Classifications

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  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
  • C07K14/70546—Integrin superfamily
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
• • 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
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
  • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
  • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
  • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P27/00—Drugs for disorders of the senses
  • A61P27/02—Ophthalmic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/12—Drugs for disorders of the metabolism for electrolyte homeostasis
  • A61P3/14—Drugs for disorders of the metabolism for electrolyte homeostasis for calcium homeostasis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
  • A61P35/04—Antineoplastic agents specific for metastasis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/76—Albumins
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• the present invention generally relates to fusion proteins comprising a rhodostomin variant having an RGD motif variant 48 ARLDDL 53 , wherein the rhodostomin variant is conjugated with a variant of Human Serum Albumin (HSA).
• HSA Human Serum Albumin
• the invention also relates to the use of these fusion proteins for the treatment and prevention of ⁇ v ⁇ 3 integrin-associated diseases.
• Bone is a complex tissue composed of several cell types which are continuously undergoing a process of renewal and repair termed "bone remodeling.”
• the two major cell types responsible for bone remodeling are osteoclasts, which resorb bone, and osteoblasts, which form new bone.
• Bone remodeling has been known to be regulated by several systemic hormones (e.g., parathyroid hormone, 1 , 25-di hydroxy vitamin D 3 , sex hormones, and calcitonin) and local factors (e.g., nitric oxide, prostaglandins, growth factors, and cytokines).
• systemic hormones e.g., parathyroid hormone, 1 , 25-di hydroxy vitamin D 3 , sex hormones, and calcitonin
• local factors e.g., nitric oxide, prostaglandins, growth factors, and cytokines.
• lntegrins are heterodimeric matrix receptors that anchor cells to substrates and transmit externally derived signals across the plasma membrane, lntegrin ⁇ v ⁇ 3 is involved in the osteoclast-mediated bone resorption, both in vivo and in vitro.
• This heterodimer molecule recognizes the amino acid motif Arg-Gly- Asp (RGD) contained in bone matrix proteins such as osteopontin and bone sialoprotein.
• lntegrin ⁇ v ⁇ 3 is expressed in an osteoclast and its expression is modulated by resorptive steroids and cytokines. Based on blocking experiments, ⁇ v ⁇ 3 integrin has been identified as a major functional adhesion receptor on osteoclasts.
• Integrin ⁇ v ⁇ 3 reduces the capacity of osteoclasts to bind to and resorb bone.
• Integrin ⁇ v ⁇ 3 plays a major role in the function of osteoclasts and inhibitors of this integrin are being considered for treating or preventing osteoporosis, osteolytic metastases, and malignancy-induced hypercalcemia.
• Osteoporosis is the most common one that is induced when resorption and formation of bone are not coordinated and bone breakdown overrides bone building. Osteoporosis is also caused by other conditions, such as hormonal imbalance, diseases, or medications (e.g., corticosteroids or anti-epileptic agents).
• Bone is one of the most common sites of metastasis by human breast, prostate, lung and thyroid cancers, as well as other cancers. Osteoporosis may also result from post-menopausal estrogen deficiency. Secondary osteoporosis may be associated with rheumatoid arthritis. Bone metastasis shows a very unique step of osteoclastic bone resorption that is not seen in metastasis of other organs. It is widely accepted that osteolysis that is associated with cancer is essentially mediated by osteoclasts, which seem to be activated and may be indirectly activated through osteoblasts or directly by tumor products. In addition, hypercalcemia (increased blood-calcium concentration) is an important complication of osteolytic bone diseases. It occurs relatively frequently in patients with extensive bone destruction, and is particularly common in breast, lung, renal, ovarian and pancreatic carcinomas and in myeloma.
• Disintegrins are a family of low-molecular-weight RGD-containing peptides that bind specifically to integrins ⁇ llb ⁇ S, ⁇ 5 ⁇ 1 and ⁇ v ⁇ 3 expressed on platelets and other cells including vascular endothelial cells and some tumor cells.
• studies of disintegrins have revealed new uses in the diagnosis of cardiovascular diseases and the design of therapeutic agents in arterial thrombosis, osteoporosis and angiogenesis-related tumor growth and metastasis.
• Rhodostomin a disintegrin derived from the venom of Colloselasma rhodostoma, has been found to inhibit platelet aggregation in vivo and in vitro through the blockade of platelet glycoprotein ⁇ !lb ⁇ 3. Furthermore, rhodostomin is reported to inhibit the adhesion of breast and prostate carcinoma cells to both unmineralized and mineralized bone extracellular matrices in a dose- dependent manner, without affecting the viability of tumor cells. In addition, rhodostomin inhibits the migration and invasion of breast and prostate carcinoma cells. Rhodostomin has also been shown to inhibit adipogenesis and obesity.
• rhodostomin non-specifically binds to integrins ⁇ llb ⁇ 3, ⁇ 5 ⁇ 1 and ⁇ v ⁇ 3, the pharmaceutical uses of rhodostomin may cause serious side effects.
• the inhibition of platelet aggregation is an undesirable side effect.
• ⁇ v ⁇ 3 integrin plays an important role in angiogenesis and tumor growth in conditions not related to bone diseases.
• polypeptides selective for ⁇ v ⁇ 3 integrin with improved stability and lasting effects.
• These polypeptides will be potentially suitable to treat diseases and conditions involving ⁇ v ⁇ 3 integrin, including but not limited to various bone diseases, cancer, and diseases involving angiogenesis.
• HSA Human serum albumin
• the invention relates to a polypeptide comprising an amino acid sequence of SEQ ID NO 1 , wherein the polypeptide is conjugated with a variant of human serum albumin (HSA) comprising an amino acid sequence of SEQ ID NO 4, or a pharmaceutically acceptable salt of said polypeptide
• HSA human serum albumin
• SEQ ID NO 1 represents an ammo acid sequence of a rhodostomin variant having an RGD motif variant 48 ARLDDL 53
• SEQ ID NO 2 and SEQ ID NO 3 represent two of the possible nucleotide sequences that encode a rhodostomin variant having an RGD motif variant 48 ARLDDL 53
• SEQ ID NO 4 represents an amino acid sequence of the HSA variant, wherein the cysteine residue at position 34 of the HSA ammo acid sequence has been replaced with serine
• HSA C34S represents an amino acid sequence of the HSA variant, wherein the cysteine residue at position 34 of the HSA ammo acid sequence has been replaced with serine
• SEQ ID NO 5 represents a nucleotide sequence that encodes HSA
• the invention relates to a polypeptide comprising an amino acid sequence of SEQ ID NO 1 , wherein the polypeptide is conjugated with a variant of HSA comprising an ammo acid sequence of SEQ ID NO 6, or a pharmaceutically acceptable salt of said polypeptide
• SEQ ID NO 6 represents an amino acid sequence of the HSA variant, wherein the cysteine residue at position 34 of the HSA ammo acid sequence has been replaced with alanine This HSA variant is referred to as HSA [018]
• SEQ ID NO: 7 represents a nucleotide sequence that encodes HSA
• the invention provides a polypeptide comprising an amino acid sequence of SEQ ID NO: 1 , wherein the polypeptide is conjugated with a variant of HSA comprising an amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 6, wherein said polypeptide further comprises a linker amino acid sequence, or a pharmaceutically acceptable salt of said polypeptide.
• the linker amino acid sequence comprises a combination of glycine and serine amino acids.
• the linker amino acid sequence comprises an amino acid sequence of SEQ ID NO: 8.
• the invention relates to a polypeptide comprising an amino acid sequence of SEQ ID NO: 9, or a pharmaceutically acceptable salt of said polypeptide.
• SEQ ID NO: 9 represents an amino acid sequence of HSA(C34S)-
• ARLDDL fusion protein wherein ARLDDL rhodostomin variant is fused to HSA C34S variant through the linker amino acid sequence of SEQ ID NO: 8.
• the invention relates to a polypeptide comprising an amino acid sequence of SEQ ID NO: 11.
• SEQ ID NO: 11 represents an amino acid sequence of HSA(C34A)-
• ARLDDL fusion protein wherein ARLDDL rhodostomin variant is fused to HSA C34A variant through the linker amino acid sequence of SEQ ID NO: 8.
• the invention relates to a polypeptide encoded by a polynucleotide comprising a nucleotide sequence of SEQ ID NO: 10.
• the invention relates to a polypeptide encoded by a polynucleotide comprising a nucleotide sequence of SEQ ID NO: 12.
• polypeptides of the present invention are generally highly selective for ⁇ v ⁇ 3 integrin and exhibit reduced binding to ⁇ llb ⁇ 3 and/or ⁇ 5 ⁇ 1 integrin as compared to a wild type disintegrin.
• polypeptides of the present invention generally exhibit at least about a 5, 50, or 100-fold decrease in affinity to ⁇ llb ⁇ 3 and/or ⁇ 5 ⁇ 1 as compared to rhodostomin.
• the polypeptides of the present invention generally exhibit at least about a 200-fold decrease in affinity to ⁇ llb ⁇ 3 integrin as compared to rhodostomin, more preferably at least about 500-fold decrease in affinity to ⁇ llb ⁇ 3 integrin as compared to rhodostomin,
• the polypeptides of the present invention generally exhibit at least about a 20-fold decrease in affinity to ⁇ 5 ⁇ 1 integrin as compared to rhodostomin, and more preferably, at least about a 70 or 90-fold decrease in affinity to ⁇ 5 ⁇ 1 integrin as compared to rhodostomin.
• polypeptides of the present invention generally exhibit at least about 5, 50, 100, or 150-fold decrease in affinity to platelets a s compared to rhodostomin.
• the polypeptides exhibit a substantially reduced activity in prolongation of blood clotting time as compared to rhodostomin and/or a wild type disintegrin.
• the invention relates to a physiologically acceptable composition
• a physiologically acceptable composition comprising a polypeptide of the invention, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
• the invention relates to a physiologically acceptable composition
• a physiologically acceptable composition comprising a polypeptide comprising an amino acid sequence of SEQ ID NO: 9, or a pharmaceutically acceptable salt of said polypeptide, and a pharmaceutically acceptable carrier.
• the invention relates to a physiologically acceptable composition
• a physiologically acceptable composition comprising a polypeptide encoded by a polynucleotide comprising a nucleotide sequence of SEQ ID NO: 10, or a pharmaceutically acceptable salt of said polypeptide, and a pharmaceutically acceptable carrier.
• the invention relates to a method for treatment and/or prevention of an ⁇ v ⁇ 3 integrin-associated disease comprising administering to a mammal in need thereof a therapeutically effective amount of a polypeptide comprising an amino acid sequence of SEQ ID NO: 1 , wherein the polypeptide is conjugated with a variant of HSA comprising an amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 6, or a pharmaceutically acceptable salt of said polypeptide.
• the invention relates to a method for the treatment and/or prevention of an ⁇ v ⁇ 3 integrin-associated disease comprising administering to a mammal in need thereof a therapeutically effective amount of a polypeptide comprising an amino acid sequence of SEQ ID NO: 9, or a pharmaceutically acceptable salt of said polypeptide.
• the invention relates to a method for the treatment and/or prevention of an ⁇ v ⁇ 3 integrin-associated disease comprising administering to a mammal in need thereof a therapeutically effective amount of a polypeptide e ncoded by a polynucleotide comprising a nucleotide sequence of SEQ ID NO: 10, or a pharmaceutically acceptable salt of said polypeptide.
• the ⁇ v ⁇ 3 integrin-associated disease includes, but is not limited to, osteoporosis, bone tumor or cancer growth and symptoms related thereto, angiogenesis-related tumor growth and metastasis, tumor metastasis in bone, malignancy-induced hypercalcemia, Paget's disease, ovariectomy-induced physiological change, rheumatic arthritis, osteoarthritis and angiogenesis-related eye disease, including but not limited to, age-related macular degeneration, diabetic retinopathy, corneal neovascuiarizing diseases, ischaemia- induced neovascuiarizing retinopathy, high myopia and retinopathy of prematurity.
• the invention relates to a method of using a polypeptide of the invention for the inhibition and/or prevention of tumor cell growth in bone or other organs and symptoms related thereto in a mammal.
• the method of treatment and/or prevention of an ⁇ v ⁇ 3 integrin-associated disease comprises administering to a mammal in need thereof a therapeutically effective amount of a polypeptide comprising an amino acid sequence of SEQ ID NO: 9, or a pharmaceutically acceptable salt of said polypeptide in combination with a therapeutically effective amount of another active agent.
• the other active agent may be administered before, during or after administering the polypeptide of the present invention.
• the other active agent is selected from the group consisting of VEGF antagonists, anti-inflammation agents, bisphosphonates and cytotoxic agents.
• the invention relates to a method for making a polypeptide of the invention, comprising (a) constructing a gene encoding the polypeptide of the invention; (b) transfecting a host cell with the gene of step (a); (c) growing said host cell in a culture medium; and (d) isolating said polypeptide.
• the invention in a preferred embodiment, relates to a method for making a polypeptide comprising an amino acid sequence of SEQ ID NO: 9, comprising (a) constructing a gene encoding said polypeptide; (b) transfecting a host cell with the gene of step (a); (c) growing said host cell in a culture medium; and (d) isolating said polypeptide.
• the methods of making the polypeptides of the present invention may further comprise growing a host cell in a culture medium free of amino acids; and collecting the supernatant to obtain said polypeptide.
• These methods may further comprise adding methanol to the culture medium to induce polypeptide expression in the host cells.
• the methods may further comprise the step of performing column chromatography to obtain said polypeptide.
• the methods may further comprise the step of performing High Performance Liquid Chromatography (HPLC) to obtain the isolated polypeptide.
• HPLC High Performance Liquid Chromatography
• FIGS. 1A and 1 B show HPLC profiles of HSA-ARLDDL
• FIGS. 1 C and 1 D show size exclusion chromatography (SEC) profiles of HSA-ARLDDL and HSA(C34S)-ARLDDL, respectively.
• FIGS. 1 E and 1 F show photographs of SDS-PAGE profiles of HSA-
• ARLDDL and HSA(C34S)-ARLDDL respectively.
• FIG. 1 G shows a photograph of 2D SDS-PAGE profiles of HSA-
• ARLDDL ARLDDL
• HSA(C34S)-ARLDDL HSA(C34S)-ARLDDL
• HSA HSA(C34S)-ARLDDL
• FIG. 1 H shows an NMR spectra of HSA(C34S)-ARLDDL and BSA.
• FIG. 2 shows an amino acid sequence SEQ ID NO: 1 of ARLDDL variant of rhodostomin.
• FIG. 3A shows a nucleotide sequence SEQ ID NO: 2 of ARLDDL variant of rhodostomin.
• FIG. 3B shows a nucleotide sequence SEQ !D NO: 3 of ARLDDL variant of rhodostomin.
• FIGS. 4A and 4B show an amino acid sequence SEQ ID NO: 4 and a nucleotide sequence SEQ ID NO: 5 of HSA C34S mutant, respectively.
• FIGS. 5A and 5B show amino acid sequence SEQ ID NO: 6 and a nucleotide sequence SEQ ID NO: 7 of HSA C34A mutant, respectively.
• FIG. 6 shows an amino acid sequence SEQ ID NO: 8 of a linker amino acid.
• FIGS. 7A and 7B show an amino acid sequence SEQ ID NO: 9 and a nucleotide sequence SEQ ID NO: 10 of HSA(C34S)-ARLDDL, respectively.
• FIGS. 8A and 8B show an amino acid sequence SEQ ID NO: 1 1 and a nucleotide sequence SEQ ID NO: 12 of HSA(C34A)-ARLDDL, respectively.
• FIGS. 9A, 9B and 9C are photographs of hematopoetic cells of bone marrow showing that HSA(C34S)-ARLDDL inhibits the differentiation of osteoclasts.
• FIGS. 10A, 10B and 10C are graphs showing that HSA-ARLDDL and
• HSA(C34S)-ARLDDL inhibit the differentiation of osteoclasts.
• FIGS. 1 1 A, 1 1 B, 11 C and 11 D are graphs showing that HSA-ARLDDL and HSA(C34S)-ARLDDL inhibit angiogenesis in a mouse model of retinopathy of prematurity (ROP).
• ROP retinopathy of prematurity
• FIGS. 11 E, 11 F and 11 G are photographs showing angiogenesis in a mouse model of oxygen-induced retinopathy. They show that HSA(C34S)-ARLDDL inhibits angiogenesis in an oxygen-induced retinopathy mouse.
• FIGS. 12A and 12B are photographs of mice injected with the human
• FIG. 12A is control and FIG. 12B shows two mice treated with HSA(C34S)-ARLDDL.
• FIGS. 12C and 12D are photographs of tumors excised, respectively, from control mice and mice treated with HSA(C34S)-ARLDDL.
• FIGS. 13 is a graph that show that HSA(C34S)-ARLDDL significantly reduced tumor size and tumor weight in mice injected with the human PC-3 tumor cells.
• FIG. 14A is a set of photographs showing a reduced blood vessel density in MATRIGELTM plugs from C57BL/6 mice treated with HSA(C34S)- ARLDDL in comparison with untreated control mice.
• FIG. 14B is a graph showing a reduced hemoglobin content in
• polynucleotide refers to polymeric forms of nucleotides of any length.
• the polynucleotides can comprise deoxyribonucleotides, ribonucleotides, and/or their analogs or derivatives.
• the term includes variants. Variants may include insertions, additions, deletions or substitutions. Nucleotide sequences are listed in the 5' to 3' direction.
• polypeptide used interchangeably to refer to a polymeric form of amino acids of any length, which can include naturally-occurring amino acids, coded and non-coded amino acids, chemically or biochemically modified, derivatized, or designer amino acids, amino acid analogs, peptidomimetics, and depsipeptides, and polypeptides having modified, cyclic, bicyclic, depsicyclic or depsibicyclic peptide backbones.
• the term includes single chain protein as well as multimers.
• the terms also include fusion proteins, including, but not limited to, glutathione S-transferase (GST) fusion proteins, fusion proteins with a heterologous amino acid sequence such as bioiuminescent proteins, for example, luciferin, or aequorin (green fluorescent protein), with heterologous and homologous leader sequences, fusion proteins with or without N-terminal methionine residues, pegylated proteins, and immunologically tagged, or his-tagged proteins.
• GST glutathione S-transferase
• fusion proteins with a heterologous amino acid sequence such as bioiuminescent proteins, for example, luciferin, or aequorin (green fluorescent protein)
• heterologous and homologous leader sequences such as fusion proteins with or without N-terminal methionine residues, pegylated proteins, and immunologically tagged, or his-tagged proteins.
• Such fusion proteins also include fusions to epitopes.
• Such fusion proteins
• hybridizes specifically in the context of a polynucleotide, refers to hybridization under stringent conditions. Conditions that increase stringency of both DNA/DNA and DNA/RNA hybridization reactions are widely known and published in the art. Examples of stringent hybridization conditions include hybridization in 4 X sodium chloride/sodium citrate (SSC), at about 65-70 °C, or hybridization in 4 X SSC plus 50% formamide at about 42-50 °C, followed by one or more washes in 1X SSC, at about 65-70 0 C.
• SSC sodium chloride/sodium citrate
• ligand refers to a molecule that binds to another molecule, including a receptor.
• mammal includes, but is not limited to, a human.
• host cell is an individual cell or cell culture which can be or has been a recipient of any recombinant vector(s) or polynucleotide.
• Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in total DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation and/or change.
• a host cell includes cells transfected or infected in vivo or in vitro with a recombinant vector or a polynucleotide of the invention.
• a host cell which comprises a recombinant vector of the invention may be called a "recombinant host cell.”
• treatment refers to any administration or application of remedies for disease in a mammal and includes inhibiting the disease, arresting its development, relieving the disease, for example, by causing regression, or restoring or repairing a lost, missing, or defective function; or stimulating an inefficient process.
• the term includes obtaining a desired pharmacologic and/or physiologic effect, covering any treatment of a pathological condition or disorder in a mammal.
• the effect may be prophylactic in terms of completely or partially preventing a disorder or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disorder and/or adverse affect attributable to the disorder.
• It includes (1 ) preventing the disorder from occurring or recurring in a subject who may be predisposed to the disorder but is not yet symptomatic, (2) inhibiting the disorder, such as arresting its development, (3) stopping or terminating the disorder or at least its associated symptoms, so that the host no longer suffers from the disorder or its symptoms, such as causing regression of the disorder or its symptoms, for example, by restoring or repairing a lost, missing or defective function, or stimulating an inefficient process, or (4) relieving, alleviating or ameliorating the disorder, or symptoms associated therewith, where ameliorating is used in a broad sense to refer to at least a reduction in the magnitude of a parameter, such as inflammation, pain and/or tumor size.
• a parameter such as inflammation, pain and/or tumor size.
• pharmaceutically acceptable carrier refers to a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material, formulation auxiliary, or excipient of any conventional type.
• a pharmaceutically acceptable carrier is nontoxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the formulation.
• composition refers to a mixture that usually contains a carrier, such as a pharmaceutically acceptable carrier or excipient that is conventional in the art and which is suitable for administration into a subject for therapeutic, diagnostic, or prophylactic purposes. It may include a cell culture in which the polypeptide or polynucleotide is present in the cells or in the culture medium.
• a carrier such as a pharmaceutically acceptable carrier or excipient that is conventional in the art and which is suitable for administration into a subject for therapeutic, diagnostic, or prophylactic purposes. It may include a cell culture in which the polypeptide or polynucleotide is present in the cells or in the culture medium.
• compositions for oral administration can form solutions, suspensions, tablets, pills, capsules, sustained release formulations, oral rinses or powders.
• disease refers to any condition, infection, disorder or syndrome that requires medical intervention or for which medical intervention is desirable. Such medical intervention can include treatment, diagnosis and/or prevention.
• Rho means "rhodostomin,” which is a disintegrin derived from the venom of Colloselasma rhodostorna. Rhodostomin non- specifically binds to integrins ⁇ llb ⁇ S, ⁇ 5 ⁇ 1 and ⁇ v ⁇ 3, and prolongs blood clotting time by inhibiting platelet aggregation through the blockade of platelet glycoprotein ⁇ llb ⁇ 3.
• IC50 the half maximal inhibitory concentration refers to the concentration of Rho or its variant that is required for 50% inhibition of its receptor. IC 50 is a measure of how much of Rho or its variant is needed to inhibit a biological process by 50%, such as the variant's affinity to its receptor.
• therapeutically effective amount refers to an amount which, when administered to a living subject, achieves a desired effect on the living subject.
• an effective amount of the polypeptide of the invention for administration to the living subject is an amount that prevents and/or treats an integrin ⁇ v ⁇ 3-mediated disease. The exact amount will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques.
• receptor antagonist refers to a binding ligand of a receptor that inhibits the function of a receptor by blocking the binding of an agonist to the receptor, or which allows agonist binding, but inhibits the ability of the agonist to activate the receptor.
• substantially reduced integrin ⁇ llb ⁇ 3 and/or ⁇ 5 ⁇ 1 receptor- blocking activity refers to a reduced activity of at least five fold in blocking integrin ⁇ llb ⁇ 3 and/or ⁇ 5 ⁇ 1 receptor as compared to wild type rhodostomin or other disintegrins.
• the IC 5O of a rhodostomin variant for inhibition of integrin ⁇ llb ⁇ 3 and/or ⁇ 5 ⁇ 1 binding to a matrix protein, such as fibrinogen is compared to of the IC 50 of Rho.
• RGD motif variant refers to a peptide comprising a modification in the amino acid sequence that spans the RGD sequence of a corresponding wild type sequence, such as the sequence comprising RGD in Rhodostomin.
• ARLDDL refers to a rhodostomin variant having an RGD motif variant 48 ARLDDL 53 .
• the numbers "48" and “53” refer to positions of these amino acids in the amino acid sequence of wild type rhodostomin.
• HSA C34S refers to a human serum albumin (HSA) variant wherein the cysteine residue at position 34 of wild type HSA amino acid sequence has been replaced with serine.
• HSA C34S comprises SEQ ID NO: 4.
• HSA C34A refers to an HSA variant wherein the cysteine residue at position 34 of wild type HSA amino acid sequence has been replaced with alanine HSA C34A comprises SEQ ID NO 6
• HSA(C34S)-ARLDDL refers to a fusion protein comprising a) a human serum albumin (HSA) variant wherein the cysteine residue at position 34 of wild type HSA amino acid sequence has been replaced with serine, b) the linker amino acid sequence of SEQ ID NO 8, and c) a rhodostomin variant having an RGD motif variant 48 ARLDDL 53
• HSA(C34S)-ARLDDL is represented by SEQ ID NO 9
• HSA(C34A)-ARLDDL refers to a fusion protein comprising a) a human serum albumin (HSA) variant wherein the cysteine residue at position 34 of wild type HSA amino acid sequence has been replaced with alanine, b) the linker amino acid sequence of SEQ ID NO 8, and a rhodostomin variant having an RGD motif variant 48 ARLDDL 53
• HSA(C34A)-ARLDDL is represented by SEQ ID NO 1 1
• inhibitory selectivity for integ ⁇ n ⁇ v ⁇ 3 relative to ⁇ llb ⁇ 3 and/or ⁇ 5 ⁇ 1 receptors refers to a polypeptide's binding selectivity toward integrin ⁇ v ⁇ 3 over ⁇ llb ⁇ 3 and/or ⁇ 5 ⁇ 1 receptors, which is expressed as a ratio of the IC 50 of the variant for inhibition of ⁇ llb ⁇ 3 and/or ⁇ 5 ⁇ 1 receptors over that for inhibition of ⁇ v ⁇ 3 receptor
• Dleeding time' refers to a polypeptide's reduced ability to inhibit blood clotting in a statistically significant manner as measured by the bleeding time experiment described in the specification
• pegylated-ARLDDL or peg-ARLDDL refer to a pegylated product of ARLDDL protein
• albumin-ARLDDL or "HSA-ARLDDL” refer to a human albumin-conjugated product of ARLDDL protein OVERVIEW OF THE INVENTION
• U.S. Patent Application Serial No. 12/004,045 describes various polypeptides selective for ⁇ v ⁇ 3 integrin and exhibiting reduced integrin ⁇ llb ⁇ 3 and/or ⁇ 5 ⁇ 1 receptor-blocking activity as compared to a wild type disintegrin. These polypeptides are encoded by modified disintegrin nucleotide sequences that encode modified amino acid sequences. As a result, polypeptides are created which have substantially reduced integrin ⁇ llb ⁇ 3 and/or ⁇ 5 ⁇ 1 receptor-blocking activity.
• Disintegrin variants such as RD-related compounds potently inhibit osteoclast differentiation in vitro. They also inhibit osteoclast resorbing activity and ovariectomy-induced increase in osteoclast formation in animal studies.
• RD inhibits the tumor growth of human prostate and breast cancer cells in bone. Malignancy-induced hypercalcemia was also effectively blocked by RD-related proteins.
• Paget's disease also known as osteitis deformans
• Bisphosphonates have been approved for the treatment of Paget's disease.
• Osteoarthritis is also related to the increase in osteoclast activity.
• RD derivatives should also be effective for treatment of these bone disorders.
• long term administration of PGP (I.V., 0.5 mg/kg/day) for 6 weeks did not affect serum level of creatinine, GOT, and GPT, suggesting lack of side effects on kidney and liver. Therefore, RD and its derivatives, especially ARLDDL, are potential drug candidates for treatment of osteoporosis, bone tumor, malignancy-induced hypercalcemia, Paget's disease, rheumatic arthritis, osteoarthritis and angiogenesis-related eye diseases.
• the present invention is generally related to polypeptides comprising an amino acid sequence of SEQ ID NO: 1 , wherein these polypeptides are conjugated with a variant of human serum albumin (HSA), wherein the cysteine residue at position 34 of the HSA amino acid sequence has been replaced either with serine to create HSA C34S mutant protein, or with alanine to create HSA C34A mutant protein.
• HSA human serum albumin
• SEQ ID NO: 1 represents an amino acid sequence of a rhodostomin variant having an RGD motif variant 48 ARLDDL 53 .
• SEQ ID NO: 2 and SEQ ID NO: 3 represent two of the possible nucleotide sequences that encode a rhodostomin variant having an RGD motif variant 48 ARLDDL 53 .
• SEQ ID NO: 4 represents an amino acid sequence of the HSA C34S mutant protein.
• SEQ ID NO: 5 represents a nucleotide sequence that encodes HSA
• SEQ ID NO: 6 represents an amino acid sequence of the HSA C34A mutant protein.
• SEQ ID NO: 7 represents a nucleotide sequence that encodes HSA
• the invention provides a polypeptide comprising an amino acid sequence of SEQ ID NO: 1 , wherein the polypeptide is conjugated with a variant of HSA comprising an amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 6, wherein said polypeptide further comprises a linker amino acid sequence, or a pharmaceutically acceptable salt of said polypeptide.
• the linker amino acid sequence comprises a combination of glycine and serine amino acids.
• the linker amino acid sequence comprises an amino acid sequence of SEQ ID NO: 8.
• the invention relates to a polypeptide comprising an amino acid sequence of SEQ ID NO: 9, or a pharmaceutically acceptable salt of said polypeptide.
• SEQ ID NO: 9 represents an amino acid sequence of HSA(C34S)-
• ARLDDL fusion protein wherein ARLDDL rhodostomin variant is fused to HSA C34S variant through the linker amino acid sequence of SEQ ID NO: 8.
• the invention relates to a polypeptide comprising an amino acid sequence of SEQ ID NO: 11.
• SEQ ID NO: 1 1 represents an amino acid sequence of HSA(C34A)-
• ARLDDL fusion protein wherein ARLDDL rhodostomin variant is fused to HSA C34A variant through the linker amino acid sequence of SEQ ID NO: 8.
• the invention relates to a polypeptide encoded by a polynucleotide comprising a nucleotide sequence of SEQ ID NO: 10.
• a polypeptide of the invention is encoded by a polynucleotide comprising a nucleotide sequence of SEQ ID NO: 12.
• polypeptides of the invention generally are highly selective for ⁇ v ⁇ 3 integrin and exhibit reduced binding to ⁇ llb ⁇ 3 and/or ⁇ 5 ⁇ 1 integrin compared to a wild type disintegrin.
• polypeptides of the present invention generally exhibit at least about a 5, 50, or 100-fold decrease in affinity to ⁇ llb ⁇ 3 and/or ⁇ 5 ⁇ 1 as compared to rhodostomin.
• the polypeptides of the present invention generally exhibit at least about a 200-fold decrease in affinity to ⁇ llb ⁇ 3 integrin as compared to rhodostomin, more preferably at least about 500-fold decrease in affinity to ⁇ llb ⁇ S integrin as compared to rhodostomin.
• the polypeptides of the present invention generally exhibit at least about a 20-fold decrease in affinity to ⁇ 5 ⁇ 1 integrin as compared to rhodostomin, and more preferably, at least about a 70 or 90-fold decrease in affinity to ⁇ 5 ⁇ 1 integrin as compared to rhodostomin.
• polypeptides of the present invention generally exhibit at least about 5, 50, 100, or 150-fold decrease in affinity to platelets as compared to rhodostomin.
• the polypeptide exhibits a substantially reduced activity in prolongation of blood clotting time as compared to rhodostomin and/or a wild type disintegrin.
• the polypeptides of the invention can be created and expressed using methods known in the art.
• Cell-based methods and cell-free methods are suitable for producing the polypeptides of the invention.
• Cell-based methods generally involve introducing a nucleic acid construct into a host cell in vitro and culturing the host cell under conditions suitable for expression, then harvesting the peptide, either from the culture medium or from the host cell, (for example, by disrupting the host cell), or both.
• the invention also provides methods of producing a peptide using cell-free in vitro transcription/translation methods, which are well known in the art.
• Suitable host cells include prokaryotic or eukaryotic cells, including, for example, bacterial, yeast, fungal, plant, insect and mammalian cells.
• Example 1 describes the construction and expression of one polypeptide according to the invention, HSA(C34S)-ARLDDL.
• a polypeptide of the invention may be expressed on its own and may include secretion signals and/or a secretory leader sequence.
• a secretory leader sequence of the invention may direct certain proteins to the endoplasmic reticulum (ER).
• the ER separates the membrane-bound proteins from other proteins. Once localized to the ER, proteins can be further directed to the Golgi apparatus for distribution to vesicles, including secretory vesicles, the plasma membrane, lysosomes and other organelles.
• peptide moieties and/or purification tags may be added to the polypeptides of the invention, in addition to a variant HSA. These peptide moieties and/or purification tags may be removed prior to final preparation of the polypeptide. Suitable purification tags include, for example, V5, polyhistidines, avidin and biotin. Conjugation of peptides to compounds, such as biotin, can be accomplished using techniques well known in the art. (Hermanson ed. (1996) Bioconjugate Techniques; Academic Press).
• Polypeptides of the invention can also be conjugated with radioisotopes, toxins, enzymes, fluorescent labels, colloidal gold, nucleic acids, vinorelbine and doxorubicin using techniques known in the art. (Hermanson ed. (1996) Bioconjugate Techniques; Academic Press; Stefano et al. (2006).
• HSA-disintegrin fusion proteins of the present invention are further fused with other proteins.
• Fusion partners suitable for such use include, for example, fetuin, Fc and/or one or more of their fragments.
• Polyethylene glycol conjugates with the fusion proteins of the present invention are also provided.
• polypeptides of the invention can also be chemically synthesized using techniques known in the art (e.g., see Hunkapiller et al., Nature, 310:105 111 (1984); Grant ed. (1992) Synthetic Peptides, A Users Guide, W.H. Freeman and Co.; U.S. Patent No. 6,974,884)).
• a polypeptide corresponding to a fragment of a polypeptide can be synthesized by use of a peptide synthesizer or through the use of solid-phase methods known in the art.
• nonclassical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the polypeptide sequence.
• Non-classical amino acids include, but are not limited to, to the D- isomers of the common amino acids, 2,4-diaminobutyric acid, a-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, g-Abu, e-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyprohne, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, b-alanine, fluoro-amino acids, designer
• polypeptides of the invention can be recovered and purified from chemical synthesis and recombinant cell cultures by standard methods which include, but are not limited to, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography
• HPLC high performance liquid chromatography
• HPLC high performance liquid chromatography
• Well known techniques for refolding protein may be employed to regenerate active conformation when the polypeptide is denatured during isolation and/or purification
• a polypeptide or peptidomimetic of the invention can be further modified with or covalently coupled to one or more of a variety of hydrophilic polymers to increase solubility and circulation half-life of the polypeptide
• Suitable nonproteinaceous hydrophilic polymers for coupling to a peptide include, but are not limited to, polyalkylethers as exemplified by polyethylene glycol and polypropylene glycol, polylactic acid, polyglycolic acid, polyoxyalkenes, polyvinylalcohol, polyvinylpyrrolidone, cellulose and cellulose derivatives, dextran, and dextran derivatives
• such nydrophilic polymers have an average molecular weight ranging from about 500 to about 100,000 daltons, from about 2,000 to about 40,000 daitons, or from about 5,000 to about 20,000 daltons
• Tne peptide can be de ⁇ vatized with or coupled to such polymers using any of the methods set forth in Zallipsky, S (19
• Polypeptides of the invention can include naturally-occurring and non- naturally occurring amino acids
• Polypeptides can comprise D-amino acids, a combination of D- and L-amino acids, and various "designer” or “synthetic” amino acids (for example, ⁇ -methyl amino acids, C ⁇ -methyl amino acids, and N ⁇ -methyl amino acids, etc.) to convey special properties.
• polypeptides can be cyclic.
• Polypeptides can include any known non-classical amino acids.
• amino acid analogs and peptidomimetics can be incorporated into a polypeptide to induce or favor specific secondary structures, including, but not limited to, LL-Acp (LL-3-amino-2-propenidone-6-carboxylic acid), a ⁇ -turn inducing dipeptide analog; ⁇ -sheet inducing analogs; ⁇ -turn inducing analogs; ⁇ -helix inducing analogs; ⁇ -tum inducing analogs; GIy-AIa turn analogs; amide bond isostere; or tretrazol, and the like.
• LL-Acp LL-3-amino-2-propenidone-6-carboxylic acid
• a ⁇ -turn inducing dipeptide analog ⁇ -sheet inducing analogs
• ⁇ -turn inducing analogs ⁇ -helix inducing analogs
• ⁇ -tum inducing analogs GIy-AIa turn analogs
• amide bond isostere or
• desamino or descarboxy residues can be incorporated at the terminal ends of the polypeptide to decrease susceptibility to proteases or to restrict conformation.
• C-terminal functional groups of the polypeptides of the present invention include amide, amide lower alkyl, amide di-lower alkyl, lower alkoxy, hydroxy, carboxy, the lower ester derivatives thereof, and the pharmaceutically acceptable salts thereof.
• the invention relates to a physiologically acceptable composition
• a physiologically acceptable composition comprising a polypeptide of the invention, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
• the invention relates to a physiologically acceptable composition
• a physiologically acceptable composition comprising a polypeptide comprising an amino acid sequence of SEQ ID NO: 9, or a pharmaceutically acceptable salt of said polypeptide, and a pharmaceutically acceptable carrier.
• the invention relates to a physiologically acceptable composition
• a physiologically acceptable composition comprising a polypeptide encoded by a polynucleotide comprising a nucleotide sequence of SEQ ID NO: 10, or a pharmaceutically acceptable salt of said polypeptide, and a pharmaceutically acceptable carrier.
• compositions of the invention may be provided as formulations with pharmaceutically acceptable carriers, excipients and diluents, which are known in the art These pharmaceutical carriers, excipients and diluents include those listed in the USP pharmaceutical excipients listing USP and NF Excipients, Listed by Categories, p 2404-2406, USP 24 NF 19, United States Pharmacopeial Convention lnc , Rockville, Md (ISBN 1-889788-03-1) Pharmaceutically acceptable excipients, such as vehicles, adjuvants, carriers or diluents, are readily available to the public Moreover, pharmaceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents and the like, are readily available to the public
• Suitable carriers include, but are not limited to, water, dextrose, glycerol, saline, ethanol, and combinations thereof
• the carrier can contain additional agents such as wetting or emulsifying agents, pH buffering agents, or adjuvants which enhance the effectiveness of the formulation
• Topical carriers include liquid petroleum, isopropyl palmitate, polyethylene glycol, ethanol (95%), polyoxyethylene monolaurate (5%) in water, or sodium lauryl sulfate (5%) in water
• Other materials such as antioxidants, humectants, viscosity stabilizers, and similar agents can be added as necessary
• Percutaneous penetration enhancers such as Azone can also be included
• Polypeptides of the invention can be formulated into preparations for injection by dissolving, suspending or emulsifying them in an aqueous or nonaqueous solvent, such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol, and if desired, with conventional additives such as solubilizers isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives
• an aqueous or nonaqueous solvent such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol, and if desired, with conventional additives such as solubilizers isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives
• an aqueous or nonaqueous solvent such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or prop
• compositions of the invention can be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants and aerosols [0153]
• the compositions of the invention can be administered in the form of their pharmaceutically acceptable salts, or they can also be used alone or in appropriate association, as well as in combination, with other pharmaceutically active compounds.
• the subject compositions are formulated in accordance to the mode of potential administration.
• the invention relates to a method for the treatment and/or prevention of an ⁇ v ⁇ 3 integrin-associated disease comprising administering to a mammal in need thereof a therapeutically effective amount of a polypeptide comprising an amino acid sequence of SEQ ID NO: 1 , wherein the polypeptide is conjugated with a variant of HSA comprising an amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 6, or a pharmaceutically acceptable salt of said polypeptide.
• the invention relates to a method for the treatment and/or prevention of an ⁇ v ⁇ 3 integrin-associated disease comprising administering to a mammal in need thereof a therapeutically effective amount of a polypeptide comprising an amino acid sequence of SEQ ID NO: 9, or a pharmaceutically acceptable salt of said polypeptide.
• the invention relates to a method for the treatment and/or prevention of an ⁇ v ⁇ 3 integrin-associated disease comprising administering to a mammal in need thereof a therapeutically effective amount of a polypeptide encoded by a polynucleotide comprising a nucleotide sequence of SEQ ID NO: 10, or a pharmaceutically acceptable salt of said polypeptide.
• An ⁇ v ⁇ 3 integrin-associated disease includes, but is not limited to, osteoporosis, bone tumor or cancer growth and symptoms related thereto, angiogenesis-related tumor growth and metastasis, tumor metastasis in bone, malignancy-induced hypercalcemia, multiple myeloma, Paget's disease, ovariectomy-induced physiological change, rheumatic arthritis, osteoarthritis and angiogenesis-related eye disease, including but not limited to, age-related macular degeneration, diabetic retinopathy, corneal neovascularizing diseases, ischaemia- induced neovascularizing retinopathy, high myopia and retinopathy of prematurity.
• the osteoporosis may be associated with a pathological condition chosen from post-menopausal estrogen deficiency, secondary osteoporosis, rheumatoid arthritis, ovariectomy, Paget's disease, bone cancer, bone tumor, osteoarthritis, increased osteoclast formation and increased osteoclast activity.
• the osteoporosis includes, but is not limited to, an ovariectomy- induced or post-menopausal osteoporosis, physiological change or bone loss.
• the invention relates to a method of using a polypeptide of the invention for inhibition and/or prevention of tumor cell growth in bone or other organs and symptoms related thereto in a mammal in need thereof.
• the pathological symptoms related to tumor cell growth in bone include an increased osteoclast activity, an increased bone resorption, bone lesion, hypercalcemia, a body weight loss, and any combinations thereof.
• the tumor cell growth in bone includes bone cancer cells and metastasized cancer cells originating from prostate cancer, breast cancer, lung cancer, renal cancer, ovarian cancer, pancreatic cancer or myeloma cancer.
• Polypeptides of the invention may be administered to a subject in need of treatment by injection systemically, such as by intravenous injection; or by injection or application to the relevant site, such as by direct injection, or direct application to the site when the site is exposed in surgery; or by topical application.
• Polypeptides of the invention can be used as monotherapy. Alternatively, the polypeptides of the invention can be used in combination with another active agent to treat ⁇ v ⁇ 3 integrin associated diseases.
• the method of treatment and/or prevention of an ⁇ v ⁇ 3 integrin-associated disease comprises administering to a mammal in need thereof a therapeutically effective amount of a polypeptide comprising an amino acid sequence of SEQ ID NO: 9, or a pharmaceutically acceptable salt of said polypeptide in combination with a therapeutically effective amount of another active agent.
• the other active agent may be administered before, during, or after administering the polypeptide of the present invention.
• the other active agent is selected from the group consisting of VEGF antagonists, anti-inflammation agents, bisphosphonates and cytotoxic agents.
• Administration of the active agents can be achieved in various ways, including oral, buccal, nasal, rectal, parenteral, intraperitoneal, intradermal, transdermal, subcutaneous, intravenous, intra-arterial, intracardiac, intraventricular, intracranial, intratracheal, and intrathecal administration, intramuscular injection, intravitreous injection, topical application, including but not limited to eye drops, creams, and emulsions, implantation and inhalation.
• the invention relates to a method for making a polypeptide of the invention, comprising (a) constructing the gene encoding the polypeptide of the invention; (b) transfecting a host cell with the gene of step (a); (c) growing said host cell in a culture medium; and (d) isolating said polypeptide.
• the invention relates to a method for making a polypeptide comprising an amino acid sequence of SEQ ID NO: 9, comprising (a) constructing a gene encoding said polypeptide; (b) transfecting a host ceil with the gene of step (a); (c) growing said host cell in a culture medium; and (d) isolating said polypeptide.
• the methods of making the polypeptides of the present invention may further comprise growing host cell in a culture medium free of amino acids; and collecting supernatant to obtain said polypeptide.
• These methods may further comprise adding methanol to the culture medium to induce the polypeptide expression in the host cells.
• the methods may further comprise the step of performing a column chromatography to obtain said polypeptide.
• the methods may further comprise the step of performing a high performance liquid chromatography (HPLC) to obtain the isolated polypeptide.
• HPLC high performance liquid chromatography
• RANKL and M-CSF Human recombinant RANKL and M-CSF were purchased from R&D Systems (Minneapolis, MN). The C-terminal telopeptides of type-l collagen ELISA kit was obtained from Cross Laps (Herlev, Denmark). All other chemicals were obtained from Sigma.
• HSA C34S The structural gene of HSA C34S was constructed using HSA (Invitrogen®, clone ID: IOH23065) as a template.
• the mutation of C34S was produced by two-step polymerase chain reaction (PCR). The first PCR was amplified with the sense primer containing C34S mutation site and with the antisense primer containing Kpn I, Sac Il restriction sites and a TAA stop codon. The second PCR was amplified with the sense primer containing BstB I restriction site and the secretion signal sequence and with the antisense primer containing Kpn I, Sac Ii restriction sites and a TAA stop codon.
• the secretion signal sequence of HSA prepro peptide, the ⁇ factor prepro peptide from Saccharomyces cerevisiae, or preHSA and pro ⁇ factor fusion peptide was used for secretory protein expression.
• the structural gene of ARLDDL was amplified by PCR with the sense primer containing Kpn I restriction site and the spacer region containing GS sequence and with the antisense primer containing Sac Il restriction site and a TAA stop codon.
• the PCR products of HSA C34S with the secretion signal peptide and Rho ARLDDL mutant with the spacer region were digested using Kpn I restriction enzyme and then were ligated.
• the resulting gene product was cloned into the BstB I and Sac Il sites of the yeast recombination vector.
• the recombinant plasmid was then transformed into an Escherichia coli XL1-blue strain, and the colonies were selected using the agar plates with low salt LB (1% tryptone, 0.5% yeast extract, 0.5% NaCI, 1.5% agar at pH 7.0) and 25 ⁇ g/ml antibiotic Zeocin.
• the E. coli XL1-blue colonies were picked and the plasmid DNA was isolated and sequenced.
• the DNA encoding secretion signal sequence HSA(C34S)-ARLDDL was synthesized.
• the secretion signal sequence of HSA prepro peptide, the ⁇ factor prepro peptide from Saccharomyces cerevisiae, or preHSA and pro ⁇ factor fusion peptide was used for secretory protein expression.
• the resulting gene product was cloned into the yeast recombination vector with proper restriction site.
• the recombinant plasmid was then transformed into an Escherichia coli XL1-blue strain, and the colonies were selected using the agar plates with low salt LB (1 % tryptone, 0.5% yeast extract, 0.5% NaCi, 1.5% agar at pH 7.0) and 25 ⁇ g/ml antibiotic Zeocin. The E. coli XL1-blue colonies were picked and the plasmid DNA was isolated and sequenced.
• HSA(C34S)-ARLDDL The colonies were selected on agar plates containing YPD (1 % yeast extract, 2% peptone, 2% glucose, and 2% agar) and 100 ⁇ g/ml Zeocin A number of clones with multiple copies of HSA(C34S)-ARLDDL gene insertions were selected to pick the clone with the highest protein expression
• the resulting recombinant HSA(C34S)-ARLDDL contained 585 amino acids of HSA, a spacer containing 17 amino acid residues, and 68 amino acids of rhodostomin ARLDDL mutant
• FIGs 1A and 1 B HPLC profiles of HSA-ARLDDL and HSA(C34S)-ARLDDL are shown in FIGs 1A and 1 B, respectively.
• Table 1 shows the reduction protein aggregates on HSA(C34S)- ARLDDL
• FIGS 1 E and 1 F show photographs of SDS-PAGE profiles of HSA- ARLDDL and HSA(C34S)-ARLDDL, respectively
• Lane 1 contains Molecular Weight Markers
• Lane 2 contains Methanol induction
• Lane 3 contains HSA-ARLDDL or HSA(C34S)-ARLDDL purified by blue sepharose chromatography;
• Lane 4 contains HSA-ARLDDL or HSA(C34S)-ARLDDL purified by reverse phase HPLC column;
• Lane 5 contains commercial BSA
• Lane 6 contains HSA-ARLDDL or HSA(C34S)-ARLDDL purified by blue sepharose chromatography with 2Me;
• Lane 7 contains HSA-ARLDDL or HSA(C34S)-ARLDDL purified by reverse phase HPLC column with 2Me.
• HSA(C34S)-ARLDDL, HSA-ARLDDL and human serum albumin (HSA) were also analyzed by 2D SDS-PAGE. 2D SDS-PAGE of HSA(C34S)-ARLDDL, HSA-ARLDDL and HSA are shown in FIG. 1G.
• FIG. 1 H shows the NMR spectra of HSA(C34S)-ARLDDL and BSA. The analysis showed that the folding of HSA(C34S)-ARLDDL was similar to that of BSA. The arrow shows the Ha proton signals from the linker region (G 4 S) 5 .
• the cell adhesion inhibition assay was performed as described in the U.S. Patent Application Serial No. 12/004,045. Briefly, wells of 96-well lmmulon-2 microtiter plates (Costar, Corning, USA) were coated with 100 ⁇ l of phosphate- buffered saline (PBS: 10 mM phosphate buffer, 0.15M NaCI, pH 7.4) containing substrates at a concentration of 50-500 nM, and incubated overnight at 4 °C.
• PBS phosphate- buffered saline
• the substrates and their coating concentrations were fibrinogen (Fg) 200 ⁇ g/ml, vitronectin (Vn) 50 ⁇ g/ml, and fibronectin (Fn) 25 ⁇ g/ml.
• Fg fibrinogen
• Vn vitronectin
• Fn fibronectin
• Non-specific protein binding sites were blocked by incubating each well with 200 ⁇ l of heat-denatured 1% bovine serum albumin (BSA, Calbiochem) at room temperature (25° C) for 1.5 hr
• BSA bovine serum albumin
• CHO cells expressing ⁇ v ⁇ 3 (CHO- ⁇ v ⁇ 3) and ⁇ llb ⁇ 3 (CHO- ⁇ llb ⁇ 3) integrins were maintained in 100 ⁇ l of Dulbecco's Modified Eagle's Medium (DMEM) medium.
• DMEM Dulbecco's Modified Eagle's Medium
• Chinese hamster ovary (CHO) cells expressing integrins ⁇ v ⁇ 3 (CHO- ⁇ v ⁇ 3) and ⁇ llb ⁇ S (CHO- ⁇ llb ⁇ 3) were kindly provided by Dr. Y. Takada (Scripps Research Institute).
• Human erythroleukemia K562 cells were purchased from ATCC and cultured in the RPMI-1640 medium containing 5% fetal calf serum.
• CHO and K562 cells growing in log phase were detached by trypsinization and used in the assay at 3 x 105 and 2.5 x 10 5 cells/ml, respectively.
• ARLDDL, pegylated-ARLDDL, HSA-ARLDDL, and HSA(C34S)-ARLDDL were added to the cultured cells and incubated at 37° C, 5% CO2 for 15 minutes. Rho and its variants were used as inhibitors at the concentrations of 0.001-500 ⁇ M.
• the treated cells were then added into the coated plate and reacted at 37° C, 5% CO2 for 1 hour. The incubation solution was then discarded and non-adhered cells were removed by washing twice with 200 ⁇ l PBS.
• PRP platelet-rich plasma
• PRP platelet-poor plasma
• a solution of 190 ⁇ l of PRP and 10 ⁇ l of either Rho or PBS buffer were incubated for 5 min in a Hema Tracer 601 aggregometer at 37° C.
• Ten microliters of 200 ⁇ M adenosine diphosphate (ADP) were further added to monitor the response of platelet aggregation by light transmission. The lower the IC 5O , the greater the specificity or potency of the variant.
• Table 2 demonstrates the inhibitory effects of HSA(C34S)-ARLDDL and other tested proteins on integrins ⁇ v ⁇ 3, ⁇ 5 ⁇ 1 , and ⁇ llb ⁇ S and on platelet aggregation.
• Osteoclasts are specialized monocyte/macrophage family members that differentiate from bone marrow hematopoietic precursors. Cultures of osteoclast precursors in the presence of M-CSF (20 ng/ml) and sRANKL (50 ng/ml) for 8 days induced the formation of large mature osteoclasts with multi-nuclei, which were characterized by the acquisition of mature phenotypic markers, such as TRAP. The method of osteoclastogenesis from cultured hematopoietic cells of bone marrow and the effects of HSA(C34S)-ARLDDL and related proteins on osteoclastogenesis were investigated as follows.
• Bone marrow cells were prepared by removing femurs from 6 ⁇ 8-week- old SD rats and flushing the bone marrow cavity with a-MEM which was supplemented with 20 mM HEPES and 10% heat-inactivated FCS, 2 mM- glutamine, penicillin (100 U/ml) and streptomycin (100 ⁇ g/ml).
• the non-adherent cells were collected and used as osteoclast precursors after 24 hr.
• Cells were seeded at 1 x106 cells/well (0.5 ml) in 24-well plates in the presence of human recombinant soluble RANKL (50 ng/ml) and murine M-CSF (20 ng/ml). The culture medium was replaced every 3 days. Osteoclast formation was confirmed by an assay of tartrate-resistant acid phosphatase (TRAP) on day-8. In brief, adherent cells were fixed with 10% formaldehyde in phosphate-buffered saline for 3 min.
• TRIP tartrate-resistant acid phosphatase
• HSA(C34S)-ARLDDL and HSA-ARLDDL markedly inhibited the differentiation of osteoclasts.
• FIG. 9A is control. It demonstrates osteoclasts in cells which were not treated with any polypeptides.
• FIG. 9B shows cells treated with 10 nM of HSA(C34S)-ARLDDL.
• FIG. 9C shows cells treated with 30 nM of HSA(C34S)-ARLDDL.
• FIG. 10A is a graph demonstrating that as the concentration of alendronate increases, the number of osteoclasts decreases.
• the IC 50 for alendronate was measured to be 1.9 ⁇ M.
• FIG. 10B is a graph demonstrating that as the concentration of HSA- ARLDDL increases, the number of osteoclasts decreases. The IC 50 for HSA- ARLDDL was measured to be 11.7 nM.
• FIG. 1 OC is a graph demonstrating that as the concentration of HSA(C34S)-ARLDDL increases, the number of osteoclasts decreases. The IC 50 for HSA(C34S)-ARLDDL was measured to be 6.7 nM.
• mice An animal model for retinopathy of prematurity in mice was generated by using hypoxic-induced angiogenesis as described in Wilkinson-Berka et al. (Wilkinson-Berka, J. L., Alousis, N. S., Kelly DJ. , et al (2003) COX-2 inhibition and retinal angiogenesis in a mouse model of retinopathy of prematurity. Invest Ophthalmol Vis Sci 44: 974-979.28). Briefly, seven-day-old pups and their mother were housed in sealed chambers containing 75% O 2 and air.
• mice remained in the chamber for five days (hyperoxic period, P7 to P12) and were then housed in room air for a further seven days (hypoxic-induced angiogenic period, postnatal 12 days to postnatal 19 days, or P12 to P19).
• Either HSA-ARLDDL or HSA(C34S)-ARLDDL at various amounts was administered via an intravitreous route on day-12 and the mice were sacrificed on day-19.
• BVPs Blood vessel profiles
• HSA-ARLDDL inhibited angiogenesis in a mouse model of retinopathy of prematurity (ROP).
• HSA-ARLDDL at doses of 0.001 , 0.1 and 10 pg/eye reduced the vessel number per retinal section as compared to normal saline treated group. Data are presented as Mean ⁇ SE. With the exception of the group administered 0.001 pg/eye of HSA-ARLDDL, p was less than 0.001.
• Endothelial cells were counted in the anterior part of the ganglion cell layer and on inner limiting membrane of the retina by a person blinded to the same identity. The results are shown in FIG. 11 B.
• HSA(C34S)-ARLDDL also inhibited angiogenesis in a mouse model of retinopathy of prematurity (ROP).
• HSA(C34S)- ARLDDL at doses of 0.1 , 10 and 1000 pg/eye reduced the vessel number per retinal section as compared to normal saline treated group. Data are presented as Mean ⁇ SE. In all instances, p was less than 0.001.
• Endothelial cells were counted in the anterior part of the ganglion cell layer and on inner limiting membrane of the retina by a person blinded to the same identity. The results are shown in FIG. 1 1 D.
• FIGS. 11 E, 11 F and 1 1 G are photographs showing angiogenesis in a mouse model of oxygen-induced retinopathy.
• FIG. 1 1 E shows normoxia (control group)
• FIG. 11 F shows angiogenesis in an oxygen-induced retinopathy mouse
• FIG. 11 G shows reduction of angiogenesis in an oxygen-induced retinopathy mouse treated with 10 pg of HSA(C34S)-ARLDDL.
• the human PC-3 (prostate cancer) cells were implanted in Non-Obese Diabetic Severe Combined Immune Deficiency (NOD-SCID) mice as follows. Each mouse was injected subcutaneously in the right flank with 1 x 10 7 cells. Tumors were monitored every two days. On day 27 of the study, the animals were divided into two groups. One group was treated with saline and another group was treated with HSA(C34S)-ARLDDL (20 mg/kg, intravenously, twice per week).
• NOD-SCID Non-Obese Diabetic Severe Combined Immune Deficiency
• Tumor size in mm 3 was calculated as follows:
• Tumor Volume w 2 x 111, where w is width (mm) and / is length (mm) of the tumor.
• Tumor weight was estimated based on the assumption that 1 mg is equivalent to 1 mm 3 of tumor volume.
• FIG. 12A is a control which shows a photograph of two mice injected with the human PC-3 cells.
• FIG. 12B is a photograph of two mice injected with the human PC-3 cells and treated with HSA(C34S)-ARLDDI_ (20 mg/kg, intravenously, twice per week).
• FIG. 12C is a photograph of tumors excised from the control mice and FIG. 12D is a photograph of tumors excised from the mice treated with HSA(C34S)- ARLDDL.
• FIG. 13 is graph that shows that HSA(C34S)-ARLDDL significantly inhibited growth of tumors in the mice treated with this protein as measured by tumor size.
• the arrows on the graph indicate injections of HSA(C34S)-ARLDDL.
• Neovessels were quantified by measuring the hemoglobin of the plugs as an indication of blood vessel formation with the Drabkin method and Drabkin reagent kit 525 (Sigma) (FIG. 14B).
• FIGS. 14A and 14B show that HSA(C34S)-ARLDDL was effective in inhibiting angiogenesis using MATRIGELTM plug assays. * : P ⁇ 0.05 versus control
• SEQ ID NO: 1 is an amino acid sequence of ARLDDL variant of rhodostomin. It is set forth in FIG. 2.
• SEQ ID NO: 2 is one nucleotide sequence that encodes ARLDDL variant of rhodostomin. It is set forth in FIG. 3A.
• SEQ ID NO: 3 is another nucleotide sequence that encodes that encodes ARLDDL variant of rhodostomin. It is set forth in FIG. 3B.
• SEQ ID NOS: 4 and 5 are, respectively, amino acid and nucleotide sequences of HSA C34S mutant. They are set forth in FIGS. 4A and 4B.
• SEQ ID NOS: 6 and 7 are, respectively, amino acid and nucleotide sequences of HSA C34A mutant. They are set forth in FIGS. 5A and 5B.
• SEQ ID NO: 8 is an amino acid sequence of a linker amino acid. It is set forth in FIG. 6.
• SEQ ID NOS: 9 and 10 are, respectively, amino acid and nucleotide sequences of HSA(C34S)-ARLDDL mutant. They are set forth in FIGS. 7A and 7B.
• SEQ ID NOS: 11 and 12 are, respectively, amino acid and nucleotide sequences of HSA(C34A)-ARLDDL mutant. They are set forth in FIGS. 8A and 8B.

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