WO2010014689A1 - Formulations pharmaceutiques comprenant des protéines analogues à de l'élastine - Google Patents

Formulations pharmaceutiques comprenant des protéines analogues à de l'élastine Download PDF

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Publication number
WO2010014689A1
WO2010014689A1 PCT/US2009/052080 US2009052080W WO2010014689A1 WO 2010014689 A1 WO2010014689 A1 WO 2010014689A1 US 2009052080 W US2009052080 W US 2009052080W WO 2010014689 A1 WO2010014689 A1 WO 2010014689A1
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Prior art keywords
elp
glp
composition
insulin
active ingredient
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PCT/US2009/052080
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English (en)
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Sue Dagher
Chris Prior
Homayoun Sadeghi
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Phasebio Pharmaceuticals, Inc.
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Publication of WO2010014689A1 publication Critical patent/WO2010014689A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/39—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions

Definitions

  • the present invention relates to pharmaceutically-active ingredients, including
  • ELP fusion proteins stabilized by formulation with one or more Elastin-Like Proteins (ELPs).
  • ELPs Elastin-Like Proteins
  • 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.
  • 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.
  • 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.
  • ELP Elastin- Like Peptide
  • 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.
  • 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.
  • the pharmaceutical compositions of the invention may be lyophilized.
  • the formulations of the invention are in solution phase.
  • 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.
  • 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.
  • the ELP is designed so as to have a transition temperature above the storage temperature, and under the storage conditions.
  • 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.
  • the ELP has a transition temperature above the refrigeration temperature under the storage conditions (which may include pH, salt, ELP concentration, and pressure).
  • formulation with ELPs may serve as a replacement to expensive lyophilization or introduction of plasma-derived excipients such as albumin.
  • the compositions of the invention may lack such plasma-derived proteins.
  • 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.
  • 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.
  • a non-solution state e.g., in a vial or ampule, which may be sealed for storage.
  • 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.
  • the product will be protected from degradative events during storage.
  • the insoluble state 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.
  • the ELP is designed so as to have a transition temperature below the storage temperature, under the storage conditions.
  • the ELP has a transition temperature below the room temperature under the storage conditions (which may include pH, salt, ELP concentration, and pressure).
  • the ELP has a transition temperature below the refrigerated temperature, under the storage conditions (which may include pH, salt, ELP concentration, and pressure).
  • the pharmaceutically active ingredient is an ELP fusion protein designed for administration by injection.
  • the ELP fusion component is designed to remain soluble under physiological conditions.
  • the ELP carrier component 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.
  • 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.
  • the pharmaceutically active ingredient is a small drug compound.
  • 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.
  • 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
  • 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.
  • components other than ELP for extending half-life such as albumin, transferrin, or PEG.
  • 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.
  • 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.
  • chemotherapeutic agent including methotrexate, daunomycin, mitomycin, cisplatin, vincristine, epirubicin, fluorouracil, verapamil, cyclophosphamide, cytosine arabinoside, aminopterin,
  • 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.
  • 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.
  • 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)am ⁇ de, 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, Gly 8 -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.
  • Lys 34 (N ⁇ - tetradecanoyl)-GLP-1 (7-37) designates GLP-1 (7-37) wherein the ⁇ -amino group of the Lys residue in position 34 has been tetradecanoylated.
  • 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
  • the optional amino acid residue in position 40 is Asp unless otherwise indicated.
  • 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.
  • 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]NH 2 (PG[72-107]) in the pancreas, and GLP-1 [7-37] (PG[78- 108]) and GLP-1 [7-36]NH 2 (PG [78-107]) in the L cells of the intestine where GLP-1 [7- 36]NH 2 (78-107 PG) is the major product.
  • glucagon PG[32-62]
  • GLP-1 [7-36]NH 2 PG[72-107]
  • GLP-1 [7-37] PG[78- 108]
  • GLP-1 [7-36]NH 2 PG [78-107]
  • 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)am ⁇ de, 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)am ⁇ de, 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.
  • 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.
  • 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.
  • 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 ⁇ 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).
  • NIDDM non-insulin-dependent diabetes mellitus
  • GLP-1 insulinotropic action of GLP-1 is dependent on plasma glucose concentration (HoIz et al., 1993, Nature 361 :362-5).
  • the loss of GLP-1 -mediated insulin release at low plasma glucose concentration protects against severe hypoglycemia.
  • GLP-1 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).
  • GLP-1 [7-36]NH 2 has the following amino acid sequence:
  • HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR (SEQ ID NO: 13), which may be employed as the GLP-1 in accordance with the invention.
  • the GLP-1 component may contain glycine (G) at the second position, giving, for example, the sequence HGEGTFTSDVSSYLEGQAAKEFIAWLVKGR (SEQ ID NO: 17).
  • G glycine
  • 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.
  • 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).
  • exenatide marketed as Byetta®
  • exenatide is structurally analogous to native GLP-1 , it has a longer half-life after injection.
  • 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 ⁇ -cell growth and replication, suppression of pancreatic glucagon release, delayed gastric emptying, and reduced appetite - all of which function to lower blood glucose.
  • exenatide increases insulin synthesis and secretion in the presence of glucose only, thus lessening the risk of hypoglycemia.
  • 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).
  • 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 ⁇ -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 ).
  • 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.
  • the lipophilic substituent may be an acyl group selected from CH 3 (CH 2 ) n CO-, 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).
  • the GLP-1 is Arg 26 -GLP-1 (7-37), Arg 34 -GLP-1 (7-37),
  • the GLP-1 receptor agonist may have the sequence/structure Arg 34 Lys 26 -(N- ⁇ -( ⁇ - Glu(N- ⁇ -hexadecanoyl)))-GLP-l(7-37).
  • 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).
  • the GLP-1 receptor agonist is as described in
  • the GLP-1 receptor agonist has the sequence [Aib 8 ' 35 ]hGLP-1 (7- 36)NH 2 (e.g. taspoglutide), wherein Aib is alpha-aminoisobutyric acid.
  • CJC-1 131 is a GLP-1 analog that consists of a DPP-IV-resistant form of GLP-
  • the GLP-1 receptor agonist is as described in
  • 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.
  • the GLP-1 receptor agonist is modified with a reactive group selected from the group consisting of succinimidyl and maleimido groups.
  • the GLP-1 receptor agonist has the sequence/structure: D- Ala 8 Lys 37 -(2-(2-(2-maleimidopropionamido(ethoxy)ethoxy)acetamide))-GLP-1 (7-37) (e.g. CJC-1 131 ).
  • AVE0010 also known as ZP-10, is a GLP-1 receptor agonist that may be employed in connection with the invention.
  • HbAI c levels a GLP-1 receptor agonist that may be employed in connection with the invention.
  • the percentages of patients with HbAIc ⁇ 7% ranged from 47-69% for once daily dosing compared to 32% for placebo.
  • AVE0010 treated patients showed dose-dependent reductions in weight and post-prandial plasma glucose.
  • the GLP-1 receptor agonist is as described in
  • the GLP-1 receptor agonist may have the sequence:
  • HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPSKKKKKK-NH2 (SEQ ID NO: 18) (e.g. AVEOOI O).
  • 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.
  • DPP-IV dipeptidase-peptidyl IV
  • the GLP-1 receptor agonist is as described in
  • 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, ⁇ - hydroxy-histidine, homohistidine, ⁇ -fluoromethyl-histidine, or ⁇ -methyl-histidine at position 7.
  • 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.
  • the GLP- 1 receptor agonist has the sequence: HVEGTFTSDVSSYLEEQAAKEFIAWLI KGRG-OH (SEQ ID NO: 19) (e.g. LY548806).
  • the present invention provides stable pharmaceutical formulations of
  • GLP-1 receptor agonists with an elastin-like peptide ELP
  • the GLP-1 receptor agonist is GLP-1 (SEQ ID NO:13 or 17) or a functional analog thereof.
  • 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.
  • 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.
  • 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 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.
  • 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.
  • the present invention provides methods for decreasing food intake, decreasing ⁇ -cell apoptosis, increasing ⁇ -cell function and ⁇ -cell mass, and/or for restoring glucose sensitivity to ⁇ -cells.
  • the patient may be a human or non-human animal patient (e.g., dog, cat, cow, or horse).
  • the patient is human.
  • 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.
  • 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.
  • cAMP activity or glucose dependent insulin production can also be measured.
  • a polynucleotide encoding the GLP-1 receptor is employed to transfect cells to thereby express the GLP-1 receptor protein.
  • 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.
  • a GLP-1 receptor agonist compound can be introduced into a cell, such as an immortalized ⁇ -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).
  • pancreatic ⁇ -cell proliferation may be assessed by 3 H-tymidine or BrdU incorporation assays (See e.g. Buteau et al., 2003, Diabetes 52: 124-32), wherein pancreatic ⁇ -cells such as INS(832/13) cells are contacted with an ELP/ GLP-1 receptor agonist compound and analyzed for increases in 3 H-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).
  • GLP-1 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.
  • the present invention provides a formulation of insulin with an ELP.
  • Insulin injections e.g. of human insulin
  • the insulin-making cells of the body are called ⁇ -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.
  • 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.
  • 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.
  • 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.
  • proinsulin 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.
  • insulin and free C-peptide are packaged into secretory granules, which accumulate in the cytoplasm of the ⁇ -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.
  • 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.
  • 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).
  • rapid acting analogs such as lispro, aspart and glulisine
  • glargine and detemir 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.
  • the insulin may contain the A and B chain of lispro
  • 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.
  • the insulin may contain an A and B chain of aspart
  • 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.
  • 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.
  • the insulin may contain an A and B chain of glargine
  • 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.
  • NPH bedtime neutral protamine Hagedorn
  • 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.
  • 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.
  • the insulin may be a single-chain insulin analog (SIA)
  • 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.
  • the linker has the sequence KDDNPNLPRLVR (SEQ ID NO.: 20) or GAGSSSRRAPQT (SEQ ID NO.: 21 ).
  • this sequence is 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.
  • several different amino acid residues may be added or removed from either end without substantially decreasing the activity of the produced SIA.
  • 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 NH 2 terminals of the native human serum albumin.
  • 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 NH 2 terminus of mature HSA, contained within a pSAC35 vector for expression in yeast.
  • HSA human serum albumin
  • the HSA component of abulin may also be replaced with an ELP component as described herein
  • the present invention provides pharmaceutical formulations of insulin, or a functional analog thereof, with an elastin-like peptide (ELP).
  • 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.
  • 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.
  • 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).
  • 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).
  • sequence identity between two sequences 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.
  • 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.
  • ELP elastin-like peptide
  • the patient may be a human or non-human animal (e.g., dog, cat, cow, or horse) patient.
  • the patient is human.
  • 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 125 l-labeled IGF-1 in L6 cells.
  • 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.
  • 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.
  • FVII Factor VII
  • FVIIa Factor Vila
  • TF tissue factor
  • FXa Factor Xa
  • FXa Factor Xa
  • FXa Factor Xa
  • FXa Factor IXa
  • 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.
  • 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.
  • 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 ⁇ g/ml in plasma.
  • the conversion of zymogen Factor VII into the activated two-chain molecule occurs by cleavage of an internal peptide bond.
  • the cleavage site is at Arg152-lle153 (Hagen et al., 1986, PNAS USA 83: 2412-6).
  • Fractor 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.
  • 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 ( ⁇ G) of binding to FVIIa.
  • 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.
  • FVIIa has only weak activity upon its physiological substrates FX and FIX whereas the TF:FVIIa complex rapidly activates FX and FIX.
  • 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.
  • FXa Factor Xa
  • FIXa FIX to Factor IXa
  • FVIIa Factor IXa
  • 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.
  • 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).
  • NovoSeven.TM NovoSevenTM 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 NovoSevenTM" FDA reference number 96-0597.
  • 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.
  • the Factor Vila analog that may be used in accordance with the invention is as described in WO02/077218 or WO05/074975.
  • the FVIIa analog may have a glutamine substituted for methionine at position 298 (i.e. M298Q-FVIIa).
  • 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.
  • the Factor Vila analog may have an alanine residue substitution for lysine at position 337 (i.e. V158D/E296V/M298Q/K337A-FVIIa).
  • 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.
  • 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.
  • 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).
  • the present invention provides pharmaceutical formulations of Factor Vl I/VI Ia, or functional analog, with an elastin-like peptide (ELP).
  • 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.
  • 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.
  • 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).
  • sequence identity between two sequences 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).
  • 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.
  • 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.
  • the patient is a human or non-human animal (e.g., dog, cat, cow, or horse) patient.
  • the patient is human.
  • 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 4 0 C. 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.
  • the clotting ability of a Factor Vll/Vlla analog can be measured in human FVII deficient plasma.
  • the Factor Vila analog diluted to varying concentrations directly into FVII deficient plasma.
  • InnovinTM Dide, 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.
  • 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.
  • 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.
  • the ELP may comprise or consist essentially of one or more structural unit(s) selected from SEQ ID NOS: 1-12, as defined below.
  • the ELP biopolymers comprising such structural units, may be of varying sizes.
  • 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.
  • 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.
  • 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.
  • Tt transition temperature
  • the ELP aggregates when reaching sufficient size, can be readily removed and isolated from solution by centrifugation.
  • 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.
  • 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.
  • the ELPs may be formed of structural units, including but not limited to:
  • 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.
  • the ELP is formed entirely (or almost entirely) of one or a combination of structural units selected from SEQ ID NOS: 1-12.
  • 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.
  • 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%.
  • 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.
  • the ELP biopolymers may include a ⁇ -turn.
  • Examples of polypeptides suitable for use as the ⁇ -turn are described in International Patent Application PCT/US96/05186, which is hereby incorporated by reference in its entirety.
  • the ELP component may lack a ⁇ -turn, or otherwise have a different conformation and/or folding character.
  • 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.
  • 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.
  • 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.
  • X may be a non-classical amino acid.
  • non-classical amino acids include: D- isomers of the common amino acids, 2,4-diaminobutyric acid, ⁇ -amino isobutyric acid, A- aminobutyric acid, Abu, 2-amino butyric acid, ⁇ -Abu, ⁇ -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, ⁇ -alanine, fluoro-amino acids, designer amino acids such as ⁇ -methyl amino acids, C ⁇ -methyl amino acids, N ⁇ -methyl amino acids,
  • Selection of X is independent in each ELP structural unit (for each structural unit defined herein having a guest residue X).
  • 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.
  • the ELPs may include polymeric or oligomeric repeats of the pentapeptide IPGXG or LPGXG, or a combination thereof, where X is as defined above.
  • 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.
  • 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).
  • 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.
  • ELP3 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 [K 1 V 2 F 1 ⁇ ] 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 [K 1 V 7 F 1 -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
  • the Tt at a given ELP length can be decreased by incorporating a larger fraction of hydrophobic guest residues in the ELP sequence.
  • suitable hydrophobic guest residues include valine, leucine, isoleucine, phenyalanine, tryptophan and methionine. Tyrosine, which is moderately hydrophobic, may also be used.
  • 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.
  • 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.
  • ELP carriers are selected to provide a Tt ranging from about 0 0 C to about 25°C, or from about 4°C to about 20 0 C, or from about 10 0 C to about 15°C.
  • 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.
  • ELP components for extending therapeutic half-life by recombinant fusion generally have Tt above body temperature (e.g., above about 37°C or 40 0 C), so as to maintain solution phase in the circulation.
  • the Tt can also be varied by varying ELP chain length.
  • the Tt increases with decreasing MW.
  • 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.
  • 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.
  • the ELP carrier may be added to the composition in an amount effective for inducing an insoluble phase under the storage conditions.
  • 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 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.
  • 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.
  • the formulations of the invention may comprise additional pharmaceutically acceptable carrier components, that is, in addition to the ELP.
  • 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.
  • the composition does not contain any plasma-derived proteins, such as albumin.
  • the pharmaceutical formulation may additionally comprise a solubilizing agent or solubility enhancer that contributes to the agent's solubility.
  • 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.
  • EDTA ethylenediaminetetracetic acid
  • disodium EDTA disodium EDTA
  • the EDTA acts as a scavenger of metal ions known to catalyze many oxidation reactions, thus providing an additional stabilizing effect.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.

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Abstract

L'invention fournit des compositions ou formulations pharmaceutiques stables, y compris des formulations en solution et pas en solution, et des procédés de préparation de celles-ci. Les formulations de l'invention peuvent améliorer la stabilité et/ou la solubilité de différents ingrédients pharmaceutiquement actifs, y compris des molécules biologiques et de petits composés médicamenteux, et peuvent être adaptées à diverses conditions de stockage. Les formulations de l'invention peuvent fournir une alternative à la lyophilisation, ou peuvent être utilisées ensemble avec une lyophilisation ou d'autres procédés de stabilisation. De manière générale, les compositions comprennent un ingrédient pharmaceutiquement actif, comme une molécule biologique ou un petit composé médicamenteux, et un support pharmaceutiquement acceptable. Le support comprend un peptide analogue à de l'élastine (ELP) en une quantité et une forme (par exemple forme soluble ou insoluble) efficaces pour stabiliser l'ingrédient pharmaceutiquement actif.
PCT/US2009/052080 2008-07-29 2009-07-29 Formulations pharmaceutiques comprenant des protéines analogues à de l'élastine WO2010014689A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2785367A4 (fr) * 2011-11-28 2015-06-17 Phasebio Pharmaceuticals Inc Agents thérapeutiques comprenant des séquences d'acides aminés d'insuline
WO2021030196A1 (fr) * 2019-08-09 2021-02-18 Phasebio Pharmaceuticals, Inc. Protéines de fusion elp comprenant une hormone parathyroïdienne pour une libération contrôlée et prolongée

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030059840A1 (en) * 1999-03-19 2003-03-27 Ashutosh Chilkoti Methods of using bioelastomers

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030059840A1 (en) * 1999-03-19 2003-03-27 Ashutosh Chilkoti Methods of using bioelastomers

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2785367A4 (fr) * 2011-11-28 2015-06-17 Phasebio Pharmaceuticals Inc Agents thérapeutiques comprenant des séquences d'acides aminés d'insuline
WO2021030196A1 (fr) * 2019-08-09 2021-02-18 Phasebio Pharmaceuticals, Inc. Protéines de fusion elp comprenant une hormone parathyroïdienne pour une libération contrôlée et prolongée

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