Title:  Thermosensitive biodegradable hydrogels for sustained delivery of leptin

United States Patent:  6,541,033

Issued:  April 1, 2003

Inventors:  Shah; Subodh (Newbury Park, CA)

Assignee:  Amgen Inc. (Thousand Oaks, CA)

Appl. No.:  107603

Filed:  June 30, 1998

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, such as leptin.

DETAILED DESCRIPTION OF THE INVENTION

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.

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, 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 370C.) 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 LCST are generally 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.

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^oC. 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 ¹ H-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 .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 ¹ H-NMR spectroscopy.

The process used to mix the copolymers with a biologically active agent and/or other materials involves dissolving the ABA 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 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 25 mM to 500 mM 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 10%.

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.

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).

The present leptin proteins used are preferably those with amino acid sequence of natural human leptin; see Zhang et al., Nature372:425-432 (1994); see also, the Correction at Nature374: 479 (1995), optionally with an N-terminal methionyl residue incident to bacterial expression is used. (See, Materials and Methods, infra). PCT publication No. WO 96/05309, published Feb. 22, 1996, entitled, "Modulators of Body Weight, Corresponding Nucleic Acids and Proteins, and Diagnostic and Therapeutic Uses Thereof" fully sets forth leptin protein and related compositions and methods, and is herein incorporated by reference. An amino acid sequence for human leptin protein is set forth at WO 96/05309 Seq. ID Nos. 4 and 6 (at pages 172 and 174 of that publication), and the first amino acid residue of the mature protein is at position 22 and is a valine residue. The mature protein is 146 residues (or 145 if the glutamine at position 49 is absent, Seq. ID No. 4). Specific leptin derivatives contemplated for use in the present invention include glycosylated leptins, Fc-leptin fusions, succinylated-leptin, and zinc derivatized leptin. It is desirable to have such leptin containing sustained-release compositions as such compositions could serve to enhance the effectiveness of either exogenously administered or endogenous leptin, or could be used, for example, to reduce or eliminate the need for exogenous leptin administration.

Moreover, because the materials utilized in the present invention are biocompatible and biodegradable, use of the leptin compositions of the present invention help prevent adverse injection site reactions normally associated with i.v. injections of leptin by improving the solubility properties of the leptin.

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 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.

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 leptin are set forth in greater detail in the following publications (see PCT publication Nos. 96/40912, 96/05309, 97/00128, 97/01010 and 97/06816 hereby incorporated by reference including figures).

Therapeutic uses of leptin include weight modulation, the treatment or prevention of diabetes, blood lipid reduction (and treatment of related conditions), increasing lean body mass and increasing insulin sensitivity.

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.

Claim 1 of 9 Claims

What is claimed is:

1. A pharmaceutical composition for the sustained administration of an effective amount of leptin, or a derivative, analog, fusion, conjugate, or chemically modified form thereof, comprising an injectable biodegradable polymeric matrix into which said leptin has been incorporated, said polymeric matrix having reverse thermal gelation properties; wherein said injectable polymeric matrix is maintained at a temperature below the lower critical solution temperature of said polymeric matrix, and 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 500-10,000.
 

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