrogel formulations.
ExamRle 99 This example describes the incorporation of GCSF-crystals in the PLGA/PEG hydrogel and the results of in vitro release studies using the formulation.
The block polymer described in the examples above was hydrated in 100mM MES, pH 7.5 buffer. The final pH of the hydrogel solution was maintained between 6.5 - 7.0 and then a MgClZ solution was added to the hydrogel to obtain a 0.2M MgClZ concentration in the final hydrogel solution. To this hydrogel solution, a GCSE
crystals suspension was added as described in Example 4.
The in vitro release of GCSF from the hydrogel was carried out in 20mM sodium phosphate buffer, pH 7.4, at 37°C, as described in Example 4. It was demonstrated that sustained release of GCSE could be obtained from these hydrogel formulations.
scamp l a 10 This example describes the effect of various excipients on the LCST of PLGA/PLGA, A-B-A block copolymers. As indicated in Table 4 below, the addition of various sugars, salts, surfactants, etc. can effect the rate of gelation and LOST of the hydrogels.
Excinient Added r n., rpiwr;n" f on .raT
Sugars at 5%-20% T rate of gelation Lowered LCST
(e.g. glucose, sucrose) Firm gel Salts at 0.5%-10% T rate of gelation Lowered LOST
3~ (e.g. NaCl,, ZnCl,,Na,SO,) Firm gel Surfactants .~ rate of gelation -----(e.g. Tween, SDS) Soft gel 3 5 Glycerol at 2%-10% T rate of gelation Lowered LCST
(e.g. NaCl,, ZnCl,,Na,SO,) Firm gel Polyethylene glycol ~. rate of gelation Increased LCST
at 5%-20% Soft gel 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.
Cha et al., U.S. Patent No. 5,702,717 (Dec. 30, 1997) 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 temperature and below, 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 palymer block comprising a member selected from the group consisting of poly(a-hydroxy acids) and polyethylene 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°C. Although Cha et al. propose use of their hydrogels for controlled release of drugs, no such demonstration is provided.
Martini et al., J. Chem. Soc., 90(13):1961-1966 (1994) describe low molecular weight ABA type triblock copolymers which utilize hydrophobic poly(s-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.
Stratton et al., PCT/US97/13479 (WO 98/02142) January 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, systems utilizing Pluronics suffer from the fact that they are toxic to body organs and are nonbiodegradable. Moreover, only high molecular weight Pluronics at higher concentrations (25-40 wt. o) exhibit thermoreversible gelation.
It is thus the object of the present invention to provide thermosensitive, biodegradable hydrogels for the sustained delivery of drugs. The hydrogels of the present invention utilize copolymer compositions which provide for instant gelation, and which possess the necessary rate of degradation to make use of the hydrogels commercially practical.
CRY OF THE INVENTION
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 block copolymer which is biodegradable, exhibits thermal gelation behavior, and is capable of providing for the sustained-release of the biologically active agent.
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
Figure 1 depicts the two methods by which the A-B-A block copolymers of the present invention can be prepared.
Figure 2 depicts the in vitro release characteristics of leptin released from a hydrogel (PLGA/PEG (74~/26o w/w)). ~ protein released is plotted vs. time (days).
Figure 3 is a photograph of an SDS-PAGE gel characterizing samples of leptin released from a hydrogel on various days. Lane 1 is a leptin standard;
Lane 2 and 15 contain molecular weight markers; and Lanes 3-14 represent leptin samples at day 1-12, respectively.
Figure 4 depicts the in vivo bioactivity for various leptin-containing hydrogel (PLGA/PEG (74~5/26~
w/w)) formulations. The -*- depicts a 20mM acetate, pH 4.8, buffer control, 1001 on day 0; -~- depicts a hydrogel (74~/26~) control, 1001 on day 0; -depicts leptin (20 mg/mL), 100 mg/kg, 1001 on day 0;
-~- depicts leptin (2 mg/mL), 10 mg/kg, 1001 daily; -1- depicts a leptin-containing hydrogel (PLGA/PEG
(74°s/26~ w/w)), 20 mg/mL leptin, 100 mg/kg, 1001 on day 0; and -~- depicts a leptin-containing hydrogel (PLGA/PEG (74~/26~ w/w)), 20 mg/mL leptin, 200 mg/kg, 200~t1 on day 0. ~ body weight change (from the day 0 body weight) is plotted vs. time (days).
Figure 5 depicts the pharmacokinetics for a leptin-containing hydrogel (PLGA/PEG (74%/26~ w/w)) (-~-) and leptin solution (-~-). Serum leptin concentration (ng/mL) is plotted vs. time (hours).
Figure 6 depicts the in vitro release characteristics for GCSE from a GCSF-containing hydrogel (PLGA/PEG (74~/26~ w/w)) (-~-) and Fc-OPG from a Fc-OPG-containing hydrogel (PLGA/PEG (74~/26~ w/w)) (-~-). % protein released is plotted vs. time (days).
Figure 7 is a photograph of an SDS-PAGE gel characterizing samples of GCSF released from a hydrogel on various days. Lane 1 is a leptin standard; Lane 2 and 15 contain molecular weight markers; and Lanes 3-14 represent leptin samples at day 1-12, respectively.
Figure 8 depicts the in vivo bioactivity for a Zn:leptin-containing hydrogel (PLGA/PEG (74~/26~
w/w)) formulation. -*- depicts a 20mM acetate, pH 4.8, buffer control, 100~t1 on day 0; -~- depicts Zn:leptin, 100 mg/kg, 1001 on day 0; and -1- depicts a Zn:leptin-containing hydrogel (PLGA/PEG (74~/26~ w/w)), 20 mg/mL
leptin, 100 mg/kg, 100~t1 on day 0. ~ body weight 30. change (from the day 0 body weight) is plotted vs. time ( days ) .
1?ETAILED DESCRIPTION OF THE INVENTION
_g_ As used herein, the following terms shall have the following meaning:
"Reverse thermal gelation" is defined as meaning the temperature below which a copolymer is soluble in water and above which the block copolymer forms a semi-solid, i.e. gels, emulsions, dispersions and suspensions.
"LCST", or lower critical solution temperature, is defined as meaning the temperature at which a biodegradable block copolymer undergoes reverse thermal gelation. For purposes of the present invention, the term "LSCT" can be used interchangeably with "reverse thermal gelation temperature".
"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.
"Biodegradable" is defined as meaning that the block copolymer will erode or degrade in vivo to form smaller non-toxic components.
"Parenteral administration" is defined as meaning any route of administration other than the alimentary canal, including, for example, subcutaneous and intramuscular.
The present invention involves utilization of block copolymers having hydrophobic ("A") block segments and hydrophilic ("B") block segments. The block copolymers are triblock copolymers, e.g., ABA or BAB type block copolymers, which possess reverse thermal gelation properties and are biodegradable and biocompatible. Importantly, triblock copolymers of the present invention provide instant gelation and possess the necessary rate of degradation to be commercially useful.
_g_ Biodegradable hydrophobic A block segments contemplated for use include poly(a-hydroxy acid) members derived from or selected from the group consisting of homopolymers and copolymers of poly(lactide)s (d, l- or 1- forms), poly(glycolide)s, polyanhydrides, polyorthoesters, polyetheresters, polycaprolactone, polyesteramides, polycarbonate, polycyanoacrylate, polyurethanes, polyacrylate, blends and copolymers thereof.
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), and the PLGA composition will be such that the necessary rate of gelation and rate of degradation are obtained.
The range of molecular weights contemplated for the 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.
Hydrophilic B block segments contemplated for use include polyethylene glycols having average molecular weights of between about 500 and 10,000.
The copolymer compositions for the 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°C) so as to minimize the initial burst of drug. In the hydrogels of the present invention the hydrophobic A block makes up 55~ to 90~
by weight of the copolymer and the hydrophilic B block makes up 10~ to 45~ of the copolymer.
The concentration at which the block copolymers of the present invention remain soluble below the LCBT are generally up to about 60~ by weight, with 10~-30o preferred. The concentration utilized will depend upon the copolymer composition actually used, as well as whether or not a gel or emulsion is desired.
The thermosensitive block copolymers of the present invention can be prepared by thermal condensation. In a typical experiment, 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°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 1H-NMR
spectroscopy.
Alternatively, the 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 ABA triblock copolymer is prepared by stannous octoate catalyzed ring-opening polymerization of d,l-dilactide (or PLGA) using a,w-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 hydrophobicites. The relative A and B
block lengths can be confirmed by 1H-NMR spectroscopy.
The process used to mix the copolymers with a biologically active agent and/or other materials involves dissolving the ABA block capolymers 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 mixing of the biologically active agent throughout the copolymer. Alternatively, the process can involve the dissolving of the ABA 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 or solidifies 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.
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 25mM to 500mM and in the pH range of 4.0 to 8.5.
It is also envisioned that other excipients, e.g., various sugars, salts, 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. Examples of such sugars include glucose or sucrose in the range of 5~ to 20~. Examples of such salts include sodium chloride or zinc chloride in the range of 0.5% to 100.
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. Patent 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.
Proteins contemplated for use would include but are not limited to interferon consensus (fee, U.S.
Patent Nos. 5,372,808, 5,541,293 4,897,471, and 4,695,623 hereby incorporated by reference including drawings), interleukins (see, U.S. Patent No.
5,075,222, hereby incorporated by reference including drawings), erythropoietins (see, U.S. Patent 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 ( ee, U.S. Patent 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).
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.
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 i.v. injections of various proteins such as leptin.
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), bone growth factors such as osteoprotegerin (OPG), insulin-like growth factors (IGFs), macrophage colony stimulating factor (M-CSF), granulocyte macrophage colony stimulating factor (GM-CSF), megakaryocyte derived growth factor (MGDF), 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.
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.
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)).
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 re:Lease 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.
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. Patent Nos.
5,372,808, 5,541,293, hereby incorporated by reference including drawings), interleukins (see, U.S. Patent No.
5,075,222, hereby incorporated by reference including drawings), erythropoietins (see, U.S. Patent 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. Patent 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), 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.
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 a week or more, preferably up to one month.
The following examples are offered to more fully illustrate the invention, but are not to be construed as limiting the scope thereof.
Materials 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°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, GCSF, Fc-Leptin, and Fc-OPG
were obtained from Amgen Inc. All other chemicals are from sources well known in the art.
Example 1 This example describes synthesis of a PLGA/PEG, A-B-A (PLGA-PEG-PLGA), block copolymer by thermal condensation. The thermal condensation method is generally depicted in Figure 1.
30 g PLGA (75~/25~ LA/GA ratio) (Mn 3740, MW 7050) and 10.7 g 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°C under nitrogen purging. The condensation reaction was further carried out at 160°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.
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 by 1H-NMR.
The PLGA/PEG block copolymer dissolved either in 100mM sodium acetate, pH 6.0, or 100mM 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°C to 35°C.
Example 2 This example describes the synthesis of PLGA/PEG, A-B-A (PLGA-PEG-PLGA), block copolymers using PLGA with different lactic acid to g:Lycolic acid ratios.
The synthesis and characterization procedures described in Example 1 were utilized to prepare PLGA/PEG block copolymers using PLGA with different LA
to GA ratios (see Table 1 below). The block copolymers listed below showed thermoreversibility (Sol-gel-sol) with LCST at about 30°C to 35°C.
2 5 PLGA (LA/GA ratio) pEG 1000 PLGA/PEG PLGA/PEG Molar ratio wt (a) wt (a) w wr;n calcu a d by HI~iR
PLA (100%) 458 17.558 72/28 1.56 1.48 (Mn 3480, Mw 6590) 3 0 PLGA (75/25%) 308 11.748 72/28 1.65 1.56 (Mn 3740, MW 7090) PLGA (50/50%) 308 10.718 74/26 1.8 1.78 35 (~ 3480/MW 6590) PLGA (56/44%) 408 15.608 72/28 1.71 1.66 (Mn 3480/Mw 6590) Exam~l a 3 This example describes the synthesis of PLGA/PEG, A-B-A (PLGA-PEG-PLGA), block copolymers using different weight ratios of PLGA and PEG.
The synthesis and characterization procedure described in Example 1 were utilized to prepare PLGA/PEG block copolymers with various PLGA to PEG
ratios (see Table 2 below). All of the block copolymers listed below showed thermoreversibility (sol-gel-sol) with LCST in the range of 25°C - 35°C.
Table 2 PLGA (75/25)PEG 1000 PLGA/PEG PLGA/PEG Molar ratio 1 Mri.3790.MWwt () w atio cal-ylatedby NMR
308 9.478 76/24 2.03 408 14.288 74/26 1.79 1.70 408 14.908 73/27 I.72 1.65 308 11.848 72/28 1.65 1.56 2 308 12.638 70/30 1.53 1.50 308 14.218 68/32 1.36 1.32 308 15.488 66/34 1.24 1.17 308 16.708 64/36 1.15 1.08 308 18.408 62/38 1.05 Exams This example describes the preparation of a leptin/hydrogel formulation and the methods used to determine the in vitro release kinetics, in vivo release kinetics, and pharmacokinetics of the leptin/hydrogel.
Preparation of leptin/hydrogel formulation The PLGA/PEG block copolymer described in Example 1 was dissolved in 50mM sodium acetate, pH 6Ø
Leptin solution (formulated in 20mM acetate, pH 4.8) was slowly added to the hydrogel solution and the mixture was gently swirled on an orbital shaker at 5°C
to assure a homogeneous mixing of leptin throughout the hydrogel solution. The final concentration of the copolymer in the final leptin/hydrogel formulation was 10-50 (w/w) and the leptin concentration was in the range of 0-100 mg/ml. The final leptin/hydrogel formulation was filtered through 0.2~x filter and stored either as a solution at 5°C or stored as a frozen mass at -20°C.
Alternatively, the leptin/hydrogel formulation was prepared by dissolving the PLGA/PEG
block copolymer in a leptin solution. The leptin solution concentration was varied to obtain desirable copolymer as well as the desired protein concentration in final formulation.
In vitro Release Study The in vitro release of leptin from the leptin/hydrogel was carried~out in 20mM sodium phosphate, 5~ sorbitol, pH 7.4, at 37°C. 1 ml of leptin/hydrogel solution formulation was placed in a glass vial at 37°C. Upon gelation of the leptin/hydrogel formulation, 1 ml of 20mM phosphate, 5~ sorbitol, 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 280nm as well as by SEC-HPLC at 220nm. 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 Figure 2. The integrity of the leptin released from the hydrogel formulation was confirmed by HPLC (data not shown) and gel electrophoresis (SDS-PAGE)(see Figure 3).
In vivo bioactivity The in vivo bioactivity of leptin/hydrogel formulations were evaluated in normal mice.
Mice were injected subcutaneously (s. c.) with either:
a) 0.1 ml of 20mM acetate buffer, pH 4.8, (n=5, day 0 only); (b) 0.1 ml of 20 mg/ml leptin formulated in 20mM
acetate buffer, pH 4.8 (n=5, 100 mg/kg, day 0 only);
(c) 0.1 ml of 2 mg/ml leptin formulated in 20mM acetate buffer, pH 4.8 (n=5, 10 mg/kg, daily) ; (d) 0.1 ml of a leptin/hydrogel (74/26 (PLGA/PEG)(w/w)) formulation consisting of 20 mg/ml leptin, in 20mM acetate, pH 4.8 (n=5, 100 mg/kg, day 0 only) ; (e) 0.2 ml of a leptin/hydrogel (74/26 (PLGA/PEG)(w/w)) formulation consisting of 20 mg/ml leptin, in 20mM acetate, pH 4.8 (n=5, 200 mg/kg ,day 0 only); or (f) 0.1 ml of a hydrogel (74/26 (PLGA/PEG)(w/w)) control, formulated in 50mM acetate, pH 6.0 (n=5, day 0 only).
~ 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), (d) and (e) 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 formulation (sample (d)) showed sustained weight loss in normal mice over a 10 day period. The duration of sustained weight loss effect was further extended up to 14 days when the dose was increased to 200 mg/kg (sample (e)). It was also observed that a single injection of 100 mg/kg or 200 mg/kg leptin/hydrogel at day '0' was more efficacious up to 14 days than daily injections of 10 mg/kg leptin without hydrogel. These results are depicted in Figure 4.
Pharmacokinetics Study A pharmacokinetics study was carried out in male rats. After a single s.c. injection of either:
1) 100 mg/kg dose of leptin (20 mg/ml) formulated in 20mM acetate buffer, pH 4.8); or 2) a leptin/hydrogel (74/260 (PLGA/PEG)(w/w)) formulation consisting of 20 mg/ml leptin, in 20mM acetate, pH 4.8, blood samples were collected at various time intervals and analyzed for leptin by ELISA assay. As shown in Figure 5, serum concentrations of leptin were detectable for up to 168 hours for animals injected with the :leptin/hydrogel formulation.
This example describes the incorporation of G-CSF into the hydrogel and the results of in vitro release studies using the formulation.
GCSF solution (formulated in lOmM acetate, 5~ sucrose, pH 4.0) was added to the copolymer hydrogel solution (formulated in 20mM acetate, pH 6.0) as described in Example 4. The final concentration of the copolymer in the GCSF/hydrogel formulation was 10-50~
(w/w) and the GCSF concentration was in the range of 1-20 mg/ml. The in vitro release of GCSE from the hydrogel was carried out in 20mM sodium phosphate buffer, pH 7.4, at 37°C as described in Example 4.
The o GCSF released over time is shown in Figure 6. As depicted in Figure 6, nearly 1000 of the GCSF is released over a 9-10 day period of time. The integrity of the GCSF released from the hydrogel formulation was confirmed by HPLC (data not shown) and gel electrophoresis (SDS-PAGE)(see Figure 7).
Example 6 This example describes the incorporation of an Fc-OPG protein into the hydrogel and the results of in vitro release studies using the Fc-OPG/hydrogel formulation.
The Fc-OPG/hydrogel formulation was prepared as described in Example 4 by adding Fc-OPG solution (formulated in lOmM sodium acetate, 5~ sorbitol, 0.02 mg/ml tween 20, pH 5.0) to the copolymer solution (formulated in 50mM acetate, pH 6.0). The in vitro release of Fc-OPG from the hydrogel was carried out in 20mM sodium phosphate buffer, pH 7.4, at 37°C as described in Example 4. The ~ Fc-OPG released over time is shown in Figure 6. As depicted in Figure 6, nearly 100 of the Fc-OPG is released over a 8-9 day period of time.
Example 7 This example describes incorporation of the Zn:leptin suspension into PLGA/PEG hydrogel and the results of in vivo release kinetics of the leptin from the Zn:leptin/hydrogel.
The PLGA/PEG block polymers described in the examples above were hydrated in 100mM Tris, pH 8.0 buffer. The final pH of the hydrogel solution was maintained between 6.5 - 7.0 and then a zinc chloride Wp 00/00222 PCT/US99/14206 solution was added to the hydrogel to obtain a O.lmM ZnClz concentration in the final hydrogel solution. To this hydrogel solution, a Zn:leptin suspension was added as described in Example 4. The final Zn:leptin concentration in the hydrogel described in this example was 20 mg/ml. The in vivo bioactivity of a Zn:leptin/hydrogel (74/26 (PLGA/PEG)(w/w)) formulation was carried out as described in Example 4. The results of the in vivo bioactivity studies are depicted in Figure 8.
Example 8 This example describes the incorporation of Zn:GCSF into the PLGA/PEG hydrogel and the results of in vitro release studies using the formulation.
The PLGA/PEG block copolymer described in the examples above was hydrated in 100mM PIPES, pH 7.5 buffer. The final pH of the hydrogel solution was maintained between 6.5 - 7.0 and then a zinc chloride solution was added to the hydrogel to obtain a 0.lmM ZnClz concentration in the final hydrogel solution. To this hydrogel solution, a Zn:GCSF suspension was added as described in Example 4. The in vitro release of GCSE
from the hydrogel was carried out in 20mM sodium phosphate buffer, pH 7.4, at 37°C, as described in Example 4. It was demonstrated that sustained release of GCSE could be obtained from these hydrogel formulations.
ExamRle 99 This example describes the incorporation of GCSF-crystals in the PLGA/PEG hydrogel and the results of in vitro release studies using the formulation.
The block polymer described in the examples above was hydrated in 100mM MES, pH 7.5 buffer. The final pH of the hydrogel solution was maintained between 6.5 - 7.0 and then a MgClZ solution was added to the hydrogel to obtain a 0.2M MgClZ concentration in the final hydrogel solution. To this hydrogel solution, a GCSE
crystals suspension was added as described in Example 4.
The in vitro release of GCSF from the hydrogel was carried out in 20mM sodium phosphate buffer, pH 7.4, at 37°C, as described in Example 4. It was demonstrated that sustained release of GCSE could be obtained from these hydrogel formulations.
scamp l a 10 This example describes the effect of various excipients on the LCST of PLGA/PLGA, A-B-A block copolymers. As indicated in Table 4 below, the addition of various sugars, salts, surfactants, etc. can effect the rate of gelation and LOST of the hydrogels.
Excinient Added r n., rpiwr;n" f on .raT
Sugars at 5%-20% T rate of gelation Lowered LCST
(e.g. glucose, sucrose) Firm gel Salts at 0.5%-10% T rate of gelation Lowered LOST
3~ (e.g. NaCl,, ZnCl,,Na,SO,) Firm gel Surfactants .~ rate of gelation -----(e.g. Tween, SDS) Soft gel 3 5 Glycerol at 2%-10% T rate of gelation Lowered LCST
(e.g. NaCl,, ZnCl,,Na,SO,) Firm gel Polyethylene glycol ~. rate of gelation Increased LCST
at 5%-20% Soft gel 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.
Claims (20)
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 wherein said injectable polymeric matrix is maintained at a temperature below the lower critical solution temperature of said polymeric matrix.
2. The composition of claim 1, wherein said polymeric matrix is a biodegradable block copolymer comprising:
(a) 55% to 90% by weight of a hydrophobic A polymer block and;
(b) 10% to 45% by weight of a hydrophilic B polymer block comprising a polyethylene glycol having an average molecular weight of between 500-10000.
(a) 55% to 90% by weight of a hydrophobic A polymer block and;
(b) 10% to 45% by weight of a hydrophilic B polymer block comprising a polyethylene glycol having an average molecular weight of between 500-10000.
3. 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.
4. The composition of claim 3, 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.
5. The composition of claim 4, wherein said poly(.alpha.-hydroxy acid) is poly lactide-co-glycolide (PLGA).
6. The composition of claim 5, wherein said block copolymer is a triblock copolymer having a configuration selected from the group consisting of ABA
or BAB block segments.
or BAB block segments.
7. The composition of claim 6, 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.
8. The composition of claim 7 further comprising an excipient which will vary the lower critical solution temperature and increase the rate of gelation of said block copolymer.
9. 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), tissue plasminogen activator (TPA), urokinase, streptokinase and kallikrein.
10. The composition of claim 1, wherein said biologically active agent is a small molecule.
11. 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 of said polymeric matrix, which comprises:
(a) providing an injectable liquid polymeric matrix comprising a biodegradable block copolymer having reverse thermal gelation 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 drug 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.
(a) providing an injectable liquid polymeric matrix comprising a biodegradable block copolymer having reverse thermal gelation 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 drug 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.
12. The method of claim 11, wherein said polymeric matrix is a biodegradable block copolymer comprising:
(a) 55% to 90% by weight of a hydrophobic A polymer block and;
(b) 10% to 45% by weight of a hydrophilic B polymer block comprising a polyethylene glycol having an average molecular weight of between 500-10000.
(a) 55% to 90% by weight of a hydrophobic A polymer block and;
(b) 10% to 45% by weight of a hydrophilic B polymer block comprising a polyethylene glycol having an average molecular weight of between 500-10000.
13. The method of claim 12, wherein said hydrophobic A polymer block is a poly(.alpha.-hydroxy acid) having an average molecular weight of between 1000-20,000.
14. The method of claim 13, wherein said poly(.alpha.-hydroxy acid) is poly lactide-co-glycolide (PLGA).
15. The method of claim 14, wherein said block copolymer is a triblock copolymer having a configuration selected from the group consisting of ABA
or BAB block segments.
or BAB block segments.
16. The method of claim 15, 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.
17. The method of claim 16 further comprising an excipient which will vary the lower critical solution temperature and increase the rate of gelation of said block copolymer.
18. The method of claim 11, wherein said biologically active agent is a protein selected from the group consisting of interferon consensus, interleukins, erythropoietins, granulocyte-colony stimulating factor (GCSE), 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), tissue plasminogen activator (TPA), urokinase, streptokinase and kallikrein.
19. The method of claim 11, wherein said biologically active agent is a small molecule.
20. A method for the preparation of a medicament of a biologically active, or a derivative, analog, fusion, conjugate, or chemically modified form thereof, in a biodegradable polymeric matrix which comprises:
(a) providing an injectable liquid polymeric matrix comprising a biodegradable block copolymer having reverse thermal gelation properties, and into which a biologically active agent has been incorporated; and (b) maintaining said liquid polymeric matrix at a temperature below the lower critical solution temperature of said polymeric matrix;
wherein said medicament is intended for use in a warm blooded animal with the resultant sustained release of said agent concomitant with biodegradation of said polymeric matrix.
(a) providing an injectable liquid polymeric matrix comprising a biodegradable block copolymer having reverse thermal gelation properties, and into which a biologically active agent has been incorporated; and (b) maintaining said liquid polymeric matrix at a temperature below the lower critical solution temperature of said polymeric matrix;
wherein said medicament is intended for use in a warm blooded animal with the resultant sustained release of said agent concomitant with biodegradation of said polymeric matrix.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US10733498A | 1998-06-30 | 1998-06-30 | |
| US09/107,334 | 1998-06-30 | ||
| PCT/US1999/014206 WO2000000222A1 (en) | 1998-06-30 | 1999-06-25 | Thermosensitive biodegradable hydrogels for sustained delivery of biologically active agents |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2335852A1 true CA2335852A1 (en) | 2000-01-06 |
Family
ID=22316103
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
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| CA002335852A Abandoned CA2335852A1 (en) | 1998-06-30 | 1999-06-25 | Thermosensitive biodegradable hydrogels for sustained delivery of biologically active agents |
Country Status (9)
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|---|---|
| EP (1) | EP1091761A1 (en) |
| JP (1) | JP2002519333A (en) |
| KR (1) | KR20010053259A (en) |
| CN (1) | CN1321092A (en) |
| AU (1) | AU754003B2 (en) |
| CA (1) | CA2335852A1 (en) |
| HU (1) | HUP0102803A3 (en) |
| IL (1) | IL140268A0 (en) |
| WO (1) | WO2000000222A1 (en) |
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| GB0417401D0 (en) | 2004-08-05 | 2004-09-08 | Controlled Therapeutics Sct | Stabilised prostaglandin composition |
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| PT1034207E (en) * | 1997-10-03 | 2005-07-29 | Macromed Inc | COPOLYMERS OF POLY (LACTIDO-CO-GLYCOLIDES) AND POLYETHYLENE GLYCOLIDES, OF LOW MOLECULAR WEIGHT, BIODEGRADABLE, WITH PROPERTIES OF REVERSE THERMAL GELIFICATION |
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1999
- 1999-06-25 EP EP99933564A patent/EP1091761A1/en not_active Withdrawn
- 1999-06-25 HU HU0102803A patent/HUP0102803A3/en unknown
- 1999-06-25 CN CN99810059A patent/CN1321092A/en active Pending
- 1999-06-25 CA CA002335852A patent/CA2335852A1/en not_active Abandoned
- 1999-06-25 WO PCT/US1999/014206 patent/WO2000000222A1/en not_active Application Discontinuation
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- 1999-06-25 JP JP2000556807A patent/JP2002519333A/en not_active Withdrawn
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| HUP0102803A3 (en) | 2004-06-28 |
| HUP0102803A2 (en) | 2001-11-28 |
| AU754003B2 (en) | 2002-10-31 |
| WO2000000222A1 (en) | 2000-01-06 |
| CN1321092A (en) | 2001-11-07 |
| EP1091761A1 (en) | 2001-04-18 |
| AU4959999A (en) | 2000-01-17 |
| JP2002519333A (en) | 2002-07-02 |
| IL140268A0 (en) | 2002-02-10 |
| WO2000000222A9 (en) | 2000-07-20 |
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| Date | Code | Title | Description |
|---|---|---|---|
| EEER | Examination request | ||
| FZDE | Discontinued |