WO2010014689A1 - Pharmaceutical formulations comprising elastin-like proteins - Google Patents

Abstract

The present invention provides stable pharmaceutical compositions or formulations, including solution and non-solution phase formulations, and methods of preparing the same. The formulations of the invention may improve the stability and/or solubility of various pharmaceutically-active ingredients, including biological molecules and small drug compounds, and may be tailored to a variety of storage conditions. The formulations of the invention may provide an alternative to lyophilization, or may be employed together with lyophilization or other methods of stabilization. Generally, the compositions comprise a pharmaceutically active ingredient, such as a biological molecule or small drug compound, and a pharmaceutically acceptable carrier. The carrier comprises an Elastin-Like Peptide (ELP) in an amount and form (e.g., soluble or insoluble form) effective for stabilizing the pharmaceutically active ingredient.

Description

PHARMACEUTICAL FORMULATIONS COMPRISING ELASTIN-LIKE PROTEINS

PRIORITY

[01] This application claims priority to US Provisional Application No. 61/084,499, filed on July 29, 2008, which is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

[02] The present invention relates to pharmaceutically-active ingredients, including

ELP fusion proteins, stabilized by formulation with one or more Elastin-Like Proteins (ELPs).

BACKGROUND

[03] Conventional methods for stabilizing pharmaceutically active ingredients include lyophilization and/or formulation with certain biological or chemical molecules. Lyophilization can be a complex and expensive procedure, and some pharmaceutically- active ingredients, including certain proteins, peptides, and mammalian cells, are difficult to lyophilize. Further, such active ingredients may be difficult to reconstitute after lyophilization, in part due to denaturation during the drying process. Other methods for stabilizing pharmaceutically-active ingredients include formulation with plasma-derived albumin or chemical polymers such as PEG. However, such conventional formulation methods cannot be sufficiently tailored for a given active agent and anticipated storage conditions.

[04] Alternative means for stabilizing pharmaceutically active ingredients, e.g., as an alternative or in addition to lyophilization or other method of stabilization, are needed.

SUMMARY OF THE INVENTION

[05] The present invention provides stable pharmaceutical compositions or formulations, including liquid and non-solution phase formulations, and methods of preparing and using the same. The formulations of the invention improve the stability and/or solubility of various pharmaceutically-active ingredients, including biological molecules (e.g., ELP fusion proteins) and small drug compounds, and may be tailored to a variety of storage conditions. The formulations of the invention may provide an alternative to lyophilization, or may be employed together with lyophilization or other methods of stabilization. [06] Generally, the compositions comprise a pharmaceutically active ingredient, such as a biological molecule or small drug compound, and a pharmaceutically acceptable carrier. The pharmaceutically active ingredient may be a fusion protein between a biologically active protein (e.g., GLP-1 , Factor-VII, or insulin) and an Elastin-Like Protein (as described herein). The ELP component of the fusion generally will have transition properties suitable for the intended therapeutic effect, such as a transition property to maintain a solution form in circulation (e.g., a transition temperature above about 37° C under physiological conditions). The carrier may comprise, or may also comprise, an ELP in an amount and form (e.g., soluble or insoluble form) effective for stabilizing the pharmaceutically active ingredient. The ELP carrier, for example, may have transition properties suitable for maintaining the composition in a non-solution phase under the storage conditions, such as a transition temperature above about 4°C. The pharmaceutical composition may be provided within sealed vials or ampules, or other suitable container, in either solution or non-solution phase.

[07] These and other aspects and embodiments will be apparent in view of the following detailed description.

DETAILED DESCRIPTION OF INVENTION

[08] The present invention provides stable pharmaceutical compositions or formulations, including solution and non-solution phase formulations, and methods of preparing and using the same. The compositions of the invention comprise a pharmaceutically active ingredient, such as a biologic (e.g., ELP fusion protein) or small drug compound, and a pharmaceutically acceptable carrier. The carrier comprises an Elastin- Like Peptide (ELP) in an amount and form (e.g., solution or non-solution form) effective for stabilizing the pharmaceutically active ingredient under the storage conditions. The pharmaceutically active ingredient and the ELP carrier may or may not be covalently associated. The formulations of the invention improve the stability and/or solubility of various pharmaceutically-active ingredients, and may be tailored to a variety of storage conditions.

[09] The pharmaceutical compositions of the invention may be formulated for a variety of delivery routes, including parenteral, subcutaneous or intravenous delivery. The compositions may be in solution form, including in concentrated form, or in an insoluble form. In certain other embodiments, the pharmaceutical compositions of the invention may be lyophilized. [010] In some embodiments, the formulations of the invention are in solution phase.

For example, the pharmaceutically-active ingredient may be stabilized by adding ELP biopolymers, as described in detail below, to the active ingredient. The active ingredient and the ELP carrier may be combined by recombinant fusion and/or may be separate (non- covalently associated ingredients). The properties of the ELP may be tailored to optimize stabilization by altering amino acid content and/or length of the biopolymers as described herein. For example, amino acid content and length of the ELP(s) may be selected so as to maintain the ELP in solution phase under the anticipated storage conditions. Thus, in these embodiments, the ELP is designed so as to have a transition temperature above the storage temperature, and under the storage conditions. For example, where the product is to be stored at about room temperature (e.g., in the range of 15°C to about 25°C, or about 20° C), the ELP has a transition temperature above room temperature under the storage conditions. The storage conditions account for the pH, salt, ELP concentration, and pressure of the pharmaceutical product.

[011] Alternatively, where the solution-phase product is to remain refrigerated (e.g., from about 2°C to about 10⁰C, or about 4°C), the ELP has a transition temperature above the refrigeration temperature under the storage conditions (which may include pH, salt, ELP concentration, and pressure). In these embodiments, formulation with ELPs may serve as a replacement to expensive lyophilization or introduction of plasma-derived excipients such as albumin. Thus, the compositions of the invention may lack such plasma-derived proteins. In some embodiments, the solution is further lyophilized, with the ELP improving the properties of the lyophilized composition, including with respect to reconstitution of active ingredient prior to administration.

[012] Alternatively, formulations of the invention may be in a non-solution phase whereby the ELP biopolymer (e.g., as added to the active ingredient in solution phase, and/or as provided as a recombinant fusion with the active agent in solution phase) has been transitioned from a solution to a non-solution state, e.g., in a vial or ampule, which may be sealed for storage. Such embodiments allow for a stable, reversible co-precipitation of the product. For example, the non-solution phase of the ELP may be induced by altering the concentration of the ELP, salt, pH, temperature, pressure, and ELP amino acid content and/or length of the biopolymer, as described more fully herein. In the non-solution state the product will be protected from degradative events during storage. Prior to administration (e.g. injection), the insoluble state is reversed to a soluble state, e.g. by adding diluent, which in effect alters the concentration of ELP, salt, pH, etc. Addition of diluent essentially reverses the conditions that induced the non-solution phase. The drug is then administered in solution form. Such embodiments can take the place of expensive and complex lyophilization procedures and nevertheless permit a room temperature formulation.

[013] For non-solution phase formulations, the ELP is designed so as to have a transition temperature below the storage temperature, under the storage conditions. For example, where the product is to be stored at room temperature (e.g., from about 2°C to about 10⁰C, or about 20° C), the ELP has a transition temperature below the room temperature under the storage conditions (which may include pH, salt, ELP concentration, and pressure). Alternatively, where the product is to remain refrigerated (e.g., in the range of 15°C to about 25°C, or about 4°C), the ELP has a transition temperature below the refrigerated temperature, under the storage conditions (which may include pH, salt, ELP concentration, and pressure).

[014] In certain embodiments, the pharmaceutically active ingredient is an ELP fusion protein designed for administration by injection. In such embodiments, the ELP fusion component is designed to remain soluble under physiological conditions. The ELP carrier component, in contrast, may be added to the fusion protein to force a non-solution phase for stable storage. The distinct transition properties of the ELP fusion component and the ELP carrier provide a means for later separation by temperature cycling if desired. See US Patent 6,852,834, which is hereby incorporated by reference in its entirety.

Pharmaceutically Active Ingredient

[015] The pharmaceutical composition may provide stable formulations of a variety of pharmaceutically active ingredients, including peptides, proteins, nucleic acids (e.g., plasmid DNA, or RNA, including siRNAs or miRNAs), and other biological products including cells (e.g., mammalian cells). Exemplary pharmaceutically active ingredients include peptide hormones, enzymes, monoclonal antibodies and antigen binding portions thereof, as well as multi-chain proteins having one or more disulfide bonds. In certain other embodiments, the pharmaceutically active ingredient is a small drug compound.

[016] The pharmaceutically-active ingredient may be a fusion between a biologically active protein and an ELP, such as an ELP designed to increase circulation half- life.

[017] Exemplary pharmaceutically active ingredients, which may be provided as

ELP fusion proteins, include GLP-1 receptor agonists such as GLP-1 and exendin-4, as well as Factor Vll/Vlla, Vasoactive Intestinal Peptide, and insulin. Additional pharmaceutically active ingredients that may be formulated in accordance with the invention include: erythropoietin, magainin, beta-defensin, interferon, monoclonal antibodies, blood factors, colony stimulating factors, growth hormones, interleukins, growth factors, calcitonin, tumor necrosis factors (TNF), receptor antagonists, corticosteroids, superoxide dismutase, asparaginease, glutamase, arginase, arginine deaminase, adenosine deaminase ribonuclease, trypsin, chromotrypsin, papin, ACTH, glucagon, somatosin, somatropin, somatomedin, parathyroid hormone, hypothalamic releasing factors, prolactin, thyroid stimulating hormones, endorphins, enkephalins, and vasopressin.

[018] The pharmaceutically active protein may contain a fusion or conjugation with components other than ELP for extending half-life, such as albumin, transferrin, or PEG. Where the biologically active protein is provided as a fusion with a half-life extending protein component, the component may be positioned at the N-terminus of the biologically active protein, the C-terminus, or both.

[019] In certain embodiments, the active agent is a drug compound, such as a chemotherapeutic agent, including methotrexate, daunomycin, mitomycin, cisplatin, vincristine, epirubicin, fluorouracil, verapamil, cyclophosphamide, cytosine arabinoside, aminopterin, bleomycin, mitomycin C, democolcine, etoposide, mithramycin, chlorambucil, melphalan, daunorubicin, doxorubicin, tamosifen, paclitaxel, vincristin, vinblastine, camptothecin, actinomycin D, and cytarabine. Such drug compounds may be conjugated to ELP polymers as described in PCT/US2006/048572, which is hereby incorporated by reference.

[020] Certain active ingredients that may be formulated with ELPs in accordance with the invention are described in detail below.

Glucagon-Like Peptide (GLP)-I Receptor Agonists

[021] In certain embodiments of the invention, the active agent is a GLP-1 receptor agonist, such as GLP-1 , exendin-4, or a functional analog thereof, each of which may optionally be provided as a fusion protein (e.g., with ELP) to extend circulation half-life or other therapeutic property.

[022] Human GLP-1 is a 37 amino acid residue peptide originating from preproglucagon which is synthesized in the L-cells in the distal ileum, in the pancreas, and in the brain. Processing of preproglucagon to give GLP-1 (7-36)amιde, GLP-1 (7-37) and GLP- 2 occurs mainly in the L-cells. A simple system is used to describe fragments and analogs of this peptide. For example, Gly⁸-GLP-1 (7-37) designates a fragment of GLP-1 formally derived from GLP-1 by deleting the amino acid residues Nos. 1 to 6 and substituting the naturally occurring amino acid residue in position 8 (Ala) by GIy. Similarly, Lys³⁴ (N^ε- tetradecanoyl)-GLP-1 (7-37) designates GLP-1 (7-37) wherein the ε-amino group of the Lys residue in position 34 has been tetradecanoylated. Where reference in this text is made to C-terminally extended GLP-1 analogues, the amino acid residue in position 38 is Arg unless otherwise indicated, the optional amino acid residue in position 39 is also Arg unless otherwise indicated and the optional amino acid residue in position 40 is Asp unless otherwise indicated. Also, if a C-terminally extended analogue extends to position 41 , 42, 43, 44 or 45, the amino acid sequence of this extension is as in the corresponding sequence in human preproglucagon unless otherwise indicated.

[023] The parent peptide of GLP-1 , proglucagon (PG), has several cleavage sites that produce various peptide products dependent on the tissue of origin including glucagon (PG[32-62]) and GLP-1 [7-36]NH₂ (PG[72-107]) in the pancreas, and GLP-1 [7-37] (PG[78- 108]) and GLP-1 [7-36]NH₂ (PG [78-107]) in the L cells of the intestine where GLP-1 [7- 36]NH₂ (78-107 PG) is the major product. The GLP-1 component in accordance with the invention may be any biologically active product or derivative of proglocagon, or functional analog thereof, including: GLP-1 (1-35), GLP-1 (1-36), GLP-1 (1-36)amιde, GLP-1 (1-37), GLP-1 (1-38), GLP-1 (1-39), GLP-1 (1-40), GLP-1 (1 -41 ), GLP-1 (7-35), GLP-1 (7- 36), GLP-1 (7-36)amιde, GLP-1 (7-37), GLP-1 (7-38), GLP-1 (7-39), GLP-1 (7-40) and GLP- 1 (7- 41 ), or a analog of the foregoing. Generally, the GLP-1 component in some embodiments may be expressed as GLP-1 (A-B), where A is an integer from 1 to 7 and B is an integer from 38 to 45, optionally with one or more amino acid substitutions as defined below.

[024] As an overview, after processing in the intestinal L-cells, GLP-1 is released into the circulation, most notably in response to a meal. The plasma concentration of GLP-1 rises from a fasting level of approximately 15 pmol/L to a peak postprandial level of 40 pmol/L. For a given rise in plasma glucose concentration, the increase in plasma insulin is approximately threefold greater when glucose is administered orally compared with intravenously (Kreymann et al., 1987, Lancet 2(8571 ): 1300-4). This alimentary enhancement of insulin release, known as the incretin effect, is primarily humoral and GLP-1 is now thought to be the most potent physiological incretin in humans. GLP-1 mediates insulin production via binding to the GLP-1 receptor, known to be expressed in pancreatic β cells. In addition to the insulinotropic effect, GLP-1 suppresses glucagon secretion, delays gastric emptying (Wettergen et al., 1993, Dig Dis Sci 38: 665-73) and may enhance peripheral glucose disposal (D'Alessio et al., 1994, J. Clin Invest 93: 2293-6).

[025] A combination of actions gives GLP-1 unique therapeutic advantages over other agents currently used to treat non-insulin-dependent diabetes mellitus (NIDDM). First, a single subcutaneous dose of GLP-1 can completely normalize post prandial glucose levels in patients with NIDDM (Gutniak et al., 1994, Diabetes Care 17: 1039-44). This effect may be mediated both by increased insulin release and by a reduction in glucagon secretion. Second, intravenous infusion of GLP-1 can delay postprandial gastric emptying in patients with NIDDM (Williams et al., 1996, J. Clin Endo Metab 81 : 327-32). Third, unlike sulphonylureas, the insulinotropic action of GLP-1 is dependent on plasma glucose concentration (HoIz et al., 1993, Nature 361 :362-5). Thus, the loss of GLP-1 -mediated insulin release at low plasma glucose concentration protects against severe hypoglycemia.

[026] When given to healthy subjects, GLP-1 potently influences glycemic levels as well as insulin and glucagon concentrations (Orskov, 1992, Diabetologia 35:701-11 ), effects which are glucose dependent (Weir et al., 1989, Diabetes 38: 338-342). Moreover, it is also effective in patients with diabetes (Gutniak, M., 1992, N. Engl J Med 226: 1316-22), normalizing blood glucose levels in type 2 diabetic subjects and improving glycemic control in type 1 patients (Nauck et al., 1993, Diabetologia 36: 741-4, Creutzfeldt et al., 1996, Diabetes Care 19:580-6).

[027] GLP-1 [7-36]NH₂ has the following amino acid sequence:

HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR (SEQ ID NO: 13), which may be employed as the GLP-1 in accordance with the invention. Alternatively, the GLP-1 component may contain glycine (G) at the second position, giving, for example, the sequence HGEGTFTSDVSSYLEGQAAKEFIAWLVKGR (SEQ ID NO: 17). The GLP-1 may be a biologically active fragment of GLP-1 , for example, as disclosed in US 2007/0041951 , which is hereby incorporated by reference in its entirety. Other fragments and modified sequences of GLP-1 are known in the art (U.S. Pat. No. 5,614,492; U.S. Pat. No. 5,545,618; European Patent Application, Publication No. EP 0658568 A1 ; WO 93/25579, which are hereby incorporated by reference in their entireties). Such fragments and modified sequences may be used in connection with the present invention, as well as those described below.

[028] Certain structural and functional analogs of GLP-1 have been isolated from the venom of the GiIa monster lizards (Heloderma suspectum and Heloderma horridum) and have shown clinical utility. Such molecules find use in accordance with the present invention. In particular, exendin-4 is a 39 amino acid residue peptide isolated from the venom of Heloderma suspectum and shares approximately 52% homology with human GLP- 1. Exendin-4 is a potent GLP-1 receptor agonist that stimulates insulin release, thereby lowering blood glucose levels. Exendin-4 has the following amino acid sequence: HGEGTFTSDLSKQMEEEAVRLFEWLKNGGPSSGAPPPS (SEQ ID NO: 14). A synthetic version of exendin-4 known as exenatide (marketed as Byetta®) has been approved for the treatment of Type-2 Diabetes. Although exenatide is structurally analogous to native GLP-1 , it has a longer half-life after injection.

[029] While exenatide has the ability to lower blood glucose levels on its own, it can also be combined with other medications such as metformin, a thiozolidinedione, a sulfonylureas, and/or insulin to improve glucose control. Exenatide is administered by injection subcutaneously twice per day using a pre-filled pen device. Typical human responses to exenatide include improvements in the initial rapid release of endogenous insulin, an increase in β-cell growth and replication, suppression of pancreatic glucagon release, delayed gastric emptying, and reduced appetite - all of which function to lower blood glucose. Unlike sulfonylureas and meglitinides, exenatide increases insulin synthesis and secretion in the presence of glucose only, thus lessening the risk of hypoglycemia.

[030] Various functional analogs of GLP-1 and exendin-4 are known, and which find use in accordance with the invention. These include liraglutide (Novo Nordisk, WO98/008871 ), R1583/taspoglutide (Roche, WO00/034331 ), CJC-1 131 (ConjuChem, WO00/06991 1 ), ZP-10/AVE0010 (Zealand Pharma, Sanofi-Aventis, WO01/004156), and LY548806 (EIi Lilly, WO03/018516).

[031] Liraglutide, also known as NN221 1 , is a GLP-1 receptor agonist analog that has been designed for once-daily injection (Harder et al., 2004, Diabetes Care 27: 1915-21 ). Liraglutide has been tested in patients with type-2 diabetes in a number of studies and has been shown to be effective over a variety of durations. In one study, treatment with liraglutide improved glycemic control, improved β-cell function, and reduced endogenous glucose release in patients with type-2 diabetes after one week of treatment (Degn et al., 2004, Diabetes 53: 1187-94). In a similar study, eight weeks of 0.6-mg liraglutide therapy significantly improved glycemic control without increasing weight in subjects with type 2 diabetes compared with those on placebo (Harder et al., 2004, Diabetes Care 27: 1915-21 ).

[032] Thus, in certain embodiments, the GLP-1 receptor agonist in accordance with the invention is as described in WO98/008871 , which is hereby incorporated by reference in its entirety. The GLP-1 receptor agonist may have at least one lipophilic substituent, in addition to one, two, or more amino acid substitutions with respect to native GLP-1. For example, the lipophilic substituent may be an acyl group selected from CH₃(CH₂)_nCO-, wherein n is an integer from 4 to 38, such as an integer from 4 to 24. The lipophilic substituent may be an acyl group of a straight-chain or branched alkyl or fatty acid (for example, as described in WO98/008871 , which description is hereby incorporated by reference).

[033] In certain embodiments, the GLP-1 is Arg²⁶-GLP-1 (7-37), Arg³⁴-GLP-1 (7-37),

Lys³⁶-GLP-1 (7-37), Arg²⁶³⁴Lys³⁶-GLP-l (7-37), Arg²⁶³⁴Lys³⁸-GLP-l (7-38), Arg²⁸'³⁴ Lys³⁹-GLP- 1 (7-39), Arg²⁶'³⁴Lys⁴⁰-GLP-1 (7-40), Arg²⁶Lys³⁶-GLP-1 (7-37), Arg³⁴Lys³⁶-GLP-1 (7-37), Arg²⁶Lys³⁹-GLP-1 (7-39), Arg³⁴Lys⁴⁰-GLP-1 (7-40), Arg²⁶'³⁴Lys³⁶'³⁹-GLP-l (7-39),

Arg²⁶'³⁴Lys³⁶'⁴⁰-GLP-1 (7-40), Gly⁸Arg²⁶-GLP-1 (7-37); Gly⁸Arg³⁴-GLP-1 (7-37); Gly⁸Lys³⁸-GLP- 1 (7-37); Gly⁸Arg²⁶'³⁴Lys³⁶-GLP-1 (7-37), Gly⁸Arg²⁶'³⁴Lys³⁹-GLP-1 (7-39), Gly⁸Arg²⁶'³⁴Lys⁴⁰- GLP-1 (7-40), Gly⁸Arg²⁶Lys³⁶-GLP-1 (7-37), Gly⁸Arg³⁴Lys³⁶-GLP-1 (7-37), Gly⁸Arg²⁶Lys³⁹-GLP- 1 (7-39); Gly⁸Arg³⁴Lys⁴⁰-GLP-1 (7-40), Gly⁸Arg²⁸'³⁴Lys³⁶'³⁹-GLP-1 (7-39) and

Gly⁸Arg²⁶'³⁴Lys³⁵'⁴⁰-GLP-1 (7-40), each optionally having a lipophilic substituent. For example, the GLP-1 receptor agonist may have the sequence/structure Arg³⁴Lys²⁶-(N-ε-(γ- Glu(N-α-hexadecanoyl)))-GLP-l(7-37).

[034] Taspoglutide, also known as R1583 or BIM 51077, is a GLP-1 receptor agonist that has been shown to improve glycemic control and lower body weight in subjects with type 2 diabetes mellitus treated with metformin (Abstract No. A-1604, June 7, 2008, 68th American Diabetes Association Meeting, San Francisco, CA).

[035] Thus, in certain embodiments, the GLP-1 receptor agonist is as described in

WO00/034331 , which is hereby incorporated by reference in its entirety. In certain exemplary embodiments, the GLP-1 receptor agonist has the sequence [Aib⁸'³⁵]hGLP-1 (7- 36)NH₂ (e.g. taspoglutide), wherein Aib is alpha-aminoisobutyric acid.

[036] CJC-1 131 is a GLP-1 analog that consists of a DPP-IV-resistant form of GLP-

1 joined to a reactive chemical linker group that allows GLP-1 to form a covalent and irreversible bond with serum albumin following subcutaneous injection (Kim et al., 2003, Diabetes 52: 751-9). In a 12-week, randomized, double-blind, placebo-controlled multicenter study, CJC-1131 and metformin treatment was effective in reducing fasting blood glucose levels in type 2 diabetes patients (Ratner et al., Abstract No. 10-OR, June 10-14th, 2005, 65th American Diabetes Association Meeting, San Francisco, CA).

[037] Thus, in certain embodiments, the GLP-1 receptor agonist is as described in

WO00/069911 , which is hereby incorporated by reference in its entirety. In some embodiments, the GLP-1 receptor agonist is modified with a reactive group which reacts with amino groups, hydroxyl groups or thiol groups on blood components to form a stable covalent bond. In certain embodiments, the GLP-1 receptor agonist is modified with a reactive group selected from the group consisting of succinimidyl and maleimido groups. In certain exemplary embodiments, the GLP-1 receptor agonist has the sequence/structure: D- Ala⁸Lys³⁷-(2-(2-(2-maleimidopropionamido(ethoxy)ethoxy)acetamide))-GLP-1 (7-37) (e.g. CJC-1 131 ).

[038] AVE0010, also known as ZP-10, is a GLP-1 receptor agonist that may be employed in connection with the invention. In a recent double-blind study, patients treated with once daily dosing of AVE0010 demonstrated significant reductions in HbAI c levels (Ratner et al., Abstract No. 433-P, 68th American Diabetes Association Meeting, San Francisco, CA.). At the conclusion of the study, the percentages of patients with HbAIc <7% ranged from 47-69% for once daily dosing compared to 32% for placebo. In addition, AVE0010 treated patients showed dose-dependent reductions in weight and post-prandial plasma glucose.

[039] Thus, in certain embodiments, the GLP-1 receptor agonist is as described in

WO01/004156, which is hereby incorporated by reference in its entirety. For example, the GLP-1 receptor agonist may have the sequence:

HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPSKKKKKK-NH2 (SEQ ID NO: 18) (e.g. AVEOOI O).

[040] LY548806 is a GLP-1 derivative designed to be resistant to proteolysis by dipeptidase-peptidyl IV (DPP-IV) (Jackson et al., Abstract No. 562, June 10-14th, 2005, 65th American Diabetes Association Meeting, San Francisco, CA). In an animal model of hyperglycemia, LY548806 has been shown to produce a significant lowering of blood glucose levels during the hyperglycemic phase (Saha et al., 2006, J. Pharm. Exp. Ther. 316: 1159-64). Moreover, LY548806 was shown to produce a significant increase in insulin levels consistent with its known mechanism of action, namely stimulation of insulin release in the presence of hyperglycemia.

[041] Thus, in certain embodiments, the GLP-1 receptor agonist is as described in

WO03/018516, which is hereby incorporated by reference in its entirety. In some embodiments, the therapeutic agents of the present invention comprise GLP-1 analogs wherein the backbone for such analogs or fragments contains an amino acid other than alanine at position 8 (position 8 analogs). The backbone may also include L-histidine, D- histidine, or modified forms of histidine such as desamino-histidine, 2-amino-histidine, β- hydroxy-histidine, homohistidine, α-fluoromethyl-histidine, or α-methyl-histidine at position 7. In some embodiments, these position 8 analogs may contain one or more additional changes at positions 12, 16, 18, 19, 20, 22, 25, 27, 30, 33, and 37 compared to the corresponding amino acid of native GLP-1. In other embodiments, these position 8 analogs may contain one or more additional changes at positions 16, 18, 22, 25 and 33 compared to the corresponding amino acid of native GLP-1. In certain exemplary embodiments, the GLP- 1 receptor agonist has the sequence: HVEGTFTSDVSSYLEEQAAKEFIAWLI KGRG-OH (SEQ ID NO: 19) (e.g. LY548806).

[042] Thus, the present invention provides stable pharmaceutical formulations of

GLP-1 receptor agonists with an elastin-like peptide (ELP). For example, in certain embodiments, the GLP-1 receptor agonist is GLP-1 (SEQ ID NO:13 or 17) or a functional analog thereof. In other embodiments, the GLP-1 receptor agonist is exendin-4 (SEQ ID NO:14) or a functional analog thereof. Such functional analogs of GLP-1 or exendin-4 include functional fragments truncated at the C-terminus by from 1 to 10 amino acids, including by 1 , 2, 3, or up to about 5 amino acids (with respect to SEQ ID NOS: 13, 14, or 17). Such functional analogs may contain from 1 to 10 amino acid insertions, deletions, and/or substitutions (collectively) with respect to the native sequence (e.g., SEQ ID NOS 13 and 14), and in each case retaining the activity of the peptide. For example, the functional analog of GLP-1 or exendin-4 may have from 1 to about 3, 4, or 5 insertions, deletions and/or substitutions (collectively) with respect to SEQ ID NOS: 13 and 14 (respectively), and in each case retaining the activity of the peptide. Such activity may be confirmed or assayed using any available assay. In these or other embodiments, the GLP-1 receptor agonist has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity with the native sequence (SEQ ID NOS: 13 and 14). The determination of sequence identity between two sequences (e.g., between a native sequence and a functional analog) can be accomplished using any alignment tool, including Tatusova et al., Blast 2 sequences - a new tool for comparing protein and nucleotide sequences, FEMS Microbiol Lett. 174:247-250 (1999). Such functional analogs may further comprise additional chemical modifications, such as those described in this section and/or others known in the art.

[043] In another aspect, the present invention provides methods for the treatment or prevention of type 2 diabetes, impaired glucose tolerance, type 1 diabetes, hyperglycemia, obesity, binge eating, bulimia, hypertension, syndrome X, dyslipidemia, cognitive disorders, atherosclerosis, non-fatty liver disease, myocardial infarction, coronary heart disease and other cardiovascular disorders. The method comprises administering the GLP-1 formulation of the invention to a patient in need of such treatment. In these or other embodiments, the present invention provides methods for decreasing food intake, decreasing β-cell apoptosis, increasing β-cell function and β-cell mass, and/or for restoring glucose sensitivity to β-cells. Generally, the patient may be a human or non-human animal patient (e.g., dog, cat, cow, or horse). Preferably, the patient is human.

[044] The ability of a GLP-1 or exendin-4 analog, or an ELP/GLP-1 receptor agonist compound, to bind the GLP-1 receptor may be determined by standard methods, for example, by receptor-binding activity screening procedures which involve providing appropriate cells that express the GLP-1 receptor on their surface, for example, insulinoma cell lines such as RINmSF cells or INS-1 cells. In addition to measuring specific binding of tracer to membrane using radioimmunoassay methods, cAMP activity or glucose dependent insulin production can also be measured. In one method, a polynucleotide encoding the GLP-1 receptor is employed to transfect cells to thereby express the GLP-1 receptor protein. Thus, these methods may be employed for testing or confirming whether a suspected GLP-1 receptor agonist is active. [045] In addition, known methods can be used to measure or predict the level of biologically activity of a GLP-1 receptor agonist in vivo (See e.g. Siegel, et al., 1999, Regul Pept 79(2-3): 93-102). In particular, GLP-1 receptor agonists can be assessed for their ability to induce the production of insulin in vivo using a variety of known assays for measuring GLP-1 activity. For example, a GLP-1 receptor agonist compound can be introduced into a cell, such as an immortalized β-cell, and the resulting cell can be contacted with glucose. If the cell produces insulin in response to the glucose, then the modified GLP- 1 is generally considered biologically active in vivo (Fehmann et al., 1992, Endocrinology 130: 159-166).

[046] The ability of an ELP/ GLP-1 receptor agonist compound to enhance β-cell proliferation, inhibit β-cell apoptosis, and regulate islet growth may also be measured using known assays. Pancreatic β-cell proliferation may be assessed by ³H-tymidine or BrdU incorporation assays (See e.g. Buteau et al., 2003, Diabetes 52: 124-32), wherein pancreatic β-cells such as INS(832/13) cells are contacted with an ELP/ GLP-1 receptor agonist compound and analyzed for increases in ³H-thymidine or BrdU incorporation. The antiapoptotic activity of an ELP/GLP-1 receptor agonist compound can be measured in cultured insulin-secreting cells and/or in animal models where diabetes occurs as a consequence of an excessive rate of beta-cell apoptosis (See e.g. Bulotta et al., 2004, Cell Biochem Biophys 40(3 suppl): 65-78).

[047] In addition to GLP-1 , other peptides of the this family, such as those derived from processing of the pro-glucagon gene, such as GLP-2, GIP, and oxyntomodulin, could be formulated with the ELP component (as described herein) to enhance the therapeutic potential.

Insulin

[048] In other embodiments, the present invention provides a formulation of insulin with an ELP. Insulin injections, e.g. of human insulin, can be used to treat diabetes. The insulin-making cells of the body are called β-cells, and they are found in the pancreas gland. These cells clump together to form the "islets of Langerhans", named for the German medical student who described them.

[049] The synthesis of insulin begins at the translation of the insulin gene, which resides on chromosome 11. During translation, two introns are spliced out of the mRNA product, which encodes a protein of 1 10 amino acids in length. This primary translation product is called preproinsulin and is inactive. It contains a signal peptide of 24 amino acids in length, which is required for the protein to cross the cell membrane. [050] Once the preproinsulin reaches the endoplasmic reticulum, a protease cleaves off the signal peptide to create proinsulin. Proinsulin consists of three domains: an amino-terminal B chain, a carboxyl-terminal A chain, and a connecting peptide in the middle known as the C-peptide. Insulin is composed of two chains of amino acids named chain A (21 amino acids - GIVEQCCASVCSLYQLENYCN) (SEQ ID NO: 15) and chain B (30 amino acids FVNQHLCGSHLVEALYLVCGERGFFYTPKA) (SEQ ID NO: 16) that are linked together by two disulfide bridges. There is a 3rd disulfide bridge within the A chain that links the 6th and 1 1th residues of the A chain together. In most species, the length and amino acid compositions of chains A and B are similar, and the positions of the three disulfide bonds are highly conserved. For this reason, pig insulin can replace deficient human insulin levels in diabetes patients. Today, porcine insulin has largely been replaced by the mass production of human proinsulin by bacteria (recombinant insulin).

[051] Insulin molecules have a tendency to form dimers in solution, and in the presence of zinc ions, insulin dimers associate into hexamers. Whereas monomers of insulin readily diffuse through the blood and have a rapid effect, hexamers diffuse slowly and have a delayed onset of action. In the design of recombinant insulin, the structure of insulin can be modified in a way that reduces the tendency of the insulin molecule to form dimers and hexamers but that does not interrupt binding to the insulin receptor. In this way, a range of preparations are made, varying from short acting to long acting.

[052] Within the endoplasmic reticulum, proinsulin is exposed to several specific peptidases that remove the C-peptide and generate the mature and active form of insulin. In the Golgi apparatus, insulin and free C-peptide are packaged into secretory granules, which accumulate in the cytoplasm of the β-cells. Exocytosis of the granules is triggered by the entry of glucose into the beta cells. The secretion of insulin has a broad impact on metabolism.

[053] There are two phases of insulin release in response to a rise in glucose. The first is an immediate release of insulin. This is attributable to the release of preformed insulin, which is stored in secretory granules. After a short delay, there is a second, more prolonged release of newly synthesized insulin.

[054] Once released, insulin is active for a only a brief time before it is degraded by enzymes, lnsulinase found in the liver and kidneys breaks down insulin circulating in the plasma, and as a result, insulin has a half-life of only about 6 minutes. This short duration of action results rapid changes in the circulating levels of insulin.

[055] Insulin analogs have been developed with improved therapeutic properties

(Owens et al., 2001 , Lancet 358: 739-46; Vajo et al., 2001 , Endocr Rev 22: 706-17), and such analogs may be employed in connection with the present invention. Various strategies, including elongation of the COOH-terminal end of the insulin B-chain and engineering of fatty acid-acylated insulins with substantial affinity for albumin are used to generate longer-acting insulin analogs.

[056] Functional analogs of insulin that may be employed in accordance with the invention include rapid acting analogs such as lispro, aspart and glulisine, which are absorbed rapidly (< 30 minutes) after subcutaneous injection, peak at one hour, and have a relatively short duration of action (3 to 4 hours). In addition, two long acting insulin analogs have been developed: glargine and detemir, and which may be employed in connection with the invention. The long acting insulin analogs have an onset of action of approximately two hours and reach a plateau of biological action at 4 to 6 hours, and may last up to 24 hours.

[057] Thus, in one embodiment, the insulin may contain the A and B chain of lispro

(also known as Humalog, EIi Lilly). Insulin lispro differs from human insulin by the substitution of proline with lysine at position 28 and the substitution of lysine with proline at position 29 of the insulin B chain. Although these modifications do not alter receptor binding, they help to block the formation of insulin dimers and hexamers, allowing for larger amounts of active monomeric insulin to be available for postprandial injections.

[058] In another embodiment, the insulin may contain an A and B chain of aspart

(also known as Novolog, Novo Nordisk). Insulin aspart is designed with the single replacement of the amino acid proline by aspartic acid at position 28 of the human insulin B chain. This modification helps block the formation for insulin hexamers, creating a faster acting insulin.

[059] In yet another embodiment, the insulin may contain an A and B chain of glulisine (also known as Apidra, Sanofi-Aventis). Insulin glulisine is a short acting analog created by substitution of asparagine at position 3 by lysine and lysine at position 29 by glutamine of human insulin B chain. Insulin glulisine has more rapid onset of action and shorter duration of action compared to regular human insulin.

[060] In another embodiment, the insulin may contain an A and B chain of glargine

(also known as Lantus, Sanofi-Aventis). Insulin glargine differs from human insulin in that the amino acid asparagine at position 21 of the A chain is replaced by glycine and two arginines are added to the C-terminus of the B-chain. Compared with bedtime neutral protamine Hagedorn (NPH) insulin (an intermediate acting insulin), insulin glargine is associated with less nocturnal hypoglycemia in patients with type 2 diabetes.

[061] In yet another embodiment, the insulin may contain an A and B chain from detemir (also known as Levemir, Novo Nordisk). Insulin detemir is a soluble (at neutral pH) long-acting insulin analog, in which the amino acid threonine at B30 is removed and a 14- carbon, myristoyl fatty acid is acetylated to the epsilon-amino group of LysB29. After subcutaneous injection, detemir dissociates, thereby exposing the free fatty acid which enables reversible binding to albumin molecules. So at steady state, the concentration of free unbound insulin is greatly reduced resulting in stable plasma glucose levels.

[062] In some embodiments, the insulin may be a single-chain insulin analog (SIA)

(e.g. as described in 6,630,438 and WO08/019368, which are hereby incorporated by reference in their entirety). Single-chain insulin analogs encompass a group of structurally- related proteins wherein the A and B chains are covalently linked by a polypeptide linker. The polypeptide linker connects the C-terminus of the B chain to the N-terminus of the A chain. The linker may be of any length so long as the linker provides the structural conformation necessary for the SIA to have a glucose uptake and insulin receptor binding effect. In some embodiments, the linker is about 5-18 amino acids in length. In other embodiments, the linker is about 9-15 amino acids in length. In certain embodiments, the linker is about 12 amino acids long. In certain exemplary embodiments, the linker has the sequence KDDNPNLPRLVR (SEQ ID NO.: 20) or GAGSSSRRAPQT (SEQ ID NO.: 21 ). However, it should be understood that many variations of this sequence are possible such as in the length (both addition and deletion) and substitutions of amino acids without substantially compromising the effectiveness of the produced SIA in glucose uptake and insulin receptor binding activities. For example, several different amino acid residues may be added or removed from either end without substantially decreasing the activity of the produced SIA.

[063] An exemplary single-chain insulin analog currently in clinical development is albulin (Duttaroy et al., 2005, Diabetes 54: 251-8). Albulin can be produced in yeast or in mammalian cells. It consists of the B and A chain of human insulin (100% identity to native human insulin) linked together by a dodecapeptide linker and fused to the NH₂ terminals of the native human serum albumin. For expression and purification of albulin, Duttaroy et al. constructed a synthetic gene construct encoding a single-chain insulin containing the B- and A- chain of mature human insulin linked together by a dodecapeptide linker using four overlapping primers and PCR amplification. The resulting PCR product was ligated in-frame between the signal peptide of human serum albumin (HSA) and the NH₂ terminus of mature HSA, contained within a pSAC35 vector for expression in yeast. In accordance with the present invention, the HSA component of abulin may also be replaced with an ELP component as described herein

[064] Thus, in one aspect, the present invention provides pharmaceutical formulations of insulin, or a functional analog thereof, with an elastin-like peptide (ELP). For example, in certain embodiments, the insulin is a mammalian insulin, such as human insulin or porcine insulin. The insulin may comprise each of chains A, B, and C (SEQ ID NOS: 51 and 52), or may contain a processed form, containing only chains A and B. In some embodiments, chains A and B are connected by a short linking peptide, to create a single chain insulin. The insulin may be a functional analog of human insulin, including functional fragments truncated at the N-terminus and/or C-terminus (of either or both of chains A and B) by from 1 to 10 amino acids, including by 1 , 2, 3, or about 5 amino acids. Functional analogs may contain from 1 to 10 amino acid insertions, deletions, and/or substitutions (collectively) with respect to the native sequence (e.g., SEQ ID NOS 15 and 16), and in each case retaining the activity of the peptide. For example, functional analogs may have 1 , 2, 3, 4, or 5 amino acid insertions, deletions, and/or substitutions (collectively) with respect to the native sequence (which may contain chains A and B, or chains A, B, and C). Such activity may be confirmed or assayed using any available assay, including those described herein. In these or other embodiments, the insulin has at least about 75%, 80%, 85%, 90%, 95%, or 98% identity with each of the native sequences for chains A and B (SEQ ID NOS: 15 and 16). The determination of sequence identity between two sequences (e.g., between a native sequence and a functional analog) can be accomplished using any alignment tool, including Tatusova et a I., Blast 2 sequences - a new tool for comparing protein and nucleotide sequences, FEMS Microbiol Lett. 174:247-250 (1999). The insulin component may contain additional chemical modifications known in the art.

[065] In another aspect, the present invention provides methods for the treatment or prevention of diabetes, including type I and Il diabetes. The method comprises administering an effective amount of the formulation comprising an elastin-like peptide (ELP) and an insulin (or functional analog thereof) to a patient in need thereof. Generally, the patient may be a human or non-human animal (e.g., dog, cat, cow, or horse) patient. Preferably, the patient is human.

[066] To characterize the in vitro binding properties of an insulin analog, competition binding assays may be performed in various cell lines that express the insulin receptor (Jehle et al., 1996, Diabetologia 39: 421-432). For example, competition binding assays using CHO cells overexpressing the human insulin receptor may be employed. Insulin can also bind to the IGF-1 receptor with a lower affinity than the insulin receptor. To determine the binding affinity of an insulin analog, a competition binding assay can be performed using ¹²⁵l-labeled IGF-1 in L6 cells.

[067] The activities of insulin include stimulation of peripheral glucose disposal and inhibition of hepatic glucose production. The ability of a particular insulin analog to mediate these biological activities can be assayed in vitro using known methodologies. For example, the effect of an ELP-containing analog on glucose uptake in 3T3-L1 adipocytes can be measured and compared with that of insulin. Pretreatment of the cells with a biologically active analog will generally produce a dose-dependent increase in 2-deoxyglucose uptake. The ability of an insulin analog to regulate glucose production may be measured in any number of cells types, for example, H4lle hepatoma cells. In this assay, pretreatment with a biologically active analog will generally result in a dose-dependent inhibition of the amount of glucose released.

Factor Vl I (Vila)

[068] In certain embodiments, the invention provides formulations of Factor Vll/Vlla with an ELP. Coagulation is the biological process of blood clot formation involving many different serine proteases as well as their essential cofactors and inhibitors. It is initiated by exposure of Factor VII (FVII) and Factor Vila (FVIIa) to its membrane bound cofactor, tissue factor (TF), resulting in production of Factor Xa (FXa) and more FVIIa. The process is propagated upon production of Factor IXa (FIXa) and additional FXa that, upon binding with their respective cofactors FVIIIa and FVa, form platelet bound complexes, ultimately resulting in the formation of thrombin and a fibrin clot. Thrombin also serves to further amplify coagulation by activation of cofactors such as FV and FVII and zymogens such as Factor Xl. Moreover, thrombin activates platelets leading to platelet aggregation, which is necessary for the formation of a hemostatic plug.

[069] Factor VII circulates in the blood in a zymogen form, and is converted to its active form, Factor Vila, by either factor IXa, factor Xa, factor XIIa, or thrombin by minor proteolysis. Factor Vila is a two-chain, 50 kilodalton (kDa) plasma serine protease. The active form of the enzyme comprises a heavy chain (254 amino acid residues) containing a catalytic domain and a light chain (152 residues) containing 2 epidermal growth factor (EGF)-like domains. The mature factor Vll/Vlla that circulates in plasma is composed of 406 amino acid residues (SEQ ID NO: 33). The light and heavy chains are held together by a disulfide bond.

[070] As noted above, Factor Vila is generated by proteolysis of a single peptide bond from its single chain zymogen, Factor VII, which is present at approximately 0.5 μg/ml in plasma. The conversion of zymogen Factor VII into the activated two-chain molecule occurs by cleavage of an internal peptide bond. In human Factor VII, the cleavage site is at Arg152-lle153 (Hagen et al., 1986, PNAS USA 83: 2412-6).

[071] "Factor Vll/Vlla" as used in this application means a product consisting of either the unactivated form (factor VII) or the activated form (factor Vila) or mixtures thereof. "Factor Vll/Vlla" within the above definition includes proteins that have an amino acid sequence of native human factor Vll/Vlla. It also includes proteins with a slightly modified amino acid sequence, for instance, a modified N-terminal end including N-terminal amino acid deletions or additions so long as those proteins substantially retain the activity of factor Vila. "Factor VII" within the above definition also includes natural allelic variations that may exist and occur from one individual to another. Also, degree and location of glycosylation or other post-translation modifications may vary depending on the chosen host cells and the nature of the host cellular environment.

[072] In the presence of calcium ions, Factor Vila binds with high affinity to TF. TF is a 263 amino acid residue glycoprotein composed of a 219 residue extracellular domain, a single transmembrane domain, and a short cytoplasmic domain (Morrissey et al., 1987, Cell 50: 129-35). The TF extracellular domain is composed of two fibronectin type III domains of about 105 amino acids each. The binding of FVIIa is mediated entirely by the TF extracellular domain (Muller et al., 1994, Biochem. 33:10864-70). Residues in the area of amino acids 16-26 and 129-147 contribute to the binding of FVIIa as well as the coagulant function of the molecule. Residues Lys20, Trp45, Asp58, Tyr94, and Phe140 make a large contribution (1 kcal/mol) to the free energy (ΔG) of binding to FVIIa.

[073] TF is expressed constitutively on cells separated from plasma by the vascular endothelium. Its expression on endothelial cells and monocytes is induced by exposure to inflammatory cytokines or bacterial lipopolysaccharides (Drake et al., 1989, J. Cell Biol. 109: 389). Upon tissue injury, the exposed extracellular domain of TF forms a high affinity, calcium dependent complex with FVII. Once bound to TF, FVII can be activated by peptide bond cleavage to yield serine protease FVIIa. The enzyme that catalyzes this step in vivo has not been elucidated, but in vitro FXa, thrombin, TF:FVIIa and FIXa can catalyze this cleavage. FVIIa has only weak activity upon its physiological substrates FX and FIX whereas the TF:FVIIa complex rapidly activates FX and FIX.

[074] The TF:FVIIa complex constitutes the primary initiator of the extrinsic pathway of blood coagulation. The complex initiates the extrinsic pathway by activation of FX to Factor Xa (FXa), FIX to Factor IXa (FIXa), and additional FVII to FVIIa. The action of TF:FVIIa leads ultimately to the conversion of prothrombin to thrombin, which carries out many biological functions. Among the most important activities of thrombin is the conversion of fibrinogen to fibrin, which polymerizes to form a clot. The TF:FVIIa complex also participates as a secondary factor in extending the physiological effects of the contact activation system.

[075] The initiation and subsequent regulation of coagulation is complex, since maintenance of hemostasis is crucial for survival. There is an exquisite balance between hemostasis (normal clot formation and dissolution) and thrombosis (pathogenic clot formation). Serious clinical conditions involving aberrations in coagulation include deep vein thrombosis, myocardial infarction, pulmonary embolism, stroke and disseminated intravascular coagulation (in sepsis). There are also many bleeding coagulopathies where there is insufficient clot formation. These include hemophilia A (FVIII deficiency) or hemophilia B (FIX deficiency), where procoagulant therapy is required. The challenge in this therapeutic area is to operate in the narrow window between too much and too little coagulation.

[076] The use of exogenous FVIIa as a therapeutic agent has been shown to induce hemostatsis in patients with hemophilia A and B (Hedner, 2001 , Seminars Hematol. 38 (suppl. 12): 43-7; Hedner, 2004, Seminars Hematol. 41 (suppl. 1 ): 35-9). It also has been used to treat bleeding in patients with liver disease, anticoagulation-induced bleeding, surgery, thrombocytopenia, thrombasthenia, Bemard-Soulier syndrome, von Willebrand disease, and other bleeding disorders (See e.g. Roberts et al., 2004, Blood 104: 3858-64).

[077] Commercial preparations of human recombinant FVIIa are sold as

NovoSeven.™ NovoSeven™ is indicated for the treatment of bleeding episodes in hemophilia A or B patients and is the only recombinant FVIIa effective for bleeding episodes currently available. A circulating recombinant FVIIa half-life of 2.3 hours was reported in "Summary Basis for Approval for NovoSeven™" FDA reference number 96-0597. Moreover, the half-life of recombinant FVIIa is shorter in pediatric patients (~ 1.3 hours), suggesting that higher doses of recombinaint FVIIa may be required in this population (Roberts et al., 2004, Blood 104: 3858-64). Accordingly, relatively high doses and frequent administration are necessary to reach and sustain the desired therapeutic or prophylactic effect.

[078] Recombinant human coagulation factor Vila (rFVIIa, NovoSeven; Novo

Nordisk A/S, Copenhagen, Denmark) has proven to be efficacious for the treatment of bleeding episodes in hemophilia patients with inhibitors. A small fraction of patients may be refractory to rFVIIa treatment and could potentially benefit from genetically modified FVIIa molecules with increased potencies. To this end, FVIIa analogs with increased intrinsic activity have been investigated that exhibit superior hemostatic profiles in vitro (see e.g. WO02/077218 or WO05/074975, which are hereby incorporated by reference in their entirety, and Tranholm et al., 2003, Blood 102(10): 3615-20, which is also incorporated by reference). These analogs may also be used as more efficacious hemostatic agents in other indications where efficacy of rFVIIa has been observed, including in thrombocytopenia and trauma. [079] Thus, in some embodiments, the Factor Vila analog that may be used in accordance with the invention is as described in WO02/077218 or WO05/074975. For example, the FVIIa analog may have a glutamine substituted for methionine at position 298 (i.e. M298Q-FVIIa). In certain exemplary embodiments, the FVIIa analog contains two additional mutations, valine at position 158 replaced by aspartic acid and glutamic acid at position 296 replaced by valine (i.e. V158D/E296V/M298Q-FVIIa). Additionally or alternatively, the Factor Vila analog may have an alanine residue substitution for lysine at position 337 (i.e. V158D/E296V/M298Q/K337A-FVIIa). In still other embodiments, the Factor Vila analog has a substitution or insertion selected from Q250C; P406C; and 407C, wherein a cysteine has also been introduced in the C-terminal sequence (see, e.g. US 7,235,638, which is hereby incorporated by reference in its entirety). The Factor Vila analog may further comprise a substitution or insertion at one or more of positions 247, 260, 393, 396, and/or 405.

[080] In these or other embodiments, the Factor Vila analog comprises a substitution relative to the sequence of native Factor Vila selected from: (a) a substitution of Lys157 with an amino acid selected from the group consisting of GIy, VaI, Ser, Thr, Asp, and GIu; (b) a substitution of Lys337 with an amino acid selected from the group consisting of Ala, GIy, VaI, Ser, Thr, GIn, Asp, and GIu; (c) a substitution of Asp334 with any amino acid other than Ala or Asn; and (d) a substitution of Ser336 with any amino acid other than Ala or Cys (see e.g. US 7,176,288, which is hereby incorporated by reference in its entirety). Additionally or alternatively, the Factor Vila analog comprises a substitution of the Leu at position 305 of Factor VII with an amino acid residue selected from the group consisting of VaI, lie, Met, Phe, Trp, Pro, GIy, Ser, Thr, Cys, Tyr, Asn, GIu, Lys, Arg, His, Asp and GIn (see e.g. US 6,905,683, which is hereby incorporated by reference in its entirety).

[081] Thus, in one aspect, the present invention provides pharmaceutical formulations of Factor Vl I/VI Ia, or functional analog, with an elastin-like peptide (ELP). For example, in certain embodiments, the Factor Vll/Vlla is human Factor Vll/Vlla (e.g., SEQ ID NO: 33). The Factor Vll/Vlla may be a functional analog of human Factor Vll/Vlla, including functional fragments truncated at the N-terminus and/or C-terminus by from 1 to 10 amino acids, including by 1 , 2, 3, or about 5 amino acids. Functional analogs may contain from 1 to 10 amino acid insertions, deletions, and/or substitutions (collectively) with respect to the native sequence (e.g., SEQ ID NO: 33), and in each case retaining the activity of the peptide. For example, such analogs may have from 1 to about 5 amino acid insertions, deletions, and/or substitutions (collectively) with respect to the native full length sequence, or with respect to one or both of the heavy and light chains. Such activity may be confirmed or assayed using any available assay, including those described herein. In these or other embodiments, the Factor Vll/Vlla component has at least about 75%, 80%, 85%, 90%, 95%, or 98% identity with the native sequence (SEQ ID NO:33). The determination of sequence identity between two sequences (e.g., between a native sequence and a functional analog) can be accomplished using any alignment tool, including Tatusova et al., Blast 2 sequences - a new tool for comparing protein and nucleotide sequences, FEMS Microbiol Lett. 174:247- 250 (1999).

[082] In another aspect, the present invention provides methods for the treatment or prevention of bleeding-related disorders. The method comprises administering an effective amount of the Factor Vll/Vlla formulation of the invention to a patient in need. In certain embodiments, the bleeding-related disorder is one or more of hemophilia, postsurgical bleeding, anticoagulation-induced bleeding, thrombocytopenia, Factor VII deficiency, Factor Xl deficiency, bleeding in patients with liver disease, thrombasthenia, Bemard-Soulier syndrome, von Willebrand disease, and intracranial hemorrhage. Generally, the patient is a human or non-human animal (e.g., dog, cat, cow, or horse) patient. Perferably, the patient is human.

[083] To characterize the in vitro binding properties of a suspected Factor Vll/Vlla analog, TF binding assays can be performed as described previously (See, e.g., Chaing et al., 1994, Blood 83(12): 3524-35). Briefly, recombinant human TF can be coated onto lmmulon Il plates in carbonate antigen buffer overnight at 4⁰C. BSA is also coated onto the plates for use as a control. Factor Vila analogs may be added at various concentrations in TBS-T buffer. After several washes, monospecific polyclonal rabbit anti-human FVIIa sera is added and incubated for approximately an hour at room temperature. Next, goat anti-rabbit IgG conjugated to alkaline phosphatase is added, followed by the alkaline phosphatase substrate PNPP, which is used for detection. After subtraction of background, the absorbance at ~ 405 nm is taken to be directly proportional to the degree of Factor Vila binding to the immobilized TF. These values can then be compared to control plasma containing Factor Vila.

[084] The clotting ability of a Factor Vll/Vlla analog can be measured in human FVII deficient plasma. In this assay, the Factor Vila analog diluted to varying concentrations directly into FVII deficient plasma. In a coagulometer, one part plasma ± a FVIIa analog can be mixed with 2 parts Innovin™ (Dade, Miami, FIa.) prothrombin time reagent (recombinant human tissue factor with phospholipids and CaC^). Clot formation is detected optically and time to clotting measured. Clotting time (seconds) is compared to the mean clotting time of FVII-deficient plasma alone and plotted as the fractional clotting time versus FVIIa analog concentration. ELP Fusion Components and ELP Carriers

[085] The compositions or formulations of the present invention comprise ELP biopolymers (e.g., as separate non-covalently associated components of the composition, and/or as recombinant fusions with peptide active agents). The ELPs comprise or consist of structural peptide units or sequences that are related to, or derived from, the elastin protein. The ELP biopolymer is constructed from structural units of from three to twenty amino acids, or in some embodiments, of from four to ten amino acids, such as five or six amino acids. Thus, the length of the individual structural units may vary, but in certain embodiments, the structural units are polytetra-, polypenta-, polyhexa-, polyhepta-, polyocta, and polynonapeptide units. Exemplary structural units include units defined by SEQ ID NOS: 1- 12, which may be employed as repeating structural units, including tandem-repeating units, or may be employed in some combination, to create an ELP effective for stabilizing the active agent under the anticipated storage conditions. Thus, the ELP may comprise or consist essentially of one or more structural unit(s) selected from SEQ ID NOS: 1-12, as defined below.

[086] The ELP biopolymers, comprising such structural units, may be of varying sizes. For example, the ELP may comprise or consist essentially of from 1 to 100 structural units, or in certain embodiments from 5 to 50 structural units, or from 10 to 30 structural units, or from 15 to 25 structural units, including one or a combination of units defined by SEQ ID NOS: 1-12. The ELP may have from 5 to 10 structural units, including one or a combination of structural units defined by SEQ ID NOS: 1-12. Thus, the ELP may have a length of from 5 to about 500 amino acid residues, or from about 10 to about 450 amino acid residues, or from about 15 to about 150 amino acid residues.

[087] In certain embodiments, the ELP undergoes a reversible inverse phase transition. That is, the ELPs are structurally disordered and highly soluble in water below a transition temperature (Tt), but exhibit a sharp (2-3°C range) disorder-to-order phase transition when the temperature is raised above the Tt, leading to desolvation and aggregation of the ELPs. For example, the ELP aggregates, when reaching sufficient size, can be readily removed and isolated from solution by centrifugation. Such phase transition is reversible, and isolated ELP aggregates can be completely resolubilized in buffer solution when the temperature is returned below the Tt of the ELPs.

[088] In certain embodiments, the ELP does not undergo a reversible inverse phase transition, or does not undergo such a transition at a temperature or conditions anticipated for storage, and thus the improvements in stability of the active agent may be entirely or substantially independent of any phase transition properties. [089] In certain embodiments, the ELPs may be formed of structural units, including but not limited to:

(a) the tetrapeptide Val-Pro-Gly-Gly, or VPGG (SEQ ID NO: 1 );

(b) the tetrapeptide Ile-Pro-Gly-Gly, or IPGG (SEQ ID NO: 2);

(C) the pentapeptide Val-Pro-Gly-X-Gly (SEQ ID NO: 3), or VPGXG, wherein X is any natural or non-natural amino acid residue, and wherein X optionally varies among polymeric or oligomeric repeats;

(d) the pentapeptide Ala-Val-Gly-Val-Pro, or AVGVP (SEQ ID NO: 4);

(e) the pentapeptide Ile-Pro-Gly-X-Gly, or IPGXG (SEQ ID NO: 5), wherein X is any natural or non-natural amino acid residue, and wherein X optionally varies among polymeric or oligomeric repeats;

(f) the pentapeptide Ile-Pro-Gly-Val-Gly, or IPGVG (SEQ ID NO: 6);

(g) the pentapeptide Leu-Pro-Gly-X-Gly, or LPGXG (SEQ ID NO: 7), wherein X is any natural or non-natural amino acid residue, and wherein X optionally varies among polymeric or oligomeric repeats;

(h) the pentapeptide Leu-Pro-Gly-Val-Gly, or LPGVG (SEQ ID NO: 8); (i) the hexapeptide Val-Ala-Pro-Gly-Val-Gly, or VAPGVG (SEQ ID NO: 9);

C) the octapeptide Gly-Val-Gly-Val-Pro-Gly-Val-Gly, or GVGVPGVG (SEQ ID NO: 10);

(k) the nonapeptide Val-Pro-Gly-Phe-Gly-Val-Gly-Ala-Gly, or VPGFGVGAG (SEQ ID NO: 1 1 ); and

(I) the nonapeptides Val-Pro-Gly-Val-Gly-Val-Pro-Gly-Gly, or VPGVGVPGG (SEQ ID NO: 12).

[090] Such structural units defined by SEQ ID NOS: 1-12 may form structural repeat units, or may be used in combination to form an ELP carrier of the invention. In some embodiments, the ELP is formed entirely (or almost entirely) of one or a combination of structural units selected from SEQ ID NOS: 1-12. In other embodiments, at least 75%, or at least 80%, or at least 90% of the ELP is formed from one or a combination of structural units selected from SEQ ID NOS: 1-12, and which may be present as repeating units.

[091] In certain embodiments, the ELP contains repeat units, including tandem repeating units, of the pentapeptide Val-Pro-Gly-X-Gly (SEQ ID NO:3), wherein X is as defined above, and wherein the ratio of Val-Pro-Gly-X-Gly pentapeptide units to the remainder of the ELP (which may comprise structural units other than VPGXG) is greater than about 75%, or greater than about 85%, or greater than about 95%.

[092] In certain embodiments, the ELP biopolymer contains at least one repeating unit, and may include a plurality of tandem repeating units, of a unit selected from VPGG, IPGG, AVGVP, IPGVG, LPGVG, VAPGVG, GVGVPGVG, VPGFGVGAG, and VPGVGVPGG. The ELP may include polymeric or oligomeric repeat units selected from the group consisting of LPGXG (SEQ ID NO: 11 ), IPGXG (SEQ ID NO: 12), and combinations thereof, wherein X is an amino acid residue that does not preclude phase transition of the ELP.

[093] The ELP biopolymers may include a β-turn. Examples of polypeptides suitable for use as the β-turn are described in International Patent Application PCT/US96/05186, which is hereby incorporated by reference in its entirety. Alternatively, the ELP component may lack a β-turn, or otherwise have a different conformation and/or folding character.

[094] Thus, the ELPs may include polymeric or oligomeric repeats of various tetra-, penta-, hexa-, hepta-, octa-, and nonapeptide structural units, including but not limited to VPGG, IPGG, VPGXG, AVGVP, IPGVG, LPGVG, VAPGVG, GVGVPGVG, VPGFGVGAG, and VPGVGVPGG. It will be appreciated by those of skill in the art that the ELPs need not consist of only polymeric or oligomeric sequences as listed herein, in order to enhance the stability of the active agent, and/or to exhibit a phase transition or otherwise constitute a suitable ELP carrier for use with the invention.

[095] In certain embodiments, the ELPs include polymeric or oligomeric repeats of the pentapeptide VPGXG (SEQ ID NO: 3), where the guest residue X is any amino acid, such as any amino acid that does not eliminate the phase transition characteristics of the ELP. X may be a naturally occurring or non-naturally occurring amino acid. For example, X may be selected from the group consisting of: alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine. In a specific embodiment, X is not proline.

[096] X may be a non-classical amino acid. Examples of non-classical amino acids include: D- isomers of the common amino acids, 2,4-diaminobutyric acid, α-amino isobutyric acid, A- aminobutyric acid, Abu, 2-amino butyric acid, γ-Abu, ε-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3 -amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t- butylalanine, phenylglycine, cyclohexylalanine, β-alanine, fluoro-amino acids, designer amino acids such as β-methyl amino acids, Cα-methyl amino acids, Nα-methyl amino acids, and amino acid analogs in general.

[097] Selection of X is independent in each ELP structural unit (for each structural unit defined herein having a guest residue X). For example, X may be independently selected for each structural unit as an amino acid having a positively charged side chain, an amino acid having a negatively charged side chain, or an amino acid having a neutral side chain, including in some embodiments, a hydrophobic side chain.

[098] In still other embodiments, the ELPs may include polymeric or oligomeric repeats of the pentapeptide IPGXG or LPGXG, or a combination thereof, where X is as defined above.

[099] In each embodiment, the structural units, or in some cases polymeric or oligomeric repeats, of the ELP sequences may be separated by one or more amino acid residues that do not eliminate the overall effect of the molecule, that is, in imparting certain improvements to the formulation as described. In certain embodiments, such one or more amino acids also do not eliminate or substantially affect the phase transition properties of the ELP component (relative to the deletion of such one or more amino acids).

[0100] In each repeat, X is independently selected. The structure of the resulting

ELP components may be described using the notation ELPk [XiYj-n], where k designates the specific type of ELP repeat unit, the bracketed capital letters are single letter amino acid codes and their corresponding subscripts designate the relative ratio of each guest residue X in the structural units (where applicable), and n describes the total length of the ELP in number of the structural repeats. For example, ELP3 [V₅A₂G₃-I O] designates an ELP component containing 10 repeating units of the pentapeptide VPGXG, where X is valine, alanine, and glycine at a relative ratio of 5:2:3; ELP3 [K₁V₂F₁^] designates an ELP component containing 4 repeating units of the pentapeptide VPGXG, where X is lysine, valine, and phenylalanine at a relative ratio of 1 :2:1 ; ELP3 [K₁V₇F₁-Q] designates a polypeptide containing 4 repeating units of the pentapeptide VPGXG, where X is lysine, valine, and phenylalanine at a relative ratio of 1 :7:1 ; ELP3 [V-5] designates a polypeptide containing 5 repeating units of the pentapeptide VPGXG, where X is exclusively valine; ELP3 [V-20] designates a polypeptide containing 20 repeating units of the pentapeptide VPGXG, where X is exclusively valine; ELP4 [5] designates a polypeptide containing 5 repeating units of the pentapeptide AVGVP; ELP12 [V-5] designates a polypeptide containing 5 repeating units of the pentapeptide IPGXG, where X is exclusively valine; ELP11 [V-5] designates a polypeptide containing 5 repeating units of the pentapeptide LPGXG, where X is exclusively valine. Such ELP components as described in this paragraph may be used in connection with the present invention to increase the therapeutic properties of the therapeutic component, and/or as ELP carriers.

[0101] Previous studies have shown that the fourth residue (X) in the elastin pentapeptide sequence, VPGXG, can be altered without eliminating the formation of the β- turn. These studies also showed that the Tt is a function of the hydrophobicity of the guest residue. By varying the identity of the guest residue(s) and their mole fraction(s), ELPs can be synthesized that exhibit an inverse transition over a 0-100⁰C range.

[0102] The Tt at a given ELP length can be decreased by incorporating a larger fraction of hydrophobic guest residues in the ELP sequence. Examples of suitable hydrophobic guest residues include valine, leucine, isoleucine, phenyalanine, tryptophan and methionine. Tyrosine, which is moderately hydrophobic, may also be used. Conversely, the Tt can be increased by incorporating residues, such as those selected from the group consisting of: glutamic acid, cysteine, lysine, aspartate, alanine, asparagine, serine, threonine, glysine, arginine, and glutamine; preferably selected from alanine, serine, threonine and glutamic acid.

[0103] In various embodiments, depending in part on the anticipated storage conditions of the product, ELP carriers are selected to provide a Tt ranging from about 0⁰C to about 25°C, or from about 4°C to about 20⁰C, or from about 10⁰C to about 15°C. In other embodiments, the ELPs are selected to provide a Tt ranging from about 2°C to about 15°C, or from about 2°C to about 8°C. The transition temperature further takes into account the storage conditions of the product, such as pH, salt content, and ELP concentration.

[0104] ELP components for extending therapeutic half-life by recombinant fusion generally have Tt above body temperature (e.g., above about 37°C or 40⁰C), so as to maintain solution phase in the circulation.

[0105] The Tt can also be varied by varying ELP chain length. The Tt increases with decreasing MW. For polypeptides having a molecular weight > 100,000, the hydrophobicity scale developed by Urry et al. (PCT/US96/05186, which is hereby incorporated by reference in its entirety) is preferred for predicting the approximate Tt of a specific ELP sequence. However, in some embodiments, ELP component length can be kept relatively small, while maintaining a target Tt, by incorporating a larger fraction of hydrophobic guest residues in the ELP sequence.

[0106] For polypeptides having a molecular weight <100,000, the Tt is preferably determined by the following quadratic function: Tt = MO + M1X + M2X2 where X is the MW of the fusion protein, and MO = 116.21 ; M1 = -1.7499; M2 = 0.010349. [0107] In some embodiments, the ELP carrier may be added to the composition in an amount effective for inducing an insoluble phase under the storage conditions. Thus, in some embodiments ELP is added to the composition in an amount in the range of about 0.1 mg to about 50 mg, such as from about 0.5 mg to about 20 mg, or about 1 mg to about 10 mg, or in some embodiments, about 2 mg to about 5 mg. The ratio of ELP carrier to therapeutic molecule (which may additionally be in fusion form) may be from about 100:1 to about 1 :1 , or in some embodiments, about 20:1 to about 2:1 , or about 10:1 to about 2:1. For solution phase formulations, ELP carrier may be added in an amount in the range of about 0.01 mg to about 50 mg, such as from about 1 mg to about 10 mg, or in some embodiments, about 2 mg to about 5 mg.

[0108] The formulations of the invention may comprise additional pharmaceutically acceptable carrier components, that is, in addition to the ELP. By "pharmaceutically acceptable carrier" is intended a carrier that is conventionally used in the art to facilitate the storage, administration, and/or the healing effect of the therapeutic ingredients. A suitable carrier should be stable, i.e., incapable of reacting with other ingredients in the formulation. It should not produce significant local or systemic adverse effects in recipients at the dosages and concentrations employed for treatment. Such carriers are generally known in the art. Suitable carriers for this invention are those conventionally used large stable macromolecules such as gelatin, collagen, polysaccharide, monosaccharides, polyvinylpyrrolidone, polylactic acid, polyglycolic acid, polymeric amino acids, fixed oils, ethyl oleate, liposomes, glucose, lactose, mannose, dextrose, dextran, cellulose, sorbitol, polyethylene glycol (PEG), and the like. However, in certain embodiments, the composition does not contain any plasma-derived proteins, such as albumin.

[0109] The pharmaceutical formulation may additionally comprise a solubilizing agent or solubility enhancer that contributes to the agent's solubility. Examples of such solubility enhancers include the amino acid arginine, as well as amino acid analogues of arginine. Such analogues include, without limitation, dipeptides and tripeptides that contain arginine. Additional suitable solubilizing agents are discussed in U.S. Pat. Nos. 4,816,440; 4,894,330; 5,004,605; 5,183,746; 5,643,566; and in Wang et al. (1980) J. Parenteral Drug Assoc. 34:452-462; herein incorporated by reference.

[0110] In addition to those agents disclosed above, other stabilizing agents, such as ethylenediaminetetracetic acid (EDTA) or one of its salts such as disodium EDTA, can be added to further enhance the stability of the pharmaceutical compositions. The EDTA acts as a scavenger of metal ions known to catalyze many oxidation reactions, thus providing an additional stabilizing effect. Other suitable stabilizing agents include non-ionic surfactants, including polyoxyethylene sorbitol esters such as polysorbate 80 (Tween 80) and polysorbate 20 (Tween 20); polyoxypropylene-polyoxyethylene esters such as Pluronic F68 and Pluronic F127; polyoxyethylene alcohols such as Brij 35; simethicone; polyethylene glycol such as PEG400; lysophosphatidylcholine; and polyoxyethylene-p-t-octylphenol such as Triton X-100. Classic stabilization of pharmaceuticals by surfactants is described, for example, in Levine et al.(1991 ) J. Parenteral Sci. Technol. 45(3):160-165, herein incorporated by reference.

[0111] Exemplary routes of administration for the formulations of the invention include, but are not limited to, oral administration, nasal delivery, pulmonary delivery, and parenteral administration, including transdermal, intravenous, intramuscular, subcutaneous, intraarterial, and intraperitoneal injection or infusion. In one such embodiment, the administration is by injection, such as subcutaneous injection. Injectable forms of the compositions of the invention include, but are not limited to, solutions, suspensions, and emulsions.

[0112] In one embodiment, the stabilized pharmaceutical composition is formulated in a unit dosage and may be in an injectable or infusible form such as solution, suspension, or emulsion. Furthermore, the composition can be stored frozen, refrigerated, or in some embodiments, at room temperature. The composition may be prepared in a concentrated, non-solution, gel, or dried form, any of which may be reconstituted into the liquid solution, suspension, or emulsion before administration by any of various methods including oral or parenteral routes of administration. The stabilized pharmaceutical composition may be stored in unit-dose or multi-dose container such as sealed vials or ampules. The composition may be stored under pressure.

Claims

CLAIMS:

1. A pharmaceutical composition comprising a pharmaceutically active ingredient and a pharmaceutically acceptable carrier, wherein the carrier comprises an Elastin-Like Peptide (ELP) in an amount and under conditions effective for stabilizing the pharmaceutically active ingredient.

2. The pharmaceutical composition of claim 1 , wherein said conditions are one or more of salt, pH, temperature, pressure, and concentration.

3. The pharmaceutical composition of claim 1 or 2, wherein the pharmaceutically active ingredient and the carrier are not covalently associated.

4. The pharmaceutical composition of any one of claims 1 to 3, wherein the pharmaceutically active ingredient is a peptide, optionally provided as a recombinant fusion with ELP.

5. The pharmaceutical composition of any one of claims 1 to 4, wherein the composition is formulated for parenteral delivery.

6. The pharmaceutical composition of any one of claims 1 to 4, wherein the composition is formulated for subcutaneous or intravenous delivery.

7. The pharmaceutical composition of any one of claims 1 to 6, wherein the composition is in solution form.

8. The pharmaceutical composition of any one of claims 1 to 6, wherein the composition is in a non-solution form.

9. The pharmaceutical composition of any one of claims 1 to 7, wherein the composition is in concentrated form.

10. The pharmaceutical composition of any one of claims 1 to 6, wherein the composition is lyophilized.

11. The pharmaceutical composition of any one of claims 1 to 10, wherein the pharmaceutically active ingredient is a peptide, protein, or nucleic acid.

12. The pharmaceutical composition of any one of claims 1 to 10, wherein the pharmaceutically active ingredient is a small drug compound.

13. The pharmaceutical composition of claim 7, wherein the ELP has a transition temperature above room temperature.

14. The pharmaceutical composition of claim 8, wherein the ELP has a transition temperature below room temperature.

15. The pharmaceutical composition of claim 7, wherein the ELP has a transition temperature above about 4° C.

16. The pharmaceutical composition of claim 8, wherein the ELP has a transition temperature below about 4° C.

17. The pharmaceutical composition of claim 8, wherein the composition is contained under pressure.

18. A method of stabilizing a pharmaceutically active ingredient, comprising: adding an ELP to the pharmaceutically active ingredient in an amount effective to stabilize the pharmaceutically active ingredient, thereby producing a stable composition.

19. The method of claim 18, wherein the pharmaceutically active ingredient is prepared as a recombinant fusion with ELP.

20. The method of claim 18 or 19, further comprising, concentrating the composition.

21. The method of any one of claims 18 to 20, further comprising, storing the composition at from about 2 to 20° C.

22. The method of any one of claims 18 to 20, further comprising, storing the composition at around room temperature.

23. The method of any one of claims 18 to 20, further comprising, storing the composition at around 4°C.

24. The method of any one of claims 18 to 20, further comprising, lyophilizing the composition.

25. The method of any one of claims 18 to 20, further comprising, changing the conditions of the composition so as to induce the non-solution phase of the ELP.

26. The method of claim 25, wherein the conditions comprise one or more of salt, pH, temperature, pressure, and concentration.

27. The method of claims 25 or 26, further comprising, resolubilizing the composition prior to delivery.

28. The method of any one of claims 18 and 20 to 27, wherein the pharmaceutically active ingredient and the carrier are not covalently associated.

29. The method of any one of claims 18 to 28, wherein the composition is formulated for parenteral delivery.

30. The method of any one of claims 18 to 28, wherein the composition is formulated for subcutaneous or intravenous delivery.

31. The method of any one of claims 18 to 30, wherein the pharmaceutically active ingredient is a peptide, protein, or nucleic acid.

32 The method of any one of claims 18 and 20 to 30, wherein the pharmaceutically active ingredient is a small drug compound.

33. The method of any one of claims 18 to 32, wherein the ELP has a transition temperature above room temperature.

34. The method of any one of claims 18 to 32, wherein the ELP has a transition temperature below room temperature.

35. The method of any one of claims 18 to 32, wherein the ELP has a transition temperature above about 4° C.

36. The method of any one of claims 18 to 32, wherein the ELP has a transition temperature below about 4° C.