WO2011116245A2 - Methods of treating inflammation - Google Patents

Methods of treating inflammation Download PDF

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Publication number
WO2011116245A2
WO2011116245A2 PCT/US2011/028904 US2011028904W WO2011116245A2 WO 2011116245 A2 WO2011116245 A2 WO 2011116245A2 US 2011028904 W US2011028904 W US 2011028904W WO 2011116245 A2 WO2011116245 A2 WO 2011116245A2
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WIPO (PCT)
Prior art keywords
seq
agent
peptide
rantes
amino acid
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PCT/US2011/028904
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French (fr)
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WO2011116245A3 (en
Inventor
Joshua Robert Schultz
Court Turner
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Carolus Therapeutics, Inc.
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Publication of WO2011116245A2 publication Critical patent/WO2011116245A2/en
Publication of WO2011116245A3 publication Critical patent/WO2011116245A3/en

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    • 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
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/521—Chemokines
    • C07K14/523—Beta-chemokines, e.g. RANTES, I-309/TCA-3, MIP-1alpha, MIP-1beta/ACT-2/LD78/SCIF, MCP-1/MCAF, MCP-2, MCP-3, LDCF-1, LDCF-2
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • 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
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • 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
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/521—Chemokines
    • C07K14/522—Alpha-chemokines, e.g. NAP-2, ENA-78, GRO-alpha/MGSA/NAP-3, GRO-beta/MIP-2alpha, GRO-gamma/MIP-2beta, IP-10, GCP-2, MIG, PBSF, PF-4, KC
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/52—Assays involving cytokines
    • G01N2333/521—Chemokines
    • G01N2333/522—Alpha-chemokines, e.g. NAP-2, ENA-78, GRO-alpha/MGSA/NAP-3, GRO-beta/MIP-2alpha, GRO-gamma/MIP-2beta, IP-10, GCP-2, MIG, PBSF, PF-4 or KC
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/52—Assays involving cytokines
    • G01N2333/521—Chemokines
    • G01N2333/523—Beta-chemokines, e.g. RANTES, I-309/TCA-3, MIP-1alpha, MIP-1beta/ACT-2/LD78/SCIF, MCP-1/MCAF, MCP-2, MCP-3, LDCF-1or LDCF-2
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/715—Assays involving receptors, cell surface antigens or cell surface determinants for cytokines; for lymphokines; for interferons
    • G01N2333/7158—Assays involving receptors, cell surface antigens or cell surface determinants for cytokines; for lymphokines; for interferons for chemokines
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/32—Cardiovascular disorders
    • G01N2800/329—Diseases of the aorta or its branches, e.g. aneurysms, aortic dissection

Definitions

  • Inflammatory diseases, disorders, conditions and symptoms are characterized, in part, by the migration of lymphocytes and monocytes into the affected tissue.
  • the migration of lymphocytes and monocytes induces tissue damage and exacerbates inflammatory diseases, disorders, conditions and symptoms.
  • RANTES also known as CCL5
  • PF4 are pro-inflammatory chemokines. In certain instances, they are secreted by an activated platelet in response to an inflammation or tissue injury. In certain instances, RANTES and PF4 induce chemotaxis in nearby leukocytes (e.g. monocytes) along their gradients.
  • leukocytes e.g. monocytes
  • a peptide that (a) mimics the RANTES binding domain of PF4 and (b) inhibits the formation of a PF4 and RANTES heterodimer.
  • the peptide comprises (a) 15-25 amino acids and (b) an amino acid sequence that is 85% homologous to an amino acid sequence selected from:
  • PRHITSLEVIKAGPHC SSKCSNLAWFVTRR (SEQ ID NO. 74)
  • MSVAAVFRGLRPSPE SEQ ID NO. 54
  • S SKS SNLA WFVTRCCKEYFYT SEQ ID NO. 54
  • VIKAGRHCAVPQLIA (SEQ ID NO. 59) NLAVVFVTRCCKEYFYTSSKSS (SEQ ID NO. 59) NLAVVFVTRCCKEYFYTSSKSS (SEQ ID NO. 59) NLAVVFVTRCCKEYFYTSSKSS (SEQ ID NO. 59) NLAVVFVTRCCKEYFYTSSKSS (SEQ ID NO. 59) NLAVVFVTRCCKEYFYTSSKSS (SEQ ID NO. 59) NLAVVFVTRCCKEYFYTSSKSS (SEQ ID NO. 59) NLAVVFVTRCCKEYFYTSSKSS (SEQ ID NO. 59) NLAVVFVTRCCKEYFYTSSKSS (SEQ ID NO. 59) NLAVVFVTRCCKEYFYTSSKSS (SEQ ID NO. 59) NLAVVFVTRCCKEYFYTSSKSS (SEQ ID NO.
  • LIATLKNGRKICLDR SEQ ID NO. 60
  • SFKGTTVYALSNVRSYSFVKCC SEQ ID NO. 60
  • ATALCAPASASPYSS (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO
  • SDTTPCCFAYIARPL (SEQ ID NO. 65) YSFVKCCSFKGTTVYALSNVRS (SEQ ID NO. 65)
  • EYFYTSGKCSNPAW (SEQ ID NO. 67) FVKCCSFKGTTVYALSNVRSYS (SEQ ID NO. 67)
  • the peptide comprises an amino acid sequence is at least 90% homologous to an amino acid sequence selected from: MSSAAGFCASRP (SEQ ID NO. 46) EKKWVREYINSLEMS (SEQ ID NO. 69)
  • PRHITSLEVIKAGPHC SSKCSNLAWFVTRR (SEQ ID NO. 74)
  • MSVAAVFRGLRPSPE SEQ ID NO. 54
  • S SKS SNLA WFVTRCCKEYFYT SEQ ID NO. 54
  • VIKAGRHCAVPQLIA (SEQ ID NO. 59) NLAVVFVTRCCKEYFYTSSKSS (SEQ ID NO. 59) NLAVVFVTRCCKEYFYTSSKSS (SEQ ID NO. 59) NLAVVFVTRCCKEYFYTSSKSS (SEQ ID NO. 59) NLAVVFVTRCCKEYFYTSSKSS (SEQ ID NO. 59) NLAVVFVTRCCKEYFYTSSKSS (SEQ ID NO. 59) NLAVVFVTRCCKEYFYTSSKSS (SEQ ID NO. 59) NLAVVFVTRCCKEYFYTSSKSS (SEQ ID NO. 59) NLAVVFVTRCCKEYFYTSSKSS (SEQ ID NO. 59) NLAVVFVTRCCKEYFYTSSKSS (SEQ ID NO. 59) NLAVVFVTRCCKEYFYTSSKSS (SEQ ID NO.
  • LIATLKNGRKICLDR SEQ ID NO. 60
  • SFKGTTVYALSNVRSYSFVKCC SEQ ID NO. 60
  • ATALCAPASASPYSS (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO
  • SDTTPCCFAYIARPL (SEQ ID NO. 65) YSFVKCCSFKGTTVYALSNVRS (SEQ ID NO. 65)
  • EYFYTSGKCSNPAW SEQ ID NO. 67
  • FVKCCSFKGTTVYALSNVRSYS SEQ ID NO. 89
  • the peptide comprises an amino acid sequence is at least 95% homologous to an amino acid sequence selected from:
  • MSSAAGFCASRP (SEQ ID NO. 46)
  • EKKWVREYINSLEMS (SEQ ID NO. 69)
  • PRHITSLEVIKAGPHC SSKCSNLAWFVTRR (SEQ ID NO. 74)
  • MSVAAVFRGLRPSPE SEQ ID NO. 54
  • S SKS SNLA WFVTRCCKEYFYT SEQ ID NO. 54
  • VIKAGRHCAVPQLIA (SEQ ID NO. 59) NLAVVFVTRCCKEYFYTSSKSS (SEQ ID NO. 59) NLAVVFVTRCCKEYFYTSSKSS (SEQ ID NO. 59) NLAVVFVTRCCKEYFYTSSKSS (SEQ ID NO. 59) NLAVVFVTRCCKEYFYTSSKSS (SEQ ID NO. 59) NLAVVFVTRCCKEYFYTSSKSS (SEQ ID NO. 59) NLAVVFVTRCCKEYFYTSSKSS (SEQ ID NO. 59) NLAVVFVTRCCKEYFYTSSKSS (SEQ ID NO. 59) NLAVVFVTRCCKEYFYTSSKSS (SEQ ID NO. 59) NLAVVFVTRCCKEYFYTSSKSS (SEQ ID NO. 59) NLAVVFVTRCCKEYFYTSSKSS (SEQ ID NO.
  • LIATLKNGRKICLDR SEQ ID NO. 60
  • SFKGTTVYALSNVRSYSFVKCC SEQ ID NO. 60
  • ATALCAPASASPYSS (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 87);
  • SDTTPCCFAYIARPL (SEQ ID NO. 65) YSFVKCCSFKGTTVYALSNVRS (SEQ ID NO. 65)
  • EYFYTSGKCSNPAW (SEQ ID NO. 67) FVKCCSFKGTTVYALSNVRSYS (SEQ ID NO. 67)
  • the peptide comprises an amino acid sequence selected from:
  • MSSAAGFCASRP (SEQ ID NO. 46)
  • EKKWVREYINSLEMS (SEQ ID NO. 69)
  • PRHITSLEVIKAGPHC SSKCSNLAWFVTRR (SEQ ID NO. 74)
  • MSVAAVFRGLRPSPE SEQ ID NO. 54
  • S SKS SNLA WFVTRCCKEYFYT SEQ ID NO. 54
  • VIKAGRHCAVPQLIA (SEQ ID NO. 59) NLAVVFVTRCCKEYFYTSSKSS (SEQ ID NO. 59) NLAVVFVTRCCKEYFYTSSKSS (SEQ ID NO. 59) NLAVVFVTRCCKEYFYTSSKSS (SEQ ID NO. 59) NLAVVFVTRCCKEYFYTSSKSS (SEQ ID NO. 59) NLAVVFVTRCCKEYFYTSSKSS (SEQ ID NO. 59) NLAVVFVTRCCKEYFYTSSKSS (SEQ ID NO. 59) NLAVVFVTRCCKEYFYTSSKSS (SEQ ID NO. 59) NLAVVFVTRCCKEYFYTSSKSS (SEQ ID NO. 59) NLAVVFVTRCCKEYFYTSSKSS (SEQ ID NO. 59) NLAVVFVTRCCKEYFYTSSKSS (SEQ ID NO.
  • LIATLKNGRKICLDR SEQ ID NO. 60
  • SFKGTTVYALSNVRSYSFVKCC SEQ ID NO. 60
  • ATALCAPASASPYSS (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO
  • SDTTPCCFAYIARPL (SEQ ID NO. 65) YSFVKCCSFKGTTVYALSNVRS (SEQ ID NO. 65)
  • EYFYTSGKCSNPAW (SEQ ID NO. 67) FVKCCSFKGTTVYALSNVRSYS (SEQ ID NO. 67)
  • the peptide comprises (a) N- and/or C-terminal chemical modifications to improve ADME-PK or (b) non-natural amino acids.
  • the peptide is a cyclical variant.
  • the peptide is used in the treatment of a disease characterized by inflammation mediated by the binding of PF4 and RANTES.
  • the peptide is used in the treatment of a disease characterized by inflammation mediated by the binding of PF4 and RANTES, wherein the disease is atherosclerosis, cystic fibrosis, AAA, RSV infection, emphysema, moderate asthma, severe asthma, or any combination thereof.
  • the use further comprises the co-administration of an active agent selected from: niacin, a fibrate, a statin, an Apo- Al mimetic peptide, an ApoA-I transcriptional up-regulator, an ACAT inhibitor, a CETP modulator, Glycoprotein (GP) Ilb/IIIa receptor antagonists, P2Y12 receptor antagonists, Lp-PLA2-inhibitors, an anti-TNF agent, an IL-1 receptor antagonist, an IL-2 receptor antagonist, a cytotoxic agent, an immunomodulatory agent, an antibiotic, a T-cell co-stimulatory blocker, a disorder-modifying antirheumatic agent, a B cell depleting agent, an immunosuppressive agent, an anti-lymphocyte antibody, an alkylating agent, an anti-metabolite, a plant alkaloid, a terpenoid, a topoisomerase inhibitor, an antitumor antibiotic, a monoclonal antibody, a hormone, or combinations thereof.
  • a method of diagnosing an abdominal aortic aneurysm in an individual in need thereof comprising (a) administering to the individual an agent that (i) specifically binds to all or a portion of PF4, RANTES, or a combination thereof, and (ii) is labeled with an imaging agent; (b) exposing the individual to an imaging device for a period of time sufficient to generate an image of the agent; and (c) diagnosing the individual with an abdominal aortic aneurysm if the image of the agent shows a concentration of the agent in the abdominal aorta that exceeds the concentration in a control.
  • the agent localizes to the site of an abdominal aortic aneurysm.
  • the agent is an antibody, a peptibody, a small molecule, or a peptide.
  • the agent is a peptide that (a) mimics all or a portion of PF4, or (b) all or a portion of RANTES.
  • the agent is a peptide that (a) mimics all or a portion of the RANTES binding domain of PF4, or (b) all or a portion of PF4 binding domain of RANTES.
  • the agent is a peptide that comprises an amino acid sequence that is at least 85% homologous to the sequence: CKEYFYTSSKSSNLAWFVTRC (SEQ ID NO: 13). In some embodiments, the agent is a peptide that comprises an amino acid sequence that is at least 85% homologous to the sequence: CKEYFYTSGKSSNPGTVFITRC (SEQ ID NO: 16). In some embodiments, the peptide is: CKEYFYTSSKSSNLAWFVTRC (SEQ ID NO: 13) or CKEYFYTSGKSSNPGTVFITRC (SEQ ID NO: 16). In some embodiments, the agent is an anti-PF4 antibody, an anti-RANTES antibody, or a combination thereof.
  • the agent is labeled for radio-imaging, PET imaging, MRI imaging, or fluorescent imaging.
  • the agent is imaged by use of a gamma camera, MRI, PET scanner, x-ray computed tomography (CT), functional magnetic resonance imaging (fMRI), or single photon emission computed tomography (SPECT).
  • CT computed tomography
  • fMRI functional magnetic resonance imaging
  • SPECT single photon emission computed tomography
  • the imaging agent is a radiolabel.
  • the imaging agent is a fluorescent label.
  • the imaging agent is magnetic, paramagnetic or superparamagnetic.
  • the imaging agent is 17 F, 18 F, 117 I, U8 I, U9 I, 120 I, 121 I, 122 I, 124 I, 126 I, 128 1 131 I, 10 C, U C, 13 0, 14 0, 15 0, 74 Br, 75 Br, 76 Br, 77 Br, 78 Br, 82 Br, "'In, 225 Ac, 13 N, 82 Rb, 32 C1, 33 C1, 34 C1, 64 Cu, monobromobimane (mBBr), dibromobimane, monobromotrimethylammoniobimane, indocyanine green acylthiazolidinethione (ICG-ATT), 7- amino -4 -methyl coumarin-3-acetic acid (AMCA), guanidinoethanethiol (GET), ferucarbotran, U C- dimethylamine, perfluoro-15-crown-5-ether (PFCE), perfluorooctylbromide (PFOB), Rhodamine 6G, Rh
  • a method of monitoring an abdominal aortic aneurysm in an individual in need thereof comprising (a) administering to the individual an agent that (i) specifically binds to all or a portion of PF4, RANTES, or a combination thereof, and (ii) is labeled with an imaging agent; (b) exposing the individual to an imaging device for a period of time sufficient to generate an image of the agent; wherein (i) a decrease in the concentration of the agent in the abdominal aorta as compared to an earlier image of the agent indicates that the Abdominal Aortic Aneurysm (AAA) is regressing, (ii) an increase in the concentration of the agent in the abdominal aorta as compared to an earlier image of the agent indicates that the Abdominal Aortic Aneurysm (AAA) is progressing, or (iii) no change in the concentration of the agent in the abdominal aorta as compared to an earlier
  • the agent localizes to the site of an abdominal aortic aneurysm.
  • the agent is an antibody, a peptibody, a small molecule, or a peptide.
  • the agent is a peptide that (a) mimics all or a portion of PF4, or (b) all or a portion of PvANTES.
  • the agent is a peptide that (a) mimics all or a portion of the PvANTES binding domain of PF4, or (b) all or a portion of PF4 binding domain of PvANTES.
  • the agent is a peptide that comprises an amino acid sequence that is at least 85% homologous to the sequence:
  • the agent is a peptide that comprises an amino acid sequence that is at least 85% homologous to the sequence:
  • the peptide is:
  • the agent is an anti-PF4 antibody, an anti-RANTES antibody, or a combination thereof.
  • the agent is labeled for radio-imaging, PET imaging, MRI imaging, or fluorescent imaging.
  • the agent is imaged by use of a gamma camera, MRI, PET scanner, x-ray computed tomography (CT), functional magnetic resonance imaging (fMRI), or single photon emission computed tomography (SPECT).
  • CT x-ray computed tomography
  • fMRI functional magnetic resonance imaging
  • SPECT single photon emission computed tomography
  • the imaging agent is a radiolabel.
  • the imaging agent is a fluorescent label.
  • the imaging agent is magnetic, paramagnetic or superparamagnetic. In some embodiments, the imaging agent is 17 F, 18 F, U7 I, U8 I, U9 I, 120 I, 121 I, 122 I, 124 I, 126 L 128 1 131 I, 10 C, U C, 13 0, 14 0, 15 0, 74 Br, 75 Br, 76 Br, 77 Br, 78 Br, 82 Br, m In, 225 Ac, 13 N, 82 Rb, 32 C1, 33 C1, 34 C1, 64 Cu, monobromobimane (mBBr), dibromobimane, monobromotrimethylammoniobimane, indocyanine green acylthiazolidinethione (ICG- ATT), 7-amino-4-methyl coumarin-3 -acetic acid (AMCA), guanidinoethanethiol (GET), ferucarbotran, u C-dimethylamine, perfluoro-15-crown-5-ether (PFCE)
  • a stent comprising a peptide comprising an amino acid sequence that is at least 85%> homologous to the amino acid sequence
  • the peptide is CKEYFYTSSKSSNLAVVFVTRC (SEQ ID NO: 13), or CKEYFYTSGKSSNPGIVFITRC (SEQ ID NO: 16).
  • a method of treating RSV infection comprising administering an agent that specifically binds to all or a portion of PF4, RANTES, or a combination thereof.
  • the agent is an antibody, a peptibody, a small molecule, or a peptide.
  • the agent (a) mimics all or a portion of PF4, or (b) all or a portion of RANTES.
  • the agent (a) mimics all or a portion of the RANTES binding domain of PF4, or (b) all or a portion of PF4 binding domain of RANTES.
  • the agent is a peptide comprising an amino acid sequence that is at least 85% homologous to the amino acid sequence: CKEYFYTSSKSSNLAWFVTRC (SEQ ID NO: 13). In some embodiments, the agent is a peptide comprising an amino acid sequence that is at least 85% homologous to the amino acid sequence: CKEYFYT SGKS SNPGIVFITRC (SEQ ID NO: 16). In some embodiments, the agent is CKEYFYTSSKSSNLAWFVTRC (SEQ ID NO: 13), or
  • the agent is an antibody, a peptibody, a small molecule, or a peptide.
  • the agent (a) mimics all or a portion of PF4, or (b) all or a portion of RANTES.
  • the agent (a) mimics all or a portion of the RANTES binding domain of PF4, or (b) all or a portion of PF4 binding domain of RANTES.
  • the agent is a peptide comprising an amino acid sequence that is at least 85% homologous to the amino acid sequence: CKEYFYTSSKSSNLAWFVTRC (SEQ ID NO: 13).
  • the agent is a peptide comprising an amino acid sequence that is at least 85% homologous to the amino acid sequence: CKEYFYT SGKS SNPGIVFITRC (SEQ ID NO: 16). In some embodiments, the agent is CKEYFYTSSKSSNLAWFVTRC (SEQ ID NO: 13), or
  • Figure 1 The peptide of SEQ ID NO: 13 showed a dose-dependent inhibition of the increase in aorta diameter caused by PPE infusion in a porcine pancreatic elastase (PPE) infusion mouse model of human AAA.
  • An aneurysm is defined as a 50% increase in aortic diameter or the presence of aortic dissection.
  • FIG. 4 The elastic Masson staining and the anti-smooth muscle cell alpha actin antibody immunostaining revealed that medial elastin fibers and smooth muscle cell layers were well preserved in porcine pancreatic elastase (PPE) infusion mice treated with 10 mg /kg/day of peptide of SEQ ID NO: 13 or 20 mg /kg/day of peptide of SEQ ID NO: 13, as compared to the vehicle- treated porcine pancreatic elastase (PPE) infusion mice.
  • PPE porcine pancreatic elastase
  • FIG. 6 The elastic Masson staining and the anti-smooth muscle cell alpha actin antibody immunostaining revealed that medial elastin fibers and smooth muscle cell layers were well preserved in the PPE infusion mice treated with 10 mg /kg/day of peptide of SEQ ID NO: 13 or 20 mg /kg/day of peptide of SEQ ID NO: 13, as compared to the vehicle-treated mice.
  • FIG. 7 Treatment of mice infected with RSV. Peptide of SEQ ID NO: 13 given by IV injection on Day 0-2 at 1 or 10 mg/kg. Given in a delayed administration at 10 mg/kg by IV injection on Day 5-7. All animals were harvested on day 8.
  • Figure 8 Treatment of mice to determine airway hyperreactivity after a methacholine challenge (0.6 mg/ml). Drug given by IV injection on Day 0-2 at 1 or 10 mg/kg. Given in a delayed administration at 10 mg/kg by IV injection on Day 5-7. All animals were harvested on day 8.
  • FIG. 9 Histology results of mice infected with RSV. Control mice received vehicle. Experimental mice received the peptide of SEQ ID NO: 13 at the indicated dosage.
  • Figure 10 Treatment of mice infected with RSV. Animals were given peptide of SEQ ID NO: 13 by IP injection on day 0-7 at lOmg/kg dose and harvested on day 8 post-infection, (no change in AHR was observed).
  • the terms "individual,” “patient,” or “subject” are used interchangeably. As used herein, they mean any mammal (i.e. species of any orders, families, and genus within the taxonomic classification animalia: chordata: vertebrata: mammalia). In some embodiments, the mammal is a human. In some embodiments, the mammal is a non-human. In some embodiments, the mammal is a member of the taxonomic orders: primates (e.g. lemurs, lorids, galagos, tarsiers, monkeys, apes, and humans); rodentia (e.g.
  • mice, rats, squirrels, chipmunks, and gophers mice, rats, squirrels, chipmunks, and gophers); lagomorpha (e.g. hares, rabbits, and pika); erinaceomorpha (e.g. hedgehogs and gymnures); soricomorpha (e.g. shrews, moles, and solenodons); chiroptera (e.g., bats); cetacea (e.g. whales, dolphins, and porpoises); carnivora (e.g. cats, lions, and other feliformia; dogs, bears, weasels, and seals); perissodactyla (e.g.
  • artiodactyla e.g. pigs, camels, cattle, and deer
  • proboscidea e.g. elephants
  • sirenia e.g. manatees, dugong, and sea cows
  • cingulata e.g. armadillos
  • pilosa e.g. anteaters and sloths
  • didelphimorphia e.g. american opossums
  • paucituberculata e.g. shrew opossums
  • microbiotheria e.g. Monito del Monte
  • notoryctemorphia e.g.
  • the animal is a reptile (i.e. species of any orders, families, and genus within the taxonomic classification animalia: chordata: vertebrata: reptilia). In some embodiments, the animal is a bird (i.e. animalia: chordata: vertebrata: aves).
  • a health care worker e.g. a doctor, a registered nurse, a nurse practitioner, a physician's assistant, an orderly, or a hospice worker.
  • the terms "treat,” “treating” or “treatment,” and other grammatical equivalents as used herein, include alleviating, inhibiting or reducing symptoms, reducing or inhibiting severity of, reducing incidence of, prophylactic treatment of, reducing or inhibiting recurrence of, preventing, delaying onset of, delaying recurrence of, abating or ameliorating a disease or condition symptoms, ameliorating the underlying metabolic causes of symptoms, inhibiting the disease or condition, e.g., arresting the development of the disease or condition, relieving the disease or condition, causing regression of the disease or condition, relieving a condition caused by the disease or condition, or stopping the symptoms of the disease or condition.
  • the terms further include achieving a therapeutic benefit.
  • therapeutic benefit is meant eradication or amelioration of the underlying disorder being treated, and/or the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the individual.
  • prevent include preventing additional symptoms, preventing the underlying metabolic causes of symptoms, inhibiting the disease or condition, e.g., arresting the development of the disease or condition and are intended to include prophylaxis.
  • the terms further include achieving a prophylactic benefit.
  • the compositions are optionally administered to an individual at risk of developing a particular disease, to an individual reporting one or more of the physiological symptoms of a disease, or to an individual at risk of reoccurrence of the disease.
  • combination treatments or prevention methods are contemplated, it is not intended that the agents described herein be limited by the particular nature of the combination.
  • the agents described herein are optionally administered in combination as simple mixtures as well as chemical hybrids.
  • An example of the latter is where the agent is covalently linked to a targeting carrier or to an active pharmaceutical.
  • Covalent binding can be accomplished in many ways, such as, though not limited to, the use of a commercially available cross-linking agent.
  • combination treatments are optionally administered separately or concomitantly.
  • the terms “pharmaceutical combination”, “administering an additional therapy”, “administering an additional therapeutic agent” and the like refer to a pharmaceutical therapy resulting from the mixing or combining of more than one active ingredient and includes both fixed and non-fixed combinations of the active ingredients.
  • the term “fixed combination” means that at least one of the agents described herein, and at least one co-agent, are both
  • non- fixed combination means that at least one of the agents described herein, and at least one co- agent, are administered to an individual as separate entities either simultaneously, concurrently or sequentially with variable intervening time limits, wherein such administration provides effective levels of the two or more agents in the body of the individual.
  • the co-agent is administered once or for a period of time, after which the agent is administered once or over a period of time.
  • the co-agent is administered for a period of time, after which, a therapy involving the administration of both the co-agent and the agent are administered.
  • the agent is administered once or over a period of time, after which, the co- agent is administered once or over a period of time.
  • the terms “co-administration”, “administered in combination with” and their grammatical equivalents are meant to encompass administration of the selected therapeutic agents to a single individual, and are intended to include treatment regimens in which the agents are administered by the same or different route of administration or at the same or different times.
  • the agents described herein will be co-administered with other agents. These terms encompass administration of two or more agents to an animal so that both agents and/or their metabolites are present in the animal at the same time. They include simultaneous administration in separate compositions, administration at different times in separate compositions, and/or administration in a composition in which both agents are present.
  • the agents described herein and the other agent(s) are administered in a single composition.
  • the agents described herein and the other agent(s) are admixed in the composition.
  • the terms "effective amount” or “therapeutically effective amount” as used herein, refer to a sufficient amount of at least one agent being administered which achieve a desired result, e.g., to relieve to some extent one or more symptoms of a disease or condition being treated.
  • the result is a reduction and/or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system.
  • the result is a decrease in the growth of, the killing of, or the inducing of apoptosis in at least one abnormally proliferating cell, e.g., a cancer stem cell.
  • an "effective amount” for therapeutic uses is the amount of the composition comprising an agent as set forth herein required to provide a clinically significant decrease in a disease.
  • An appropriate "effective" amount in any individual case is determined using any suitable technique, such as a dose escalation study.
  • administer refers to the methods that may be used to enable delivery of agents or compositions to the desired site of biological action. These methods include, but are not limited to oral routes, intraduodenal routes, parenteral injection (including intravenous, subcutaneous, intraperitoneal, intramuscular, intravascular or infusion), topical and rectal administration. Administration techniques that are optionally employed with the agents and methods described herein, include e.g., as discussed in Goodman and Gilman, The Pharmacological Basis of Therapeutics, current ed.; Pergamon; and Remington's, Pharmaceutical Sciences (current edition), Mack Publishing Co., Easton, Pa. In certain embodiments, the agents and compositions described herein are administered orally.
  • pharmaceutically acceptable refers to a material that does not abrogate the biological activity or properties of the agents described herein, and is relatively nontoxic (i.e., the toxicity of the material significantly outweighs the benefit of the material).
  • a pharmaceutically acceptable material may be administered to an individual without causing significant undesirable biological effects or significantly interacting in a deleterious manner with any of the components of the composition in which it is contained.
  • the term "recruiting of monocytes” as described herein includes the migration of monocytes into or out of the endothelium, their attachment and propagation, for example, into endothelial fissures.
  • the attachment of monocytes is also known as monocyte adhesion, or as monocyte arrest when the attachment occurs in shear flow as under physiological conditions, for example, in blood capillaries, microvascular or arterial streamlines.
  • a binding molecule i.e., the agent; e.g., a peptide or peptide mimetic
  • the specified antibodies or binding molecules bind to a particular polypeptide, protein or epitope yet does not bind in a significant or undesirable amount to other molecules present in a sample.
  • the specified antibody or binding molecule does not undesirably cross-react with non-target antigens and/or epitopes.
  • a variety of immunoassay formats are used to select antibodies or other binding molecule that are immunoreactive with a particular polypeptide and have a desired specificity.
  • solid-phase ELISA immunoassays, BIAcore, flow cytometry and radioimmunoassays are used to select monoclonal antibodies having a desired immunoreactivity and specificity. See, Harlow, 1988, ANTIBODIES, A LABORATORY MANUAL, Cold Spring Harbor Publications, New York (hereinafter, "Harlow”), for a description of immunoassay formats and conditions that are used to determine or assess immunoreactivity and specificity.
  • “Selective binding,” “selectivity,” and the like refer the preference of agent to interact with one molecule as compared to another. Preferably, interactions between an agent disclosed herein and proteins are both specific and selective. Note that in some embodiments, an agent is designed to "specifically bind” and “selectively bind” two distinct, yet similar targets without binding to other undesirable targets.
  • polypeptide polypeptide
  • peptide protein
  • protein protein
  • the terms apply to naturally occurring amino acid polymers as well as amino acid polymers in which one or more amino acid residues is a non-naturally occurring amino acid (e.g., an amino acid analog).
  • the terms encompass amino acid chains of any length, including full length proteins (i.e., antigens), wherein the amino acid residues are linked by covalent peptide bonds.
  • motif and domain are used interchangeably. As used herein, they mean a discrete, contiguous or non-contiguous portion of a polypeptide that folds independently of the rest of the polypeptide and possesses its own function.
  • disruption means to interfere with the function of.
  • to disrupt a motif/domain means to interfere with the function of the motif/domain.
  • an antigen refers to a substance that is capable of inducing the production of an antibody.
  • an antigen is a substance that specifically binds to an antibody variable region.
  • antibody refers to monoclonal antibodies, polyclonal antibodies, bi-specific antibodies, multispecific antibodies, grafted antibodies, human antibodies, humanized antibodies, synthetic antibodies, chimeric antibodies, camelized antibodies, single-chain Fvs (scFv), single chain antibodies, Fab fragments, F(ab') fragments, disulfide-linked Fvs (sdFv), intrabodies, and anti-idiotypic (anti-Id) antibodies and antigen-binding fragments of any of the above.
  • antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules that contain an antigen binding site.
  • immunoglobulins can be assigned to different classes.
  • the heavy-chain constant motif/domains (Fc) that correspond to the different classes of immunoglobulins are called ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ , respectively.
  • the subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.
  • Immunoglobulin molecules are of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG i; IgG 2 , IgG 3; IgG 4; IgAi and IgA 2 ) or subclass.
  • an antibody is part of a larger molecule, formed by covalent or non-covalent association of the antibody with one or more other proteins or peptides.
  • peptide mimetics are modeled after a specific peptide and they display an altered peptide backbone, altered amino acids and/or an altered primary amino acid sequence when compared to the peptide that it was designed to mimic.
  • peptide refers to a molecule comprising peptide(s) fused either directly or indirectly to an antibody or one or more antibody motif/domains (e.g., an Fc motif/domain of an antibody), where the peptide moiety specifically binds to a desired target.
  • the peptide(s) may be fused to either an Fc region or inserted into an Fc- Loop, a modified Fc molecule.
  • eptibody does not include Fc-fusion proteins (e.g., full length proteins fused to an Fc
  • isolated and purified refer to a material that is substantially or essentially removed from or concentrated in its natural environment.
  • an isolated nucleic acid is one that is separated from at least some of the nucleic acids that normally flank it or other nucleic acids or components (proteins, lipids, etc.) in a sample.
  • a polypeptide is purified if it is substantially removed from or concentrated in its natural environment. Methods for purification and isolation of nucleic acids and proteins are documented methodologies.
  • Embodiments of "substantially” include at least 20%, at least 40%, at least 50%, at least 75%, at least 85%, at least 90%, at least 95%, or at least 99%.
  • monoclonal antibody refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that are present in minor amounts.
  • monoclonal antibodies are made, for example, by the hybridoma method first described by Kohler and Milstein (1975) Nature 256:495. or are made by recombinant methods, e.g., as described in U.S. Pat. No. 4,816,567.
  • monoclonal antibodies are isolated from phage antibody libraries using the techniques described in Clackson et al, Nature 352:624-628 (1991), as well as in Marks et al, J. Mol. Biol. 222:581 -597 (1991).
  • the PF4/RANTES targeting agents, methods and compositions disclosed herein inhibit (partially or fully) the activity of RANTES.
  • RANTES also known as CCL5
  • CCL5 is a pro-inflammatory chemokine
  • human RANTES is encoded by the following nucleic acid sequence:
  • TGT GCC AAC CCA GAG AAG AAA TGG GTT CGG GAG TAC
  • mouse RANTES is encoded by the following nucleic acid sequence:
  • human RANTES is encoded by the following peptide sequence:
  • mouse RANTES is encoded by the following peptide sequence:
  • rat RANTES is encoded by the following peptide sequence:
  • pig RANTES is encoded by the following peptide sequence:
  • dog RANTES is encoded by the following peptide sequence:
  • RANTES is secreted by an activated platelet in response to an inflammation or tissue injury.
  • RANTES is a ligand for a CCR5 receptor found on the plasma membrane of a target leukocyte (e.g. monocyte).
  • RANTES induces chemotaxis in nearby leukocytes (e.g. monocytes) along a RANTES gradient.
  • RANTES induces the chemotaxis of a leukocyte to the site of an inflammation or tissue injury.
  • the chemotaxis of monocytes along a RANTES gradient results in monocyte arrest (i.e., the deposition of monocytes on epithelium) at the site of injury or inflammation.
  • the PF4/RANTES targeting agents, methods and compositions disclosed herein inhibit (partially or fully) the activity of Platelet Factor 4 (PF4).
  • PF4 Platelet Factor 4
  • PF4 (also known as CXCL4) is a chemokine.
  • human PF4 is encoded by the following nucleic acid sequence:
  • mouse PF4 is encoded by the following nucleic acid sequence:
  • AAG CAC ATC ACC AGC CTG GAG GTG ATC AAG GCA GGA CGC CAC
  • human PF4 is encoded by the following peptide sequence:
  • mouse PF4 is encoded by the following peptide sequence: MSVAAVFRGLRPSPELLLLGLLFLPAVVAVTSAGPEESDGDLSCVCVKTI
  • PF4 is secreted by the alpha granules of an activated platelet during platelet aggregation in response to tissue injury and/or inflammation.
  • PF4 is a ligand for a CXC3 receptor (i.e., CXC3RB). In certain instances, it induces directed chemotaxis in nearby leukocytes (e.g. monocytes). In certain instances, PF4 induces the chemotaxis of a leukocyte to the site of an inflammation or tissue injury.
  • RANTES and PF4 form a heteromultimer (e.g., a heterodimer).
  • a RANTES and PF4 heteromultimer e.g., a heterodimer amplifies the effects of RANTES-induced monocyte arrest.
  • inflammation results from (either partially or fully) an infection.
  • inflammation results from (either partially or fully) damage to a tissue (e.g., by a burn, by frostbite, by exposure to a cytotoxic agent, or by trauma).
  • inflammation results from (either partially or fully) an autoimmune disorder. In certain instances, inflammation results from (either partially or fully) the presence of a foreign body (e.g., a splinter). In certain instances, inflammation results from exposure to a toxin and/or chemical irritant.
  • a method of treating an inflammatory disorder comprising administering to an individual in need thereof (a) a peptide that mimics the RANTES binding domain of PF4, and inhibits the formation of a PF4 and RANTES heterodimer, or (b) a peptide that comprises an amino acid sequence that is at least 85% homologous (i.e., is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homologous, including any amount of homology between 85% and 100%) to an amino acid sequence selected from:
  • PRHITSLEVIKAGPHC SSKCSNLAWFVTRR (SEQ ID NO. 74)
  • MSVAAVFRGLRPSPE SEQ ID NO. 54
  • S SKS SNLA WFVTRCCKEYFYT SEQ ID NO. 54
  • VIKAGRHCAVPQLIA (SEQ ID NO. 59) NLA WFVTRCCKEYFYT S SKS S (SEQ ID NO. 59) NLA WFVTRCCKEYFYT S SKS S (SEQ ID NO. 59) NLA WFVTRCCKEYFYT S SKS S (SEQ ID NO. 59) NLA WFVTRCCKEYFYT S SKS S (SEQ ID NO. 59) NLA WFVTRCCKEYFYT S SKS S (SEQ ID NO. 59) NLA WFVTRCCKEYFYT S SKS S (SEQ ID NO. 59) NLA WFVTRCCKEYFYT S SKS S (SEQ ID NO. 59) NLA WFVTRCCKEYFYT S SKS S (SEQ ID NO. 59) NLA WFVTRCCKEYFYT S SKS S (SEQ ID NO. 59) NLA WFVTRCCKEYFY
  • LIATLKNGRKICLDR SEQ ID NO. 60
  • SFKGTTVYALSNVRSYSFVKCC SEQ ID NO. 60
  • ATALCAPASASPYSS (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO
  • SDTTPCCFAYIARPL (SEQ ID NO. 65) YSFVKCCSFKGTTVYALSNVRS (SEQ ID NO.
  • EYFYTSGKCSNPAW (SEQ ID NO. 67) FVKCCSFKGTTVYALSNVRSYS (SEQ ID NO. 67)
  • a peptide that mimics the RANTES binding domain of PF4, and inhibits the formation of a PF4 and RANTES heterodimer or (b) a peptide that comprises an amino acid that is 85% homologous (i.e., is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homologous, including any amount of homology between 85% and 100%) to an amino acid sequence selected from:
  • EYFYTSGKCSNPAW (SEQ ID NO. 67) FVKCCSFKGTTVYALSNVRSYS (SEQ ID NO. 67)
  • acute inflammation refers to inflammation characterized in that it develops over the course of a few minutes to a few hours, and ceases once the stimulus has been removed (e.g., an infectious agent has been killed by an immune response or administration of a therapeutic agent, a foreign body has been removed by an immune response or extraction, or damaged tissue has healed).
  • the short duration of acute inflammation results from the short half- lives of most inflammatory mediators.
  • acute inflammation begins with the activation of leukocytes (e.g., monocytes, macrophages, neutrophils, basophils, eosinophils, lymphocytes, dendritic cells, endothelial cells and mastocytes).
  • leukocytes e.g., monocytes, macrophages, neutrophils, basophils, eosinophils, lymphocytes, dendritic cells, endothelial cells and mastocytes.
  • the leukocytes release inflammatory mediators (e.g., histamines, proteoglycans, serine proteases, eicosanoids, and cytokines).
  • inflammatory mediators result in (either partially or fully) the symptoms associated with inflammation. For example, in certain instances an inflammatory mediator dilates post capillary venules, and increases blood vessel permeability.
  • the increased blood flow that follows vasodilation results in (either partially or fully) rubor and calor.
  • increased permeability of the blood vessels results in an exudation of plasma into the tissue leading to edema. In certain instances, the latter allows leukocytes to migrate along a chemotactic gradient to the site of the inflammatory stimulant.
  • structural changes to blood vessels e.g., capillaries and venules
  • the structural changes are induced (either partially or fully) by monocytes and/or macrophages.
  • the structural changes include, but are not limited to, remodeling of vessels, and angiogenesis.
  • angiogenesis contributes to the maintenance of chronic inflammation by allowing for increased transport of leukocytes.
  • histamines and bradykinin irritate nerve endings leading to itching and/or pain.
  • chronic inflammation results from the presence of a persistent stimulant (e.g., persistent acute inflammation, bacterial infection (e.g., by Mycobacterium tuberculosis), prolonged exposure to chemical agents (e.g., silica, or tobacco smoke) and autoimmune reactions (e.g., rheumatoid arthritis)).
  • a persistent stimulant e.g., persistent acute inflammation, bacterial infection (e.g., by Mycobacterium tuberculosis), prolonged exposure to chemical agents (e.g., silica, or tobacco smoke) and autoimmune reactions (e.g., rheumatoid arthritis)
  • the persistent stimulant results in continuous inflammation (e.g., due to the continuous recruitment of monocytes, and the proliferation of macrophages).
  • the continuous inflammation further damages tissues which results in the additional recruitment of mononuclear cells thus maintaining and exacerbating the inflammation.
  • physiological responses to inflammation further include angiogenesis and fibrosis.
  • Inflammatory disorders include, but are not limited to, Acute disseminated encephalomyelitis; Addison's disease; Ankylosing spondylitis; Antiphospholipid antibody syndrome; Autoimmune hemolytic anemia; Autoimmune hepatitis; Autoimmune inner ear disease; Bullous pemphigoid; Chagas disease; Chronic obstructive pulmonary disease; Coeliac disease; Dermatomyositis;
  • Diabetes mellitus type 1 Diabetes mellitus type 2; Endometriosis; Goodpasture's syndrome; Graves' disease; Guillain-Barre syndrome; Hashimoto's disease; Idiopathic thrombocytopenic purpura; Interstitial cystitis; Systemic lupus erythematosus (SLE); Metabolic syndrome, Multiple sclerosis; Myasthenia gravis; Myocarditis, Narcolepsy; Obesity; Pemphigus Vulgaris; Pernicious anaemia; Polymyositis; Primary biliary cirrhosis; Rheumatoid arthritis; Schizophrenia; Scleroderma; Sjogren's syndrome; Vasculitis; Vitiligo; Wegener's granulomatosis; Allergic rhinitis; Prostate cancer; Non- small cell lung carcinoma; Ovarian cancer; Breast cancer; Melanoma; Gastric cancer; Colorectal cancer; Brain cancer; Metastatic bone
  • cystic fibrosis comprising administering to an individual in need thereof (a) a peptide that mimics the RANTES binding domain of PF4, and inhibits the formation of a PF4 and RANTES heterodimer, or (b) a peptide that comprises an amino acid that is 85% homologous (i.e., is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homologous, including any amount of homology between 85% and 100%) to an amino acid sequence selected from: SAEAEEDGDLQC (SEQ ID NO. 49) SDTTPCCFAYLSLAL (SEQ ID NO. 72)
  • PRHITSLEVIKAGPHC SSKCSNLAWFVTRR (SEQ ID NO. 74)
  • MSVAAVFRGLRPSPE SEQ ID NO. 54
  • S SKS SNLA WFVTRCCKEYFYT SEQ ID NO. 54
  • VIKAGRHCAVPQLIA (SEQ ID NO. 59) NLA WFVTRCCKEYFYT S SKS S (SEQ ID NO. 59) NLA WFVTRCCKEYFYT S SKS S (SEQ ID NO. 59) NLA WFVTRCCKEYFYT S SKS S (SEQ ID NO. 59) NLA WFVTRCCKEYFYT S SKS S (SEQ ID NO. 59) NLA WFVTRCCKEYFYT S SKS S (SEQ ID NO. 59) NLA WFVTRCCKEYFYT S SKS S (SEQ ID NO. 59) NLA WFVTRCCKEYFYT S SKS S (SEQ ID NO. 59) NLA WFVTRCCKEYFYT S SKS S (SEQ ID NO. 59) NLA WFVTRCCKEYFYT S SKS S (SEQ ID NO. 59) NLA WFVTRCCKEYFY
  • LIATLKNGRKICLDR SEQ ID NO. 60
  • SFKGTTVYALSNVRSYSFVKCC SEQ ID NO. 60
  • ATALCAPASASPYSS (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO
  • SDTTPCCFAYIARPL (SEQ ID NO. 65) YSFVKCCSFKGTTVYALSNVRS (SEQ ID NO.
  • EYFYTSGKCSNPAW (SEQ ID NO. 67) FVKCCSFKGTTVYALSNVRSYS (SEQ ID NO. 67)
  • a peptide that mimics the RANTES binding domain of PF4, and inhibits the formation of a PF4 and RANTES heterodimer or (b) a peptide that comprises an amino acid that is 85% homologous (i.e., is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homologous, including any amount of homology between 85% and 100%) to an amino acid sequence selected from:
  • EYFYTSGKCSNPAW (SEQ ID NO. 67) FVKCCSFKGTTVYALSNVPvSYS (SEQ ID NO. 67)
  • a method of treating atherosclerosis comprising administering to an individual in need thereof (a) a peptide that mimics the RANTES binding domain of PF4, and inhibits the formation of a PF4 and RANTES heterodimer, or (b) a peptide that comprises an amino acid that is 85% homologous (i.e., is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homologous, including any amount of homology between 85% and 100%) to an amino acid sequence selected from:
  • ATALCAPASASPYSS (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO
  • SDTTPCCFAYIARPL (SEQ ID NO. 65) YSFVKCCSFKGTTVYALSNVRS (SEQ ID NO.
  • EYFYTSGKCSNPAW (SEQ ID NO. 67) FVKCCSFKGTTVYALSNVRSYS (SEQ ID NO. 67)
  • a peptide that mimics the RANTES binding domain of PF4, and inhibits the formation of a PF4 and RANTES heterodimer or (b) a peptide that comprises an amino acid that is 85% homologous (i.e., is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homologous, including any amount of homology between 85% and 100%) to an amino acid sequence selected from:
  • MSSAAGFCASRP (SEQ ID NO. 46)
  • EKKWVREYINSLEMS (SEQ ID NO. 69)
  • PRHITSLEVIKAGPHC SSKCSNLAWFVTRR (SEQ ID NO. 74)
  • MSVAAVFRGLRPSPE SEQ ID NO. 54
  • S SKS SNLA WFVTRCCKEYFYT SEQ ID NO. 54
  • VIKAGRHCAVPQLIA (SEQ ID NO. 59) NLAVVFVTRCCKEYF YT S SKS S (SEQ ID NO. 59) NLAVVFVTRCCKEYF YT S SKS S (SEQ ID NO. 59) NLAVVFVTRCCKEYF YT S SKS S (SEQ ID NO. 59) NLAVVFVTRCCKEYF YT S SKS S (SEQ ID NO. 59) NLAVVFVTRCCKEYF YT S SKS S (SEQ ID NO. 59) NLAVVFVTRCCKEYF YT S SKS S (SEQ ID NO. 59) NLAVVFVTRCCKEYF YT S SKS S (SEQ ID NO. 59) NLAVVFVTRCCKEYF YT S SKS S (SEQ ID NO. 59) NLAVVFVTRCCKEYF YT S SKS S (SEQ ID NO. 59)
  • ATALCAPASASPYSS (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO
  • SDTTPCCFAYIARPL (SEQ ID NO. 65) YSFVKCCSFKGTTVYALSNVRS (SEQ ID NO.
  • EYFYTSGKCSNPAW (SEQ ID NO. 67) FVKCCSFKGTTVYALSNVRSYS (SEQ ID NO. 67)
  • a method of treating asthma comprising administering to an individual in need thereof (a) a peptide that mimics the RANTES binding domain of PF4, and inhibits the formation of a PF4 and RANTES heterodimer, or (b) a peptide that comprises an amino acid that is 85% homologous (i.e., is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homologous, including any amount of homology between 85% and 100%) to an amino acid sequence selected from:
  • MSVAAVFPvGLRPSPE SEQ ID NO. 54
  • S SKS SNLA WFVTRCCKEYFYT SEQ ID NO. 54
  • VIKAGRHCAVPQLIA (SEQ ID NO. 59) NLA WFVTRCCKEYFYT S SKS S (SEQ ID NO. 59) NLA WFVTRCCKEYFYT S SKS S (SEQ ID NO. 59) NLA WFVTRCCKEYFYT S SKS S (SEQ ID NO. 59) NLA WFVTRCCKEYFYT S SKS S (SEQ ID NO. 59) NLA WFVTRCCKEYFYT S SKS S (SEQ ID NO. 59) NLA WFVTRCCKEYFYT S SKS S (SEQ ID NO. 59) NLA WFVTRCCKEYFYT S SKS S (SEQ ID NO. 59) NLA WFVTRCCKEYFYT S SKS S (SEQ ID NO. 59) NLA WFVTRCCKEYFYT S SKS S (SEQ ID NO. 59) NLA WFVTRCCKEYFY
  • ATALCAPASASPYSS (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO
  • SDTTPCCFAYIARPL (SEQ ID NO. 65) YSFVKCCSFKGTTVYALSNVRS (SEQ ID NO.
  • EYFYTSGKCSNPAW (SEQ ID NO. 67) FVKCCSFKGTTVYALSNVRSYS (SEQ ID NO. 67)
  • the asthma is moderate asthma. In some embodiments, the asthma is sever asthma.
  • RANTES binding domain of PF4 inhibits the formation of a PF4 and RANTES heterodimer, or (b) a peptide that comprises an amino acid that is 85% homologous (i.e., is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homologous, including any amount of homology between 85% and 100%) to an amino acid sequence selected from:
  • EYFYTSGKCSNPAW (SEQ ID NO. 67) FVKCCSFKGTTVYALSNVPvSYS (SEQ ID NO. 67)
  • the asthma is moderate asthma. In some embodiments, the asthma is severe asthma.
  • a method of treating respiratory syncytial virus comprising administering to an individual in need thereof a peptide disclosed herein.
  • the peptide is (a) a peptide that mimics the RANTES binding domain of PF4, and inhibits the formation of a PF4 and RANTES heterodimer, or (b) a peptide that mimics the PF4 binding domain of RANTES, and inhibits the formation of a PF4 and RANTES heterodimer.
  • the peptide comprises an amino acid that is 85% homologous (i.e., is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homologous, including any amount of homology between 85% and 100%) to an amino acid sequence CKEYFYTSSKSSNLAVVFVTRC (SEQ ID NO: 13).
  • the peptide comprises an amino acid that is 85% homologous (i.e., is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homologous, including any amount of homology between 85% and 100%) to an amino acid sequence CKEYFYTSGKSSNPGIVFITRC (SEQ ID NO: 16).
  • the peptide is CKEYFYTSSKSSNLAVVFVTRC (SEQ ID NO: 13) or CKEYFYTSGKSSNPGIVFITRC (SEQ ID NO: 16).
  • RSV respiratory syncytial virus
  • the peptide comprises an amino acid that is 85% homologous (i.e., is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%), 99%, or 100% homologous, including any amount of homology between 85% and 100%) to an amino acid sequence CKEYFYTSSKSSNLAVVFVTRC (SEQ ID NO: 13).
  • the peptide comprises an amino acid that is 85% homologous (i.e., is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homologous, including any amount of homology between 85% and 100%) to an amino acid sequence
  • a method of treating emphysema comprising administering to an individual in need thereof a peptide disclosed herein.
  • the peptide is (a) a peptide that mimics the RANTES binding domain of PF4, and inhibits the formation of a PF4 and RANTES heterodimer, or (b) a peptide that mimics the PF4 binding domain of RANTES, and inhibits the formation of a PF4 and RANTES heterodimer.
  • the peptide comprises an amino acid that is 85% homologous (i.e., is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homologous, including any amount of homology between 85% and 100%) to an amino acid sequence CKEYFYTSSKSSNLAVVFVTRC (SEQ ID NO: 13).
  • the peptide comprises an amino acid that is 85% homologous (i.e., is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%), 99%, or 100% homologous, including any amount of homology between 85% and 100%) to an amino acid sequence CKEYFYTSGKSSNPGIVFITRC (SEQ ID NO: 16).
  • the peptide is CKEYFYTSSKSSNLAVVFVTRC (SEQ ID NO: 13) or
  • a peptide disclosed herein for use in the treatment of emphysema wherein the peptide is (a) a peptide that mimics the RANTES binding domain of PF4, and inhibits the formation of a PF4 and RANTES heterodimer, or (b) a peptide that mimics the PF4 binding domain of RANTES, and inhibits the formation of a PF4 and RANTES heterodimer .
  • the peptide for use is CKEYFYTSSKSSNLAVVFVTRC (SEQ ID NO: 13).
  • the peptide comprises an amino acid that is 85% homologous (i.e., is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) homologous, including any amount of homology between 85% and 100%) to an amino acid sequence CKEYFYTSGKSSNPGIVFITRC (SEQ ID NO: 16).
  • the peptide is CKEYFYTSSKSSNLAVVFVTRC (SEQ ID NO: 13) or CKEYFYTSGKSSNPGIVFITRC (SEQ ID NO: 16).
  • the PF4/RANTES targeting agents, methods and compositions described herein treat a T-cell mediated autoimmune disorder.
  • a T-cell mediated autoimmune disorder is characterized by a T-cell mediated immune response against self (e.g., native cells and tissues).
  • T-cell mediated autoimmune disorders include, but are not limited to colitis, multiple sclerosis, arthritis, rheumatoid arthritis, osteoarthritis, juvenile arthritis, psoriatic arthritis, acute pancreatitis, chronic pancreatitis, diabetes, insulin-dependent diabetes mellitus (IDDM or type I diabetes), insulitis, inflammatory bowel disease, Crohn's disease, ulcerative colitis, autoimmune hemolytic syndromes, autoimmune hepatitis, autoimmune neuropathy, autoimmune ovarian failure, autoimmune orchitis, autoimmune thrombocytopenia, reactive arthritis, ankylosing spondylitis, silicone implant associated autoimmune disease, Sjogren's syndrome, systemic lupus erythematosus (SLE), vasculitis syndromes (e.g., giant cell arteritis, Behcet's disease & Wegener's granulomatosis), vitiligo, secondary hematologic manifestation of autoimmune diseases (e.g.,
  • the PF4/RANTES targeting agents, methods and compositions described herein treat pain. Pain includes, but is not limited to acute pain, acute inflammatory pain, chronic inflammatory pain and neuropathic pain.
  • the PF4/RANTES targeting agents, methods and compositions described herein treat hypersensitivity.
  • hypersensitivity refers to an undesirable immune system response. Hypersensitivity is divided into four categories. Type I hypersensitivity includes allergies (e.g., Atopy, Anaphylaxis, or Asthma). Type II hypersensitivity is
  • cytotoxic/antibody mediated e.g., Autoimmune hemolytic anemia, Thrombocytopenia,
  • Type III is immune complex diseases (e.g., Serum sickness, Arthus reaction, or SLE).
  • Type IV is delayed-type hypersensitivity (DTH), Cell- mediated immune memory response, and antibody-independent (e.g., Contact dermatitis, Tuberculin skin test, or Chronic transplant rejection).
  • allergy means a disorder characterized by excessive activation of mast cells and basophils by IgE.
  • the excessive activation of mast cells and basophils by IgE results (either partially or fully) in an inflammatory response.
  • the inflammatory response is local.
  • the inflammatory response results in the narrowing of airways (i.e., bronchoconstriction).
  • the inflammatory response results in inflammation of the nose (i.e., rhinitis).
  • the inflammatory response is systemic (i.e., anaphylaxis).
  • the PF4/RANTES targeting agents, methods and compositions described herein treat angiogenesis.
  • angiogenesis refers to the formations of new blood vessels. In certain instances, angiogenesis occurs with chronic inflammation. In certain instances, angiogenesis is induced by monocytes and/or macrophages.
  • the present invention comprises a method of treating a neoplasia.
  • a neoplastic cell induces an inflammatory response.
  • part of the inflammatory response to a neoplastic cell is angiogenesis.
  • angiogenesis facilitates the development of a neoplasia.
  • the neoplasia is: angiosarcoma, Ewing sarcoma, osteosarcoma, and other sarcomas, cecum carcinoma, colon carcinoma, lung carcinoma, ovarian carcinoma, pharyngeal carcinoma, rectosigmoid carcinoma, pancreatic carcinoma, renal carcinoma, endometrial carcinoma, gastric carcinoma, liver carcinoma, head and neck carcinoma, breast carcinoma and other carcinomas, Hodgkins lymphoma and other lymphomas, malignant and other melanomas, parotid tumor, chronic lymphocytic leukemia and other leukemias, astrocytomas, gliomas, hemangiomas, retinoblastoma, neuroblastoma, acoustic neuroma, neurofibroma, trachoma and pyogenic granulomas.
  • the PF4/RANTES targeting agents, methods and compositions described herein treat obesity.
  • “obesity” means an accumulation of adipose tissue with a BMI of greater than or equal to 30kg/m 2 .
  • obesity is characterized a proinflammatory state, increasing the risk of thrombosis.
  • obesity is associated with a low-grade inflammation of white adipose tissue (WAT).
  • WAT associated with obesity is characterized by an increased production and secretion of a wide range of inflammatory molecules including TNF-alpha and interleukin-6 (IL-6).
  • WAT is infiltrated by macrophages, which produce pro-inflammatory cytokines.
  • TNF- alpha is overproduced in adipose tissue.
  • IL-6 production increases during obesity.
  • the PF4/RANTES targeting agents, methods and compositions described herein treat metabolic syndrome.
  • metabolic syndrome is associated with fasting hyperglycemia; high blood pressure; central obesity; decreased HDL levels; elevated triglyceride levels; systemic inflammation; or combinations thereof.
  • metabolic syndrome is characterized by an increase in the levels of C-reactive protein, fibrinogen, IL-6, and TNFa.
  • the PF4/RANTES targeting agents, methods and compositions described herein treat a cardiovascular disorder involving inflammation.
  • cardiovascular disease refers to a disease or disorder characterized by impairment or dysfunction of the heart, an artery, and/or vein.
  • the disorder is a
  • the disorder is hyperlipidemia; hypercholesterolemia;
  • hyperglyceridemia combined hyperlipidemia; hypolipoproteinemia; hypocholesterolemia;
  • the disorder is acute coronary syndrome; unstable angina; non-ST segment elevation myocardial infarction; ST segment elevation myocardial infarction; stable angina; Prinzmetal's angina; arteriosclerosis;
  • Atherosclerosis atherosclerosis; stenosis; restenosis; venous thrombosis; arterial thrombosis; stroke; transient ischemic attack; peripheral vascular disease; coronary artery disease; obesity; diabetes; metabolic syndrome; or combinations thereof.
  • the PF4/RANTES targeting agents, methods and compositions described herein treat dyslipidemia.
  • dyslipidemia means a disruption (i.e., variation from a normal range) in the concentration of a lipid in the blood.
  • a dyslipidemia is an increase in lipid (e.g. cholesterol, glycerides, or triglyceride) concentrations over a normal range (i.e., a hyperlipidemia).
  • a hyperlipidemia involves an increase in the concentration of cholesterol (i.e., hypercholesterolemia); glycerides (i.e., hyperglyceridemia); triglycerides (i.e., hypertriglyceridemia); lipoproteins (i.e.,
  • a dyslipidemia is a decrease in lipid concentrations below a normal range (i.e., a hypolipidemia).
  • a hypolipidemia involves a decrease in the concentration of lipoproteins (i.e., hypolipoproteinemia); cholesterol (i.e., hypocholesterolemia); beta lipoproteins (i.e., abetalipoproteinemia); HDL (i.e., Tangier disease); or combinations thereof.
  • a dyslipidemia results from environmental factors (e.g., lack of exercise or food intake).
  • a dyslipidemia results from genetic factors (e.g., aberrant expression of ApoAl, Apo B, ApoC2, LPL, or LDL receptor).
  • blood comprises lipoproteins.
  • a lipoprotein is a complex of proteins (e.g., ApoAl, ApoA2, ApoA4, ApoA5, ApoCl, ApoC2, ApoC3, ApoD, ApoE, LCAT, PAF-AH, PON1, GPX, serum amyloid A, a-1 antitrypsin, and amyloid- ⁇ ) and lipids.
  • a lipoprotein is a high density lipoprotein (HDL).
  • a lipoprotein is a low density lipoprotein (LDL).
  • HDL is a type of lipoprotein that transports cholesterol and triglycerides to the liver.
  • HDL comprises ApoAl and ApoA2.
  • ApoAl and ApoA2 are expressed in the liver.
  • HDL transport cholesterol from cells to the liver, adrenals, ovary and/or testes.
  • cholesterol transported to the liver is excreted as bile.
  • cholesterol transported to adrenals, ovaries and/or testes are used to synthesize steroid hormones.
  • HDL comprises multiple subclasses of lipoprotein. In certain instances, the subclasses of HDL differ in size, density, protein and lipid composition.
  • some HDL are protective, anti-oxidative, anti-inflammatory and/or anti-atherogenic. In certain instances, some HDL are neutral. In certain instances, some HDL enhance oxidation, increase inflammation and/or are pro-atherogenic. In certain instances, increasing the concentration of HDL across all or most sub-classes results in the production of reactive oxygen species (ROS).
  • ROS reactive oxygen species
  • an enzyme associated with HDL modifies a phospholipid into an oxidized phospholipid. In certain instances, an enzyme associated with HDL modifies cholesterol into an oxidized sterol. In certain instances, an oxidized sterol and/or an oxidized phospholipid results in pro-inflammatory and/or pro-atherogenic HDL.
  • cholesteryl ester transfer protein exchanges triglycerides transported by VLDL (very low density lipoprotein) for cholesteryl esters transported by HDL.
  • VLDL very low density lipoprotein
  • the exchange of triglycerides for cholesteryl esters results in VLDL being processed into LDL.
  • LDL is removed from circulation by the LDL receptor pathway.
  • the triglycerides are degraded by hepatic lipase.
  • delipidified HDL recirculate in the blood and transport additional lipids to the liver.
  • inhibiting CETP disrupts the metabolism of HDL.
  • inhibiting CETP prevents transfer of HDL-cholesterol and increases circulating levels of cholesteryl-ester enriched (larger) HDL subfractions.
  • inhibiting (partially or fully) CETP treat CVD.
  • slowing the catabolism of HDL increases total circulating HDL levels.
  • increasing total circulating HDL levels treats atherogenesis.
  • inhibiting (partially or fully) CETP results (partially or fully) in inflammation and/or worsening of CVD.
  • increasing total circulating HDL levels generates a lipid pool with reduced clearance (kinetics).
  • reduced clearance of lipids increases HDL capacity to harbor oxidizable and potentially inflammatory lipid stores.
  • LDL Low-density lipoprotein
  • LDL comprises an
  • Apolipoprotein B (ApoB).
  • ApoB is expressed as two isoforms, ApoB48 and ApoBlOO.
  • ApoB48 is synthesized by intestinal cells.
  • ApoB 100 is synthesized in the liver.
  • Hspl 10 stabilizes ApoB.
  • the PF4/RANTES targeting agents, methods and compositions described herein treat atherosclerosis.
  • atherosclerosis means inflammation of an arterial wall.
  • the inflammation results from (partially or fully) the accumulation of macrophage white blood cells.
  • the inflammation results from (partially or fully) the presence of oxidized LDL.
  • oxidized LDL damages an arterial wall.
  • monocytes respond to (i.e., follow a chemotactic gradient to) the damaged arterial wall.
  • the monocytes differentiate into macrophages.
  • macrophages endocytose the oxidized-LDL (cells such as macrophages with endocytosed LDL are called "foam cells").
  • a foam cell dies.
  • the rupture of a foam cell deposits oxidized cholesterol into the artery wall.
  • the arterial wall becomes inflamed due to the damaged caused by the oxidized LDL.
  • cells form a hard covering over the inflamed area.
  • the cellular covering narrows an artery.
  • an atheromatous plaque is divided into three distinct components: (a) the atheroma (i.e., a nodular accumulation of a soft, flaky, yellowish material comprised of macrophages nearest the lumen of the artery; (b) areas of cholesterol crystals; and (c) calcification at the outer base.
  • the atheroma i.e., a nodular accumulation of a soft, flaky, yellowish material comprised of macrophages nearest the lumen of the artery.
  • areas of cholesterol crystals calcification at the outer base.
  • an atherosclerotic plaque results (partially or fully) in stenosis (i.e., the narrowing of blood vessel). In certain instances, stenosis results (partially or fully) in decreased blood flow. In some embodiments, the PF4/RANTES targeting agents, methods and compositions described herein treat stenosis and/or restenosis. In certain instances, an atherosclerotic plaque results (partially or fully) in the development of an aneurysm. In some embodiments, the PF4/RANTES targeting agents, methods and compositions described herein treat an aneurysm.
  • the rupture of an atherosclerotic plaque results (partially or fully) in an infarction (i.e., the deprivation of oxygen) to a tissue.
  • an infarction i.e., the deprivation of oxygen
  • the PF4/RANTES targeting agents, methods and compositions described herein treat an infarction.
  • the PF4/RANTES targeting agents, methods and compositions described herein treat a myocardial infarction.
  • Myocardial infarction and "heart attack” are used interchangeably.
  • both terms refer to an interruption in the blood supply to the heart.
  • an interruption in the blood supply to the heart results from (partially or fully) the occlusion of a coronary artery by a ruptured atherosclerotic plaque.
  • occlusion of an artery results in the infarction of myocardium.
  • the infarction of myocardium results in the scarring of myocardial tissue.
  • scarred of myocardial tissue conducts electrical impulses more slowly than unscarred tissue.
  • the difference in conduction velocity between scarred and unscarred tissue results (partially or fully) in ventricular fibrillation or ventricular tachycardia.
  • the PF4/RANTES targeting agents, methods and compositions described herein treat an angina (e.g., stable or unstable).
  • angina pectoris refers chest pain resulting from (partially or fully) ischemia of the heart.
  • the PF4/RANTES targeting agents, methods and compositions described herein treat a thrombosis (venous or arterial).
  • thrombosis refers to the formation of a blood clot.
  • the blood clot forms in a vein (i.e., venous thrombosis).
  • the blood clot forms in an artery (i.e., arterial thrombosis).
  • a piece of or the entire blood clot is transported (i.e., an embolism) to the lungs (i.e., a pulmonary embolism).
  • the PF4/RANTES targeting agents, methods and compositions described herein treat an embolism.
  • the PF4/RANTES targeting agents, methods and compositions described herein treat a stroke.
  • stroke refers to a loss of brain function (e.g., necrosis of brain tissue) resulting from (partially or fully) a disturbance in blood supply (e.g., ischemia). In certain instances, a stroke results from (partially or fully) a thrombosis or an embolism.
  • an atherosclerotic plaque results (partially or fully) in the development of an aneurysm.
  • the PF4/RANTES targeting agents, methods and compositions described herein treat an aneurysm.
  • the PF4/RANTES targeting agents, methods and compositions described herein treat an abdominal aortic aneurysm ("AAA").
  • AAA abdominal aortic aneurysm
  • an "abdominal aortic aneurysm" is a localized dilatation of the abdominal aorta.
  • the rupture of an AAA results in bleeding, leading to hypovolemic shock with hypotension, tachycardia, cyanosis, and altered mental status.
  • AAA abdominal aortic aneurysms
  • a method of treating AAA comprising administering to an individual in need thereof (a) a peptide that mimics the RANTES binding domain of PF4, and inhibits the formation of a PF4 and RANTES heterodimer, or (b) a peptide that comprises an amino acid that is 85% homologous (i.e., is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homologous, including any amount of homology between 85% and 100%) to an amino acid sequence selected from:
  • ATALCAPASASPYSS (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO
  • SDTTPCCFAYIARPL (SEQ ID NO. 65) YSFVKCCSFKGTTVYALSNVRS (SEQ ID NO.
  • EYFYTSGKCSNPAW (SEQ ID NO. 67) FVKCCSFKGTTVYALSNVRSYS (SEQ ID NO. 67)
  • RANTES binding domain of PF4 inhibits the formation of a PF4 and RANTES heterodimer, or (b) a peptide that comprises an amino acid that is 85% homologous (i.e., is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homologous, including any amount of homology between 85% and 100%) to an amino acid sequence selected from:
  • MSSAAGFCASRP (SEQ ID NO. 46)
  • EKKWVREYINSLEMS (SEQ ID NO. 69)
  • PRHITSLEVIKAGPHC SSKCSNLAWFVTRR (SEQ ID NO. 74)
  • MSVAAVFRGLRPSPE SEQ ID NO. 54
  • S SKS SNLA WFVTRCCKEYFYT SEQ ID NO. 54
  • VIKAGRHCAVPQLIA (SEQ ID NO. 59) NLA WFVTRCCKEYFYT S SKS S (SEQ ID NO. 59) NLA WFVTRCCKEYFYT S SKS S (SEQ ID NO. 59) NLA WFVTRCCKEYFYT S SKS S (SEQ ID NO. 59) NLA WFVTRCCKEYFYT S SKS S (SEQ ID NO. 59) NLA WFVTRCCKEYFYT S SKS S (SEQ ID NO. 59) NLA WFVTRCCKEYFYT S SKS S (SEQ ID NO. 59) NLA WFVTRCCKEYFYT S SKS S (SEQ ID NO. 59) NLA WFVTRCCKEYFYT S SKS S (SEQ ID NO. 59) NLA WFVTRCCKEYFYT S SKS S (SEQ ID NO. 59) NLA WFVTRCCKEYFY
  • LIATLKNGRKICLDR SEQ ID NO. 60
  • SFKGTTVYALSNVRSYSFVKCC SEQ ID NO. 60
  • ATALCAPASASPYSS (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO
  • SDTTPCCFAYIARPL (SEQ ID NO. 65) YSFVKCCSFKGTTVYALSNVRS (SEQ ID NO.
  • EYFYTSGKCSNPAW (SEQ ID NO. 67) FVKCCSFKGTTVYALSNVRSYS (SEQ ID NO. 67)
  • the PF4/RANTES targeting agents, methods and compositions described herein prevents (partially or fully) or reduces inflammation at the site of an AAA. In some embodiments, the PF4/RANTES targeting agents, methods and compositions described herein decreases the dilation of the abdominal aorta. In some embodiments, the PF4/RANTES targeting agents, methods and compositions described herein partially or fully inhibit the breakdown of a structural protein (e.g., elastin and collagen) in an AAA. In some embodiments, the PF4/RANTES targeting agents, methods and compositions described herein facilitate the regeneration of a structural protein (e.g., elastin and collagen) in an AAA.
  • a structural protein e.g., elastin and collagen
  • the PF4/RANTES targeting agents, methods and compositions described herein partially or fully inhibit the activation of an MMP in an AAA. In some embodiments, the PF4/RANTES targeting agents, methods and compositions described herein inhibit the upregulation of MMP-1, MMP-9 or MMP-12 in an AAA.
  • the method comprises (a) administering to the individual an agent that (i) specifically binds to all or a portion of PF4, RANTES, or a combination thereof, and (ii) is labeled with an imaging agent; (b) exposing the individual to an imaging device for a period of time sufficient to generate an image of the agent.
  • the agent localizes to the site of an abdominal aortic aneurysm.
  • the agent is an antibody, a peptibody, a small molecule, or a peptide.
  • the agent is a peptide that (a) mimics all or a portion of PF4, or (b) all or a portion of PvANTES. In some embodiments, the agent is a peptide that (a) mimics all or a portion of the PvANTES binding domain of PF4, or (b) all or a portion of PF4 binding domain of PvANTES.
  • the peptide comprises an amino acid that is 85% homologous (i.e., is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homologous, including any amount of homology between 85% and 100%) to an amino acid sequence: CKEYFYTSSKSSNLAVVFVTRC (SEQ ID NO: 13).
  • the peptide comprises an amino acid that is 85% homologous (i.e., is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homologous, including any amount of homology between 85% and 100%) to an amino acid sequence
  • the agent is an anti-PF4 antibody, an anti-RANTES antibody, or a combination thereof.
  • a method of diagnosing an abdominal aortic aneurysm in an individual in need thereof comprising (a) administering to the individual an agent that (i) specifically binds to all or a portion of PF4, RANTES, or a combination thereof, and (ii) is labeled with an imaging agent; (b) exposing the individual to an imaging device for a period of time sufficient to generate an image of the agent; and (c) diagnosing the individual with an abdominal aortic aneurysm if the image of the agent shows a concentration of the agent in the abdominal aorta that exceeds the concentration in a control.
  • the agent localizes to the site of an abdominal aortic aneurysm.
  • the agent is an antibody, a peptibody, a small molecule, or a peptide.
  • the agent is a peptide that (a) mimics all or a portion of PF4, or (b) all or a portion of RANTES.
  • the agent is a peptide that (a) mimics all or a portion of the RANTES binding domain of PF4, or (b) all or a portion of PF4 binding domain of RANTES.
  • the peptide comprises an amino acid that is 85% homologous (i.e., is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%), 99%, or 100% homologous, including any amount of homology between 85% and 100%) to an amino acid sequence: CKEYFYTSSKSSNLAVVFVTRC (SEQ ID NO: 13).
  • the peptide comprises an amino acid that is 85% homologous (i.e., is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homologous, including any amount of homology between 85% and 100%) to an amino acid sequence
  • the agent is an anti-PF4 antibody, an anti-PvANTES antibody, or a combination thereof.
  • a method of monitoring an abdominal aortic aneurysm in an individual in need thereof comprising (a) administering to the individual an agent that (i) specifically binds to all or a portion of PF4, PvANTES, or a combination thereof, and (ii) is labeled with an imaging agent; (b) exposing the individual to an imaging device for a period of time sufficient to generate an image of the agent; wherein a decrease in the concentration of the agent in the abdominal aorta as compared to an earlier image of the agent indicates that the Abdominal Aortic Aneurysm (AAA) is regressing, (ii) an increase in the concentration of the agent in the abdominal aorta as compared to an earlier image of the agent indicates that the Abdominal Aortic Aneurysm (AAA) is progressing, or (iii) no change in the concentration of the agent in the abdominal aorta as compared to an earlier image of
  • the agent localizes to the site of an abdominal aortic aneurysm.
  • the agent is an antibody, a peptibody, a small molecule, or a peptide.
  • the agent is a peptide that (a) mimics all or a portion of PF4, or (b) all or a portion of PvANTES.
  • the agent is a peptide that (a) mimics all or a portion of the PvANTES binding domain of PF4, or (b) all or a portion of PF4 binding domain of RANTES.
  • the peptide comprises an amino acid that is 85% homologous (i.e., is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%o, 97%o, 98%o, 99%o, or 100% homologous, including any amount of homology between 85% and 100%) to an amino acid sequence: CKEYFYTSSKSSNLAVVFVTRC (SEQ ID NO: 13).
  • the peptide comprises an amino acid that is 85% homologous (i.e., is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homologous, including any amount of homology between 85% and 100%) to an amino acid sequence
  • the agent that specifically binds to all or a portion of PF4, RANTES, or a combination thereof is administered to identify and/or locate a site of inflammation. In some embodiments, the agent that specifically binds to all or a portion of PF4, RANTES, or a combination thereof disclosed herein is administered to identify and/or locate a site of chronic inflammation. In some embodiments, the agent that specifically binds to all or a portion of PF4, RANTES, or a combination thereof disclosed herein is administered to identify and/or locate a site of acute inflammation.
  • the agent that specifically binds to all or a portion of PF4, RANTES, or a combination thereof disclosed herein is administered to identify and/or locate an atherosclerotic plaque.
  • the agent that specifically binds to all or a portion of PF4, RANTES, or a combination thereof disclosed herein is administered to identify and/or locate an AAA.
  • the agent that specifically binds to all or a portion of PF4, RANTES, or a combination thereof disclosed herein is administered to identify and/or locate an occlusion of an artery.
  • the agent that specifically binds to all or a portion of PF4, RANTES, or a combination thereof disclosed herein is administered to identify and/or locate a thrombus. In some embodiments, the agent that specifically binds to all or a portion of PF4, RANTES, or a combination thereof disclosed herein is administered to identify and/or locate an embolism.
  • the agent that specifically binds to all or a portion of PF4, RANTES, or a combination thereof disclosed herein localizes to areas of a body with high concentrations of leukocytes. In some embodiments, the agent that specifically binds to all or a portion of PF4, RANTES, or a combination thereof disclosed herein localizes to areas of a circulatory system with high concentrations of leukocytes. In some embodiments, an area of the circulatory system with high concentrations of leukocytes is an atherosclerotic plaque. In some embodiments, an area of the circulatory system with high concentrations of leukocytes is an AAA.
  • an area of the circulatory system with high concentrations of leukocytes is an occlusion of an artery. In some embodiments, an area of the circulatory system with high concentrations of leukocytes is a thrombus. In some embodiments, an area of the circulatory system with high concentrations of leukocytes is an embolism.
  • the agent that specifically binds to all or a portion of PF4, RANTES, or a combination thereof disclosed herein is labeled for imaging (e.g., molecular imaging). In some embodiments, the agent that specifically binds to all or a portion of PF4, RANTES, or a combination thereof disclosed herein is labeled for medical imaging.
  • the agent that specifically binds to all or a portion of PF4, RANTES, or a combination thereof disclosed herein is labeled for radio-imaging, PET imaging, SPECT imaging, MRI imaging, ultrasound imaging, optical imaging modalities (e.g., near-infrared, fluorescent, optical coherence tomography (OCT)), and combination imaging modalities (e.g., CT/SPECT, CT/PET, MR/optical, PET/optical).
  • optical imaging modalities e.g., near-infrared, fluorescent, optical coherence tomography (OCT)
  • combination imaging modalities e.g., CT/SPECT, CT/PET, MR/optical, PET/optical.
  • the label is a radiolabel. In some embodiments, the label is a fluorescent label. In some embodiments, the label is magnetic, paramagnetic or superparamagnetic. In some embodiments, the agent is labeled with 17 F, 18 F, 117 I, U8 I, U9 I, 120 I, 121 I, 122 I, 124 I, 126 L 128 1 131 I, 10 C, U C, 13 0, 14 0, 15 0, 74 Br, 75 Br, 76 Br, 77 Br, 78 Br, 82 Br, m In, 225 Ac, 13 N, 82 Rb, 32 C1, 33 C1, 34 C1, M Cu, monobromobimane (mBBr), dibromobimane, monobromotrimethylammoniobimane, indocyanine green acylthiazolidinethione (ICG- ATT), 7-amino-4-methyl coumarin-3 -acetic acid (AMCA), guanidinoethanethiol (mBBr), dibromobi
  • a labeled agent disclosed herein is detected by any suitable method (e.g., by use of a gamma camera, MRI, PET scanner, x-ray computed tomography (CT), functional magnetic resonance imaging (fJVIRI), and single photon emission computed tomography (SPECT)).
  • CT computed tomography
  • fJVIRI functional magnetic resonance imaging
  • SPECT single photon emission computed tomography
  • the agent that specifically binds to all or a portion of PF4, RANTES, or a combination thereof disclosed herein is labeled by conjugating the imaging agent to a portion of the PF4/RANTES targeting agent (e.g., a peptide residue) that has minimal effect on the binding affinity of the PF4/RANTES targeting agent.
  • a portion of the PF4/RANTES targeting agent e.g., a peptide residue
  • an Abdominal Aortic Aneurysm is diagnosed by (a) administering to an individual the agent that specifically binds to all or a portion of PF4, RANTES, or a combination thereof that has been labeled (the "labeled agent"), (b) exposing the individual to an imaging device for a period of time sufficient to generate an image of the labeled agent, and (c) indicating that the individual should be selected for treatment of a Abdominal Aortic Aneurysm (AAA) if the image of the labeled agent shows a concentration of the labeled agent in the abdominal aorta that exceeds the level in a control.
  • the labeled agent the agent that specifically binds to all or a portion of PF4, RANTES, or a combination thereof that has been labeled
  • control is the image produced from a healthy individual that (a) was administered the agent that specifically binds to all or a portion of PF4, RANTES, or a combination thereof that had been labeled; and (b) exposed to an imaging device for a period of time sufficient to generate an image of the labeled agent.
  • image of the labeled agent shows a concentration of the labeled agent in the abdominal aorta that exceeds the level in a control
  • the individual is administered an agent disclosed herein.
  • the PF4/RANTES targeting agent is
  • the PF4/RANTES targeting agent is CKEYF YTS GKS SNPGIVFITRC (SEQ ID NO: 16).
  • the peptide is CKEYF YTSSKSSNLAVVFVTRC (SEQ ID NO: 13) or
  • high concentrations of PF4 or RANTES are found at the site of an AAA.
  • the PF4/RANTES targeting agent localizes to areas of the circulatory system with high concentrations of PF4 or RANTES.
  • the PF4/RANTES targeting agent localizes to an AAA.
  • the progression or regression of an Abdominal Aortic Aneurysm is determined by (a) administering to an individual the agent that specifically binds to all or a portion of PF4, RANTES, or a combination thereof that has been labeled (the "labeled agent"), and (b) exposing the individual to an imaging device for a period of time sufficient to generate an image of the labeled agent.
  • the labeled agent the agent that specifically binds to all or a portion of PF4, RANTES, or a combination thereof that has been labeled
  • a decrease in the concentration of the labeled agent in the abdominal aorta as compared to an earlier image of the label agent indicates that the Abdominal Aortic Aneurysm (AAA) is regressing.
  • the dose of the agent administered to the individual is maintained. In some embodiments, where an Abdominal Aortic Aneurysm (AAA) is regressing, the dose of the agent administered to the individual is decreased.
  • an increase in the concentration of the labeled agent in the abdominal aorta as compared to an earlier image of the labeled agent indicates that the Abdominal Aortic Aneurysm (AAA) is progressing.
  • AAA Abdominal Aortic Aneurysm
  • the dose of the agent administered to the individual is increased.
  • an alternative agent is administered to the individual.
  • the alternative agent is administered in combination with the agent administered previously.
  • no change in the concentration of the labeled agent in the abdominal aorta as compared to an earlier image of the labeled agent indicates that the Abdominal Aortic Aneurysm (AAA) is neither regressing nor progressing.
  • AAA Abdominal Aortic Aneurysm
  • the dose of the agent administered to the individual is increased.
  • an alternative agent is administered to the individual.
  • the alternative agent is administered in combination with the agent administered previously.
  • the agent that specifically binds to all or a portion of PF4, RANTES, or a combination thereof disclosed herein is labeled for imaging (e.g., molecular imaging). In some embodiments, the agent that specifically binds to all or a portion of PF4, RANTES, or a combination thereof disclosed herein is labeled for medical imaging.
  • the agent that specifically binds to all or a portion of PF4, RANTES, or a combination thereof disclosed herein is labeled for radio-imaging, PET imaging, SPECT imaging, MRI imaging, ultrasound imaging, optical imaging modalities (e.g., near-infrared, fluorescent, optical coherence tomography (OCT)), and combination imaging modalities (e.g., CT/SPECT, CT/PET, MR/optical, PET/optical).
  • optical imaging modalities e.g., near-infrared, fluorescent, optical coherence tomography (OCT)
  • combination imaging modalities e.g., CT/SPECT, CT/PET, MR/optical, PET/optical.
  • the label is a radiolabel. In some embodiments, the label is a fluorescent label. In some embodiments, the label is magnetic, paramagnetic or superparamagnetic. In some embodiments, the agent is labeled with 17 F, 18 F, 117 I, U8 I, U9 I, 120 I, 121 I, 122 I, 124 I, 126 I, 128 1 131 I, 10 C, U C, 13 0, 14 0, 15 0, 74 Br, 75 Br, 76 Br, 77 Br, 78 Br, 82 Br, m In, 225 Ac, 13 N, 82 Rb, 32 C1, 33 C1, 34 C1, M Cu, monobromobimane (mBBr), dibromobimane, monobromotrimethylammoniobimane, indocyanine green acylthiazolidinethione (ICG- ATT), 7-amino-4-methyl coumarin-3 -acetic acid (AMCA), guanidinoethanethiol
  • a labeled agent disclosed herein is detected by any suitable method (e.g., by use of a gamma camera, MRI, PET scanner, x-ray computed tomography (CT), functional magnetic resonance imaging (fJVIRI), and single photon emission computed tomography (SPECT)).
  • CT computed tomography
  • fJVIRI functional magnetic resonance imaging
  • SPECT single photon emission computed tomography
  • the agent that specifically binds to all or a portion of PF4, RANTES, or a combination thereof disclosed herein is labeled by conjugating the imaging agent to a portion of the PF4/RANTES targeting agent (e.g., a peptide residue) that has minimal effect on the binding affinity of the PF4/RANTES targeting agent.
  • a portion of the PF4/RANTES targeting agent e.g., a peptide residue
  • an individual in need thereof displays one or more risk factors for developing an Abdominal Aortic Aneurysm (AAA) (e.g., 60 years of age or older; male; cigarette smoking; high blood pressure; high serum cholesterol; diabetes mellitus; atherosclerosis).
  • AAA Abdominal Aortic Aneurysm
  • the ability of RANTES and PF4 to bind together is disrupted by use of a small molecule that binds to the PF4 binding domain of RANTES. In some embodiments, the ability of PF4 and RANTES to bind together is disrupted by use of a small molecule that binds to the RANTES binding domain of PF4.
  • the function of a RANTES/PF4 heteromultimer is disrupted by use of a small molecule that binds to a RANTES/PF4 heterodimer.
  • an inflammatory disease, disorder, condition, or symptom is treated by disrupting the ability of PF4 and RANTES to bind each other.
  • an inflammatory disease, disorder, condition, or symptom is treated by administering to an individual in need an antibody that binds to PF4, one or more PF4 motifs. In some embodiments, an inflammatory disease, disorder, condition, or symptom is treated by administering to an individual in need an antibody that binds to the RANTES binding domain of PF4.
  • an inflammatory disease, disorder, condition, or symptom is treated by administering to an individual in need an antibody that binds to RANTES, one or more RANTES motifs. In some embodiments, an inflammatory disease, disorder, condition, or symptom is treated by administering to an individual in need an antibody that binds to the PF4 binding domain of RANTES. [00132] In some embodiments, an inflammatory disease, disorder, condition, or symptom is treated by administering to an individual in need an antibody that binds to a PF4/RANTES multimer, or one or more motifs on a PF4/RANTES multimer.
  • the antibody is a human antibody or a humanized antibody. In some embodiments, the antibody is a human IgG. In some embodiments, the antibody is or comprises one or more polypeptides derived from a human IgGl, IgG4, IgG2, IgD, IgA or IgM. An antibody disclosed herein is generated by any suitable method.
  • an antibody disclosed herein is generated by contacting a host (e.g., a mouse or rabbit) with an antigen.
  • a host e.g., a mouse or rabbit
  • the antigen is PF4.
  • the antigen is RANTES.
  • the antigen is a PF4/RANTES multimer.
  • the antigen is a fragment of a full-length RANTES polypeptide.
  • the antigen is a polypeptide that encompasses all or part of a RANTES alpha- helix (e.g., human RANTES 58 .67) or a peptide that is at least 85% homologous thereto.
  • the antigen is a polypeptide that encompasses all or part of a RANTES beta-strand (e.g., human RANTES 25 .30, human RANTES 38 .44, human RANTES 47 .51) or a peptide that is at least 85% homologous thereto.
  • the antigen is a polypeptide that encompasses all or part of a RANTES beta-sheet (e.g., human RANTES 25 -51) or a peptide that is at least 85% homologous thereto. In some embodiments, the antigen is a polypeptide that encompasses all or part of human RANTES 25 .44 (e.g., KEYFYTSGKCSNPAWFVTR) or a peptide that is at least 85% homologous thereto.
  • the antigen is a polypeptide that encompasses all or part of mouse RANTES 25 -44 (e.g., KEYFYTSSKCSNLAWFVTR) or a peptide that is at least 85% homologous thereto. In some embodiments, the antigen is a polypeptide that encompasses all or part of rat RANTES 25 -44 (e.g., KEYFYTSSKCSNLAWFVTR) or a peptide that is at least 85% homologous thereto.
  • the antigen is a polypeptide that encompasses all or part of pig RANTES 25 -44 (e.g., QEYFYTS SKCSMAAWFITR) or a peptide that is at least 85% homologous thereto.
  • the antigen is a polypeptide that encompasses all or part of dog RANTES 25 -44 (e.g., QEYFYT S SKC SMPA WFVTR) or a peptide that is at least 85% homologous thereto.
  • the antigen is a fragment of a full-length PF4 polypeptide.
  • the antigen is a polypeptide that encompasses all or part of the PF4 alpha-helix (e.g., human PF4 9 i_i 0 i) or a peptide that is at least 85% homologous thereto.
  • the antigen is a polypeptide that encompasses all or part of a PF4 beta-strand (e.g., human PF4 55 _ 62 , human PF4 70 _ 7 5, human PF4 80 _84) or a peptide that is at least 85% homologous thereto.
  • the antigen is a polypeptide that encompasses all or part of the PF4 beta-sheet (e.g., human PF4 5 5_8 4 ) or a peptide that is at least 85% homologous thereto.
  • the antigen is a polypeptide that encompasses all or part of a PF4 DLQ motif (e.g., human PF4 3 8_ 4 o, human PF4 85 . 87 ) or a peptide that is at least 85% homologous thereto.
  • the antigen is a polypeptide that encompasses all or part of the PF4 external loop (human PF4 51 _ 54 ) or a peptide that is at least 85% homologous thereto.
  • an antibody disclosed herein is generated by contacting a host (e.g., a mouse or rabbit) with at least two antigens selected from a peptide sequence that encompasses all or part of: human RANTES 25 -44 (i.e., KEYFYTSGKCSNPAVVFVTR), a sequence that is at least 85% homologous to human RANTES 25 -44, mouse RANTES 25 -44 (i.e., KEYFYTSSKCSNLAVVFVTR), a sequence that is at least 85% homologous to mouse RANTES 25 -44, rat RANTES 25 -44 (i.e.,
  • KEYFYTSSKCSNLAVVFVTR a sequence that is at least 85% homologous to rat RANTES 25 -44, pig RANTES 25 -44 (i.e., QEYFYTSSKCSMAAWFITR), a sequence that is at least 85% homologous to pig RANTES 25 -44, dog RANTES 25 -44 (i.e., QEYFYTSSKCSMPAWFVTR), a sequence that is at least 85% homologous to dog RANTES 25 -44, a RANTES alpha-helix (e.g., human RANTESsg-ev), a sequence that is at least 85% homologous to a RANTES alpha-helix, a RANTES beta-strand (e.g., human RANTES 25 .30, RANTES 38 .44, RANTES 47 .51), a sequence that is at least 85% homologous to a RANTES beta-strand, a RANTES beta
  • a sequence that is at least 85% homologous to a PF4 alpha-helix, a PF4 beta-strand e.g., human PF4 55 _ 62 , human PF4 70 _ 75 , human PF4 80 _84
  • a sequence that is at least 85% homologous to a PF4 beta-strand e.g., human PF4 55 _ 62 , human PF4 70 _ 75 , human PF4 80 _84
  • a sequence that is at least 85% homologous to a PF4 beta-strand e.g., human PF4 beta-sheet (e.g., human PF4 5 5_ 84 )
  • a sequence that is at least 85% homologous to a PF4 beta- sheet, a PF4 DLQ motif e.g., human PF4 38 . 4 o, human PF4 85 .
  • PF4 DLQ motif a sequence that is at least 85% homologous to a PF4 DLQ motif
  • PF4 external loop e.g., human PF4 51 _ 54
  • sequence that is at least 85% homologous to a PF4 external loop e.g., human PF4 51 _ 54
  • an antibody disclosed herein is generated by contacting a host (e.g., a mouse or rabbit) with at least three antigens selected from a peptide sequence that encompasses all or part of: human RANTES 25 -44 (i.e., KEYFYTSGKCSNPAVVFVTR), a sequence that is at least 85%) homologous to human RANTES 25 -44, mouse RANTES 25 -44 (i.e.,
  • KEYFYTSSKCSNLAVVFVTR a sequence that is at least 85% homologous to mouse RANTES 25 - 44, rat RANTES 25 -44 (i.e., KEYFYTSSKCSNLAVVFVTR), a sequence that is at least 85% homologous to rat RANTES 25 -44, pig RANTES 25 -44 (i.e., QEYFYTSSKCSMAAWFITR), a sequence that is at least 85% homologous to pig RANTES 25 -44, dog RANTES 25 -44 (i.e.,
  • QEYFYTSSKCSMPAWFVTR a sequence that is at least 85% homologous to dog RANTES 25 -44, a RANTES alpha-helix (e.g., human RANTES 58 . 6 7), a sequence that is at least 85% homologous to a RANTES alpha-helix, a RANTES beta-strand (e.g., human RANTES 25 .30, RANTES 38 . 4 4, RANTES 47 .
  • a sequence that is at least 85% homologous to a RANTES beta-strand a RANTES beta-sheet (e.g., human RANTES 25 - 51 ), a sequence that is at least 85% homologous to a RANTES beta-sheet, a PF4 alpha-helix (e.g., human PF4 91 .
  • PF4 DLQ motif a sequence that is at least 85% homologous to a PF4 DLQ motif
  • PF4 external loop e.g., human PF4 5 i_ 54
  • sequence that is at least 85% homologous to a PF4 external loop e.g., human PF4 5 i_ 54
  • an antibody disclosed herein is generated by contacting a host with a nucleic acid sequence encoding part or all of a RANTES polypeptide (alternatively, "RANTES nucleic acid sequence"). In some embodiments, an antibody disclosed herein is generated by contacting a host with a nucleic acid sequence encoding part or all of a PF4 polypeptide
  • the nucleic acid sequence has been cloned into an expression vector (e.g., a plasmid).
  • an expression vector e.g., a plasmid
  • the host is a mammal. In some embodiments, the host is a mouse, a rabbit, or a rat. In some embodiments, the host is a mammalian cell. In some embodiments, the host is a bacterial cell.
  • the nucleic acid sequence i.e., PF4 or RANTES
  • the contacting further comprises applying an electric current to the site of injection (i.e., electroporation).
  • the nucleic acid sequence i.e., PF4 or RANTES
  • the nucleic acid sequence is contacted with the host by use of a gene gun.
  • nucleic acid sequence i.e., PF4 or RANTES
  • PF4 or RANTES is expressed by a host cell (or a plurality of host cells) to generate an antigen.
  • the RANTES nucleic acid sequence is expressed by a host cell (or a plurality of host cells) to generate an expressed RANTES polypeptide.
  • the PF4 nucleic acid sequence is expressed by a host cell (or a plurality of host cells) to generate an expressed PF4 polypeptide.
  • a method of generating an antibody disclosed herein further comprises contacting the host with an adjuvant.
  • the adjuvant is administered as a nucleic acid sequence.
  • the adjuvant is administered as a polypeptide or polysaccharide.
  • the adjuvant is a cytokine, a lymphokine, or a combination thereof.
  • the adjuvant is an interleukin, a tumor necrosis factor, GM-CSF, or a combination thereof.
  • the adjuvant is B7-1, B7-2, CD40L, or a combination thereof.
  • the expression vector containing the nucleic acid sequence i.e., PF4 or RANTES
  • the host is contacted with a second expression vector encoding an adjuvant.
  • the nucleic acid sequence (a) encodes part or all of a PF4 alpha-helix (e.g., TAC AAG AAA ATA ATT AAG AAA CTT TTG GAG AGT), or (b) is a sequence that is at least 85% homologous to a PF4 alpha-helix.
  • the nucleic acid sequence (a) encodes part or all of a PF4 beta-strand (e.g., ATC ACC AGC CTG GAG GTG ATC AAG, GCC CAA CTG ATA GCC ACG, AGG AAA ATT TGC TTG), or (b) is a sequence that is at least 85% homologous to a PF4 beta-strand.
  • a PF4 beta-strand e.g., ATC ACC AGC CTG GAG GTG ATC AAG, GCC CAA CTG ATA GCC ACG, AGG AAA ATT TGC TTG
  • the nucleic acid sequence (a) encodes part or all of a PF4 beta-sheet (e.g., CTG GAG GTG ATC AAG GCC GGA CCC CAC TGC CCC ACT GCC CAA CTG ATA GCC ACG CTG AAG AAT GGA AGG AAA ATT TGC TTG), or (b) is a sequence that is at least 85% homologous to a PF4 beta-sheet.
  • the nucleic acid sequence (a) encodes part or all of a PF4 DLQ motif (e.g., GAC CTG CAG, GAC CTG CAA), or (b) is a sequence that is at least 85% homologous to a PF4 DLQ motif.
  • the nucleic acid sequence (a) encodes part or all of a PF4 external loop (e.g., CGT CCC AGG CAC), or (b) is a sequence that is at least 85% homologous to a PF4 external loop.
  • a PF4 external loop e.g., CGT CCC AGG CAC
  • the nucleic acid sequence (a) encodes part or all of the RANTES alpha-helix (e.g., TGG GTT CGG GAG TAC ATC AAC TCT TTG GAG), or (b) is a sequence that is at least 85% homologous to a RANTES alpha-helix.
  • the nucleic acid sequence (a) encodes part or all of a RANTES beta-strand (e.g., ATC AAG GAG TAT TTC TAC, CCA GCA GTC GTC TTT GTC ACC, AAC CGC CAA GTG TGT), or (b) is a sequence that is at least 85%) homologous to a RANTES beta-strand.
  • the nucleic acid sequence (a) encodes part or all of the RANTES beta-sheet (e.g., ATC AAG GAG TAT TTC TAC ACC AGT GGC AAG TGC TCC AAC CCA GCA GTC GTC TTT GTC ACC CGA AAG AAC CGC CAA GTG TGT), or (b) is a sequence that is at least 85% homologous to a RANTES beta-sheet.
  • the RANTES beta-sheet e.g., ATC AAG GAG TAT TTC TAC ACC AGT GGC AAG TGC TCC AAC CCA GCA GTC GTC TTT GTC ACC CGA AAG AAC CGC CAA GTG TGT
  • an antibody disclosed herein is generated by contacting a host with at least two nucleic acid sequences selected from sequences that encode part or all of: a PF4 alpha- helix (e.g., TAC AAG AAA ATA ATT AAG AAA CTT TTG GAG AGT), a sequence that is at least 85% homologous to a PF4 alpha-helix, a PF4 beta-strand (e.g., ATC ACC AGC CTG GAG GTG ATC AAG, GCC CAA CTG ATA GCC ACG, AGG AAA ATT TGC TTG), a sequence that is at least 85% homologous to a PF4 beta-strand, a PF4 beta-sheet (e.g., CTG GAG GTG ATC AAG GCC GGA CCC CAC TGC CCC ACT GCC CAA CTG ATA GCC ACG CTG AAG AAT GGA AGG AAA ATT TGC TTG), a sequence that is at least two nucleic acid sequences
  • an antibody disclosed herein is generated by contacting a host with at least three nucleic acid sequences selected from sequences that encode part or all of: a PF4 alpha- helix (e.g., TAC AAG AAA ATA ATT AAG AAA CTT TTG GAG AGT), a sequence that is at least 85% homologous to a PF4 alpha-helix, a PF4 beta-strand (e.g., ATC ACC AGC CTG GAG GTG ATC AAG, GCC CAA CTG ATA GCC ACG, AGG AAA ATT TGC TTG), a sequence that is at least 85% homologous to a PF4 beta-strand, a PF4 beta-sheet (e.g., CTG GAG GTG ATC AAG GCC GGA CCC CAC TGC CCC ACT GCC CAA CTG ATA GCC ACG CTG AAG AAT GGA AGG AAA ATT TGC TTG), a sequence that is at least three nucleic acid sequences
  • an antibody disclosed herein is produced via the use of a hybridoma.
  • a "hybridoma” is an immortalized antibody producing cell.
  • a host e.g., a mouse or a rabbit
  • B-cells from the host's spleen are extracted.
  • a hybridoma is generated by fusing (1) an extracted B-cell with (2) a myeloma cell (i.e., hypoxanthine -guanine -phosphoribosyl transferase negative, immortalized myeloma cells).
  • the B-cell and the myeloma cells are cultured together and exposed to an agent that renders their cell membranes more permeable (e.g., PEG).
  • the culture comprises a plurality of hybridoma, a plurality of myeloma cells, and a plurality of B-cells.
  • the cells are individual to culturing conditions that select for hybridoma (e.g., culturing with HAT media).
  • an individual hybridoma i.e., the clone
  • the hybridoma are isolated and cultured.
  • the hybridoma are injected into a laboratory animal.
  • the hybridoma are cultured in a cell culture.
  • the methods described herein comprise a humanized monoclonal antibody.
  • a humanized monoclonal antibody comprises heavy and light chain constant regions from a human source and variable regions from a murine source.
  • humanized immunoglobulins are constructed by genetic engineering.
  • humanized immunoglobulins comprise a framework that is identical to the framework of a particular human immunoglobulin chain (i.e., an acceptor or recipient), and three CDRs from a non-human (donor) immunoglobulin chain.
  • a limited number of amino acids in the framework of a humanized immunoglobulin chain are identified and chosen to be the same as the amino acids at those positions in the donor rather than in the acceptor.
  • a framework is used from a particular human immunoglobulin that is homologous to the donor immunoglobulin to be humanized. For example, comparison of the sequence of a mouse heavy (or light) chain variable region against human heavy (or light) variable regions in a data bank (for example, the National Biomedical Research Foundation Protein
  • immunoglobulin comprises light and heavy chains from different human antibody germline sequences as acceptor sequences; when such combinations are used, one can readily determine whether the VH and VL bind an epitope of interest using conventional assays (e.g., an ELISA).
  • the human antibody will be chosen in which the light and heavy chain variable regions sequences, taken together, are overall most homologous to the donor light and heavy chain variable region sequences.
  • higher affinity is achieved by selecting a small number of amino acids in the framework of the humanized immunoglobulin chain to be the same as the amino acids at those positions in the donor rather than in the acceptor.
  • the relevant framework amino acids to change are selected based on differences in amino acid framework residues between the donor and acceptor molecules.
  • the amino acid positions to change are residues known to be important or to contribute to CDR conformation (e.g., canonical framework residues are important for CDR conformation and/or structure).
  • the relevant framework amino acids to change are selected based on frequency of an amino acid residue at a particular framework position (e.g., comparison of the selected framework with other framework sequences within its subfamily can reveal residues that occur at minor frequencies at a particular position or positions).
  • the relevant framework amino acids to change are selected based on proximity to a CDR. In some embodiments, the relevant framework amino acids to change are selected based on known or predicted proximity to the antigen-CDR interface or predicted to modulate CDR activity. In some embodiments, the relevant framework amino acids to change are framework residues that are known to, or predicted to, form contacts between the heavy (VH) and light (VL) chain variable region interface. In some embodiments, the relevant framework amino acids to change are framework residues that are inaccessible to solvent.
  • amino acid changes at some or all of the selected positions are incorporated into encoding nucleic acids for the acceptor variable region framework and donor CDRs.
  • altered framework or CDR sequences are individually made and tested, or are sequentially or simultaneously combined and tested.
  • the variability at any or all of the altered positions is from a few to a plurality of different amino acid residues, including all twenty naturally occurring amino acids or functional equivalents and analogues thereof. In some embodiments, non-naturally occurring amino acids are considered.
  • the humanized antibody sequence is cloned into a vector.
  • any suitable vector is used.
  • the vector is a plasmid, viral e.g. 'phage, or phagemid, as appropriate.
  • plasmid a plasmid, viral e.g. 'phage, or phagemid, as appropriate.
  • Many known techniques and protocols for manipulation of nucleic acid for example in preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells and gene expression, and analysis of proteins, are described in detail in Short Protocols in Molecular Biology, Second Edition, Ausubel et al. eds., John Wiley & Sons, 1992. The disclosures of Sambrook et al. and Ausubel et al. are incorporated herein by reference for such disclosure.
  • any suitable host cell is transformed with the vector expressing the humanized antibody sequence.
  • the host cell is bacteria, mammalian cells, yeast and baculovirus systems.
  • the expression of antibodies and antibody fragments in prokaryotic cells such as E. coli is well established in the art. For a review, see for example Pluckthun, A.
  • a mammalian expression system is used.
  • the mammalian expression system is dehydrofolate reductase deficient ("dhfr- ") Chinese hamster ovary cells.
  • dhfr- CHO cells are transfected with an expression vector containing a functional DHFR gene, together with a gene that encodes part or all of a desired humanized antibody.
  • DNA is transformed by any suitable method.
  • suitable techniques include, for example, calcium phosphate transfection, DEAE Dextran, electroporation, liposome-mediated transfection and transduction using retrovirus or other virus, e.g., vaccinia or, for insect cells, baculovirus.
  • suitable techniques include, for example, calcium chloride transformation, electroporation and transfection using bacteriophage.
  • a DNA sequence encoding an antibody or antigen-binding fragment thereof is prepared synthetically rather than cloned.
  • the DNA sequence is designed with the appropriate codons for the antibody or antigen-binding fragment amino acid sequence. In general, one will select preferred codons for the intended host if the sequence will be used for expression.
  • the complete sequence is assembled from overlapping oligonucleotides prepared by standard methods and assembled into a complete coding sequence. See, e.g., Edge, Nature, 292:756 (1981); Nambair et al., Science, 223: 1299 (1984); Jay et al., J. Biol. Chem., 259:6311 (1984), each of which is incorporated herein by reference for such disclosure.
  • the interaction of RANTES and PF4 is disrupted by use of a peptide that mimics all or part of RANTES. In some embodiments, the interaction of RANTES and PF4 is disrupted by use of a peptide that mimics the PF4 binding domain of RANTES. In certain instances, PF4 binds to the peptide mimic and thus does not bind to RANTES.
  • the interaction of RANTES and PF4 is disrupted by use of a peptide that mimics all or part of PF4. In some embodiments, the interaction of RANTES and PF4 is disrupted by use of a peptide that mimics the RANTES binding domain of RANTES. In certain instances, RANTES binds to the peptide mimic and thus does not bind to PF4.
  • a peptide mimic described herein is an isolated peptide
  • a peptide mimic described herein does not effect (or only partially effect) other functions of the RANTES and/or PF4. In some embodiments, a selective blocking of the recruiting of monocytes is achieved, for example, on endothelium. [00168] In some embodiments, a peptide mimic described herein provides a high specificity, and does not effect (or only partially effect) the many metabolic processes mediated by the chemokines RANTES and PF4, for example, the immune or clotting systems.
  • a peptide mimic described herein comprises between 15 and 25 amino acids. In some embodiments, a peptide mimic described herein comprises between 19 and 25 amino acids. In some embodiments, a peptide mimic described herein comprises no more than 25 amino acids. In a further embodiment, a peptide mimic described herein comprises aboutl5 to about 25 amino acids. Ina further embodiment, a peptide mimic described herein comprises about 15 to about 22 amino acids. In further embodiments, a peptide mimic described herein comprises about 18 to about 23 amino acids, including in the range of about 18 to about 22 amino acids, and including, in the range of about 19 to about 22 amino acids, and also including in the range of about 20 to about 21 amino acids. In certain embodiments, a peptide mimic described herein comprises 22 amino acids.
  • a peptide mimic described herein comprises a cysteine residue at each of the amino-terminal and carboxy-terminal ends.
  • the cysteine residue at the amino-terminus and the cysteine residue at the carboxy terminus bind together, yielding a ring.
  • a cyclical peptide mimic is more stable than a non-cyclical peptide mimic.
  • peptide mimics described herein are prepared by any suitable manner (e.g., literature methods).
  • a peptide mimic described herein comprises an amino acid sequence that is at least 85% homologous (i.e., is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%), 96%), 97%), 98%), 99%, or 100% homologous, including any amount of homology between 85%o and 100%) to the amino acid sequence SEQ ID NO: 1, as indicated below:
  • XI is chosen from the group containing lysine, glutamine, arginine, histidine and asparagine, or an amino acid deletion;
  • X2 is chosen from the group containing glutamic acid, aspartic acid and glutamine, or an amino acid deletion;
  • X3 is chosen from the group containing glycine, serine and alanine
  • X4 is chosen from the group containing lysine, leucine and arginine;
  • X5 is chosen from the group containing serine, cysteine, glycine and threonine;
  • X6 is chosen from the group containing proline and alanine
  • X7 is chosen from the group containing asparagine and glutamine
  • X8 is chosen from the group containing proline, tyrosine and glycine
  • X9 is chosen from the group containing glycine, alanine and serine
  • XI 0 is chosen from the group containing isoleucine, valine and asparagine;
  • XI 1 is chosen from the group containing valine, isoleucine and asparagine;
  • XI 2 is chosen from the group containing phenylalanine, tyrosine, isoleucine, valine, leucine and methionine;
  • XI 3 is chosen from the group containing isoleucine, valine, leucine, methionine and phenylalanine
  • XI 4 is chosen from the group containing threonine, glycine, alanine, serine and tyrosine;
  • XI 5 is chosen from the group containing arginine, lysine, alanine, glutamine, histidine and asparagine, or an amino acid deletion.
  • a peptide mimic described herein is derived from a human RANTES amino acid sequence. In some embodiments, a peptide mimic described herein comprises a portion of a human RANTES amino acid sequence. In some embodiments, a peptide mimic described herein comprises an amino acid sequence that is at least 85% homologous (i.e., is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homologous, including any amount of homology between 85% and 100%) to the amino acid sequence SEQ ID NO: 2, as indicated below:
  • a peptide mimic described herein is derived from a mouse RANTES amino acid sequence. In some embodiments, a peptide mimic described herein comprises a portion of a mouse RANTES amino acid sequence. In some embodiments, a peptide mimic described herein comprises an amino acid sequence that is at least 85% homologous (i.e., is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homologous, including any amount of homology between 85% and 100%) to the amino acid sequence SEQ ID NO: 3, as indicated below:
  • a peptide mimic described herein is derived from a pig RANTES amino acid sequence.
  • a peptide mimic described herein comprises a portion of a pig RANTES amino acid sequence.
  • a peptide mimic described herein comprises an amino acid sequence that is at least 85% homologous (i.e., is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homologous, including any amount of homology between 85% and 100%) to the amino acid sequence SEQ ID NO: 4, as indicated below:
  • a peptide mimic described herein comprises an amino acid sequence that is at least 85% homologous (i.e., is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%), 98%o, 99%, or 100% homologous, including any amount of homology between 85%o and 100%) to the amino acid sequence SEQ ID NO. 5, as indicated below:
  • XI is chosen from the group containing lysine, glutamine, arginine, histidine and asparagine, or an amino acid deletion;
  • X2 is chosen from the group containing glutamic acid, aspartic acid and glutamine, or an amino acid deletion;
  • X3 is chosen from the group containing glycine, serine and alanine
  • X4 is chosen from the group containing lysine, leucine and arginine;
  • X5 is chosen from the group containing serine, cysteine, glycine and threonine;
  • X6 is chosen from the group containing proline and alanine
  • X7 is chosen from the group containing asparagine and glutamine
  • X8 is chosen from the group containing proline, tyrosine and glycine
  • X9 is chosen from the group containing glycine, alanine and serine;
  • XI 0 is chosen from the group containing isoleucine, valine and asparagine;
  • XI I is chosen from the group containing valine, isoleucine and asparagine;
  • XI 2 is chosen from the group containing phenylalanine, tyrosine, isoleucine, valine, leucine and methionine;
  • XI 3 is chosen from the group containing isoleucine, valine, leucine, methionine and phenylalanine
  • XI 4 is chosen from the group containing threonine, glycine, alanine, serine and tyrosine
  • XI 5 is chosen from the group containing arginine, lysine, alanine, glutamine, histidine and asparagine, or an amino acid deletion.
  • a peptide mimic described herein comprises an amino acid sequence that is at least 85% homologous (i.e., is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%), 96%), 97%), 98%), 99%, or 100% homologous, including any amount of homology between 85%) and 100%) to the amino acid sequence SEQ ID NO: 6, as indicated below:
  • XI is chosen from the group containing lysine, glutamine, arginine, histidine and asparagine, or an amino acid deletion;
  • X2 is chosen from the group containing glutaminic acid, asparaginic acid and glutamine, or an amino acid deletion
  • X3 is chosen from the group containing glycine, serine and alanine
  • X4 is chosen from the group containing lysine, leucine and arginine;
  • X5 is chosen from the group containing serine, cysteine, glycine and threonine;
  • X6 is chosen from the group containing serine, glycine and threonine
  • X7 is chosen from the group containing methionine, isoleucine, leucine, and phenylalanine
  • X8 is chosen from the group containing proline, tyrosine and glycine
  • X9 is chosen from the group containing glycine, alanine and serine;
  • XI 0 is chosen from the group containing isoleucine, valine and asparagine;
  • XI 1 is chosen from the group containing valine, isoleucine and asparagine;
  • XI 2 is chosen from the group containing phenylalanine, tyrosine, isoleucine, valine, leucine and methionine;
  • XI 3 is chosen from the group containing isoleucine, valine, leucine, methionine and phenylalanine
  • XI 4 is chosen from the group containing threonine, glycine, alanine, serine and tyrosine
  • XI 5 is chosen from the group containing arginine, alanine, lysine, glutamine, histidine and asparagine, or an amino acid deletion.
  • a peptide mimic described herein comprises an amino acid sequence that is at least 85% homologous (i.e., is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%o, 97%), 98%), 99%, or 100% homologous, including any amount of homology between 85%) and 100%) to the amino acid sequence SEQ ID NO: 7, as indicated below:
  • XI is chosen from the ; group containing ; lysine, glutamine, arginine, histidine and asparagine, or an amino acid deletion;
  • X2 is chosen from the ; group containing ; glutaminic acid, asparaginic acid and glutamine, or an amino acid deletion;
  • X3 is chosen from the ; group containing ; glycine, serine and alanine;
  • X4 is chosen from the ; group containing ; lysine, leucine and arginine;
  • X5 is chosen from the ; group containing ; serine, cysteine, glycine and threonine;
  • X6 is chosen from the ; group containing ; serine, glycine and threonine;
  • X7 is chosen from the ; group containing ; asparagine and glutamine;
  • X8 is chosen from the ; group containing ; leucine, isoleucine, phenylalanine, alanine, valine, threonine and methionine;
  • X9 is chosen from the ; group containing ; glycine, alanine and serine;
  • XI 0 is chosen from the group containing isoleucine, valine and asparagine;
  • XI 1 is chosen from the group containing valine, isoleucine and asparagine;
  • XI 2 is chosen from the group containing phenylalanine, tyrosine, isoleucine, valine, leucine and methionine;
  • XI 3 is chosen from the group containing isoleucine, valine, leucine, methionine and phenylalanine
  • XI 4 is chosen from the group containing threonine, glycine, alanine, serine and tyrosine
  • XI 5 is chosen from the group containing arginine, alanine, lysine, glutamine, histidine and asparagine, or an amino acid deletion.
  • a peptide mimic described herein comprises an amino acid sequence that is at least 85% homologous (i.e., is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%), 96%), 97%), 98%), 99%, or 100% homologous, including any amount of homology between 85%) and 100%) to the amino acid sequence SEQ ID NO: 8, as indicated below:
  • XI is chosen from the group containing lysine, glutamine, arginine, histidine and asparagine, or an amino acid deletion;
  • X2 is chosen from the group containing glutaminic acid, asparaginic acid and glutamine, or an amino acid deletion;
  • X3 is chosen from the group containing glycine, serine and alanine
  • X4 is chosen from the group containing lysine, leucine and arginine;
  • X5 is chosen from the group containing serine, cysteine, glycine and threonine;
  • X6 is chosen from the group containing proline and alanine
  • X7 is chosen from the group containing methionine, isoleucine, leucine, and phenylalanine;
  • X8 is chosen from the group containing proline, tyrosine and glycine
  • X9 is chosen from the group containing glycine, alanine and serine;
  • XI 0 is chosen from the group containing isoleucine, valine and asparagine;
  • XI I is chosen from the group containing valine, isoleucine and asparagine;
  • XI 2 is chosen from the group containing phenylalanine, tyrosine, isoleucine, valine, leucine and methionine;
  • XI 3 is chosen from the group containing isoleucine, valine, leucine, methionine and phenylalanine
  • XI 4 is chosen from the group containing threonine, glycine, alanine, serine and tyrosine
  • XI 5 is chosen from the group containing arginine, alanine, lysine, glutamine, histidine and asparagine, or an amino acid deletion.
  • a peptide mimic described herein comprises an amino acid sequence that is at least 85% homologous (i.e., is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%), 96%), 97%), 98%), 99%, or 100% homologous, including any amount of homology between 85%) and 100%) to the amino acid sequence SEQ ID NO: 9, as indicated below:
  • XI is chosen from the group containing lysine, glutamine, arginine, histidine and asparagine, or an amino acid deletion
  • X2 is chosen from the group containing glutaminic acid, asparaginic acid and glutamine, or an amino acid deletion
  • X3 is chosen from the group containing glycine, serine and alanine
  • X4 is chosen from the group containing lysine, leucine and arginine;
  • X5 is chosen from the group containing serine, cysteine, glycine and threonine;
  • X6 is chosen from the group containing serine, glycine and threonine
  • X7 is chosen from the group containing methionine, isoleucine, leucine, and phenylalanine;
  • X8 is chosen from the group containing leucine, isoleucine, phenylalanine, alanine, valine, threonine and methionine;
  • X9 is chosen from the group containing glycine, alanine and serine;
  • XI 0 is chosen from the group containing isoleucine, valine and asparagine;
  • XI 1 is chosen from the group containing valine, isoleucine and asparagine;
  • XI 2 is chosen from the group containing phenylalanine, tyrosine, isoleucine, valine, leucine and methionine;
  • XI 3 is chosen from the group containing isoleucine, valine, leucine, methionine and phenylalanine
  • XI 4 is chosen from the group containing threonine, glycine, alanine, serine and tyrosine
  • XI 5 is chosen from the group containing arginine, alanine, lysine, glutamine, histidine and asparagine, or an amino acid deletion.
  • a peptide mimic described herein comprises an amino acid sequence that is at least 85% homologous (i.e., is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%o, 97%), 98%), 99%, or 100% homologous, including any amount of homology between 85%) and 100%) to the amino acid sequence SEQ ID NO: 10, as indicated below:
  • XI is chosen from the ; group containing ; lysine, glutamine, arginine, histidine and asparagine, or an amino acid deletion;
  • X2 is chosen from the ; group containing ; glutaminic acid, asparaginic acid and glutamine, or an amino acid deletion;
  • X3 is chosen from the ; group containing ; glycine, serine and alanine;
  • X4 is chosen from the ; group containing ; lysine, leucine and arginine;
  • X5 is chosen from the ; group containing ; serine, cysteine, glycine and threonine;
  • X6 is chosen from the ; group containing ; proline and alanine;
  • X7 is chosen from the ; group containing ; asparagine and glutamine;
  • X8 is chosen from the ; group containing ; leucine, isoleucine, phenylalanine, alanine, valine, threonine and methionine;
  • X9 is chosen from the ; group containing ; glycine, alanine and serine; XI 0 is chosen from the group containing isoleucine, valine and asparagine;
  • XI 1 is chosen from the group containing valine, isoleucine and asparagine;
  • XI 2 is chosen from the group containing phenylalanine, tyrosine, isoleucine, valine, leucine and methionine;
  • XI 3 is chosen from the group containing isoleucine, valine, leucine, methionine and phenylalanine
  • XI 4 is chosen from the group containing threonine, glycine, alanine, serine and tyrosine
  • XI 5 is chosen from the group containing arginine, alanine, lysine, glutamine, histidine and asparagine, or an amino acid deletion.
  • a peptide mimic described herein comprises an amino acid sequence that is at least 85% homologous (i.e., is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%o, 97%), 98%), 99%, or 100% homologous, including any amount of homology between 85%) and 100%) to the amino acid sequence SEQ ID NO: 1 1 , as indicated below:
  • XI is chosen from the ; group containing ; lysine, glutamine, arginine, histidine and asparagine, or an amino acid deletion;
  • X2 is chosen from the ; group containing ; glutaminic acid, asparaginic acid and glutamine, or an amino acid deletion;
  • X3 is chosen from the ; group containing ; glycine, serine and alanine;
  • X4 is chosen from the ; group containing ; lysine, leucine and arginine;
  • X5 is chosen from the ; group containing ; serine, cysteine, glycine and threonine;
  • X6 is chosen from the ; group containing ; proline and alanine;
  • X7 is chosen from the ; group containing ; methionine, isoleucine, leucine, and phenylalanine;
  • X8 is chosen from the ; group containing ; leucine, isoleucine, phenylalanine, alanine, valine, threonine and methionine;
  • X9 is chosen from the ; group containing ; glycine, alanine and serine;
  • XI 0 is chosen from the group containing isoleucine, valine and asparagine;
  • XI 1 is chosen from the group containing valine, isoleucine and asparagine;
  • XI 2 is chosen from the group containing phenylalanine, tyrosine, isoleucine, valine, leucine and methionine;
  • XI 3 is chosen from the group containing isoleucine, valine, leucine, methionine and phenylalanine
  • XI 4 is chosen from the group containing threonine, glycine, alanine, serine and tyrosine
  • XI 5 is chosen from the group containing arginine, alanine, lysine, glutamine, histidine and asparagine, or an amino acid deletion.
  • a peptide mimic described herein comprises an amino acid sequence that is at least 85% homologous (i.e., is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%), 98%o, 99%, or 100% homologous, including any amount of homology between 85%o and 100%) to the amino acid sequence SEQ ID NO: 12, as indicated below:
  • XI is chosen from the group containing lysine, glutamine, arginine, histidine and/or asparagine, or an amino acid deletion;
  • X2 is chosen from the group containing glutaminic acid, asparaginic acid and/or glutamine, or an amino acid deletion;
  • X3 is chosen from the group containing glycine, serine and/or alanine
  • X4 is chosen from the group containing lysine, leucine and/or arginine;
  • X5 is chosen from the group containing serine, cysteine, glycine and/or threonine;
  • X6 is chosen from the group containing serine, glycine and/or threonine
  • X7 is chosen from the group containing asparagine and/or glutamine
  • X8 is chosen from the group containing proline, tyrosine and/or glycine
  • X9 is chosen from the group containing glycine, alanine and/or serine;
  • XI 0 is chosen from the group containing isoleucine, valine and/or asparagine;
  • XI I is chosen from the group containing valine, isoleucine and/or asparagine;
  • X12 is chosen from the group containing phenylalanine, tyrosine, isoleucine, valine, leucine and/or methionine;
  • XI 3 is chosen from the group containing isoleucine, valine, leucine, methionine and/or phenylalanine;
  • X14 is chosen from the group containing threonine, glycine, alanine, serine and/or tyrosine; and XI 5 is chosen from the group containing arginine, lysine, glutamine, histidine and/or asparagine, or an amino acid deletion.
  • a peptide mimic described herein comprises an amino acid sequence that is at least 85% homologous (i.e., is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%), 96%), 97%), 98%), 99%, or 100% homologous, including any amount of homology between 85%) and 100%) to the amino acid sequence SEQ ID NO: 13, as indicated below:
  • a peptide mimic described herein comprises an amino acid sequence that is at least 85% homologous (i.e., is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%), 96%), 97%), 98%), 99%, or 100% homologous, including any amount of homology between 85%) and 100%) to the amino acid sequence SEQ ID NO: 14, as indicated below:
  • a peptide mimic described herein comprises an amino acid sequence that is at least 85% homologous (i.e., is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%o, 97%o, 98%o, 99%, or 100% homologous, including any amount of homology between 85%o and 100%) to the amino acid sequence SEQ ID NO: 15, as indicated below:
  • a peptide mimic described herein comprises an amino acid sequence that is at least 85% homologous (i.e., is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%o, 96%o, 97%o, 98%o, 99%, or 100% homologous, including any amount of homology between 85%o and 100%) to the amino acid sequence SEQ ID NO: 16, as indicated below:
  • a peptide mimic described herein comprises an amino acid sequence that is at least 85% homologous (i.e., is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%o, 96%o, 97%o, 98%o, 99%, or 100% homologous, including any amount of homology between 85%o and 100%) to an amino acid sequence listed in Table 1.
  • Equus caballus QEYFYTSSKCSIPAWFVTR (SEQ ID NO. 35)
  • Loxodonta KEYFYTSGKCSMPAV (SEQ ID NO. 37)
  • Sorex araneus QDYFYTSSKCSMPAWFVTR (SEQ ID NO. 42)
  • a peptide mimic described herein comprises an amino acid sequence that is at least 85% homologous (i.e., is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%), 98%), 99%), or 100% homologous, including any amount of homology between 85% and 100%) to disclosed in Table 2.
  • MSVAAVFRGLRPSPE SEQ ID NO. 54
  • S SKS SNLA WFVTRCCKEYFYT SEQ ID NO. 54
  • ESDGDLSCVCVKTIS SEQ ID NO. 57
  • SSNLAWFVTRCCKEYFYTSSK SEQ ID NO. 57
  • VIKAGRHCAVPQLIA (SEQ ID NO. 59) NLA WFVTRCCKEYFYT S SKS S (SEQ ID NO. 59) NLA WFVTRCCKEYFYT S SKS S (SEQ ID NO. 59) NLA WFVTRCCKEYFYT S SKS S (SEQ ID NO. 59) NLA WFVTRCCKEYFYT S SKS S (SEQ ID NO. 59) NLA WFVTRCCKEYFYT S SKS S (SEQ ID NO. 59) NLA WFVTRCCKEYFYT S SKS S (SEQ ID NO. 59) NLA WFVTRCCKEYFYT S SKS S (SEQ ID NO. 59) NLA WFVTRCCKEYFYT S SKS S (SEQ ID NO. 59) NLA WFVTRCCKEYFYT S SKS S (SEQ ID NO. 59) NLA WFVTRCCKEYFY
  • LIATLKNGRKICLDR SEQ ID NO. 60
  • SFKGTTVYALSNVRSYSFVKCC SEQ ID NO. 60
  • ATALCAPASASPYSS (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO
  • SDTTPCCFAYIARPL (SEQ ID NO. 65) YSFVKCCSFKGTTVYALSNVRS (SEQ ID NO.
  • EYFYTSGKCSNPAW (SEQ ID NO. 67) FVKCCSFKGTTVYALSNVRSYS (SEQ ID NO. 67)
  • a peptide mimic described herein comprises an amino acid sequence that is at least 85% homologous (i.e., is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%), 96%), 97%), 98%), 99%, or 100% homologous, including any amount of homology between 85% and 100%) to an amino acid sequence selected from: SSKSSNLA WFVTRCCKEYFYT (SEQ ID NO. 91); SKS SNLAVVFVTRCCKEYFYTS (SEQ ID NO. 92);
  • KSSNLAWFVTRCCKEYFYTSS (SEQ ID NO. 93); SSNLAWFVTRCCKEYFYTSSK (SEQ ID NO. 94); SNLAWFVTRCCKEYFYTSSKS (SEQ ID NO. 95); NLAWFVTRCCKEYFYTSSKSS (SEQ ID NO. 96); SFKGTTVYALSNVRSYSFVKCC (SEQ ID NO. 97);
  • SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 101); YSFVKCCSFKGTTVYALSNVRS (SEQ ID NO. 102); SFVKCCSFKGTTVYALSNVRSY (SEQ ID NO. 103); or
  • FVKCCSFKGTTVYALSNVRSYS (SEQ ID NO. 104).
  • a peptide that (a) mimics the RANTES binding domain of PF4, and (b) inhibits the formation of a PF4 and RANTES heterodimer.
  • the peptide comprises an amino acid that is 85% homologous (i.e., is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homologous, including any amount of homology between 85% and 100%) to an amino acid sequence selected from:
  • ATALCAPASASPYSS (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO
  • SDTTPCCFAYIARPL (SEQ ID NO. 65) YSFVKCCSFKGTTVYALSNVRS (SEQ ID NO.
  • EYFYTSGKCSNPAW (SEQ ID NO. 67) FVKCCSFKGTTVYALSNVRSYS (SEQ ID NO. 67)
  • the peptide comprises an amino acid sequence that is at least 85% homologous (i.e., is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%o, 99%, or 100% homologous, including any amount of homology between 85% and 100%) to the sequence: CKEYFYTSGAAAAPGIVFITRC (SEQ ID NO: 91).
  • the peptide comprises N- and/or C-terminal chemical modifications to improve ADME-PK; non-natural amino acids.
  • the peptide is a cyclical variant.
  • the agent that inhibits the interaction of PF4/RANTES interaction is a fragment of any peptide sequence disclosed herein (hereinafter, "peptide fragment").
  • peptide fragment means an amino acid polymer produced by cleaving any peptide of comprising an amino acid sequence that is at least 85% homologous to the sequences SEQ ID NO. 1 through SEQ ID NO. 90.
  • a peptide comprising an amino acid sequence that is at least 85% homologous to any of SEQ ID NO. 1 through SEQ ID NO. 90 is cleaved at one site (e.g., one peptide bond is broken).
  • a peptide comprising an amino acid sequence that is at least 85% homologous to any of SEQ ID NO. 1 through SEQ ID NO. 90 is cleaved at two sites (e.g., two peptide bonds are broken).
  • the peptide fragment is produced by the metabolism of a peptide comprising an amino acid sequence that is at least 85% homologous to any of SEQ ID NO. 1 through SEQ ID NO. 90. [00193]
  • the fragment has structural features similar to a peptide disclosed herein.
  • the fragment is linear.
  • the fragment comprises between 5 and 10 amino acids. In some embodiments, the fragment comprises 5 amino acids. In some embodiments, the fragment comprises between 6 and 10 amino acids. In some embodiments, the fragment comprises 6 amino acids. In some embodiments, the fragment comprises between 7 and 10 amino acids. In some embodiments, the fragment comprises between 8 and 10 amino acids. In some embodiments, the fragment comprises between 9 and 10 amino acids.
  • the metabolite comprises a formula selected from:
  • XI is selected from serine and lysine
  • X2 is selected from glutamic acid, phenylalanine and serine;
  • X3 is selected from lysine and tyrosine
  • X4 is selected from phenylalanine and glycine
  • X5 is selected from threonine and tyrosine
  • X6 is selected from serine and valine
  • X7 is selected from serine and tyrosine
  • X8 is selected from alanine and lysine
  • X9 is selected from leucine and serine
  • XI 0 is selected from leucine and valine
  • XI I is selected from alanine and arginine
  • XI 2 is selected from serine and valine
  • XI 3 is selected from valine and tyrosine
  • XI 4 is selected from phenylalanine and serine
  • XI 5 is selected from phenylalanine and valine.
  • XI 6 is selected from threonine and valine.
  • any of the aforementioned peptides and/or peptide fragments is used as a "model" to do structure-activity relationship (SAR) chemistry.
  • the SAR chemistry yields smaller peptides.
  • the smaller peptides yield small molecules that disrupt the activity of RANTES and/or PF4 (e.g., by figuring out the amino acid residues involved in disrupting the activity of RANTES and/or PF4).
  • a peptide mimetic is used in place of the peptides described herein, including for use in the treatment or prevention of the diseases disclosed herein.
  • such peptide mimetics have greater chemical stability, enhanced pharmacological properties (half-life, absorption, potency, efficacy, etc.), altered specificity (e.g., a broad-spectrum of biological activities), reduced antigenicity, and are more economically prepared.
  • peptide mimetics include covalent attachment of one or more labels or conjugates, directly or through a spacer (e.g., an amide group), to non-interfering positions(s) on the analog that are predicted by quantitative structure-activity data and/or molecular modeling. Such non-interfering positions generally are positions that do not form direct contacts with the receptor(s) to which the peptide mimetic binds to produce the therapeutic effect.
  • a peptide mimetic is generated by use of phage display peptide libraries.
  • phage display peptide libraries For disclosure regarding the creation of a phage display peptide library see Scott, J. K. et al. (1990) Science 249:386; Devlin, J. J. et al. (1990) Science 249:404; US5,223,409, US5,733,731 ; US5,498,530; US5,432,018;US5,338,665;US5,922,545; WO 96/40987and WO 98/15833 each of which is incorporated by reference for such disclosure.
  • random peptide sequences are displayed by fusion with coat proteins of filamentous phage.
  • the displayed peptides are affinity-eluted against an antibody-immobilized extracellular domain (in this case PF4 or RANTES).
  • peptide mimetics are isolated by biopanning.
  • whole cells expressing PF4 or RANTES are used to screen the library utilizing FACs to isolate phage bound cells.
  • the retained phages are enriched by successive rounds of biopanning and repropagation.
  • the best binding peptides are sequenced to identify key residues within one or more structurally related families of peptides.
  • the peptide sequences also suggest which residues to replace by alanine scanning or by mutagenesis at the DNA level.
  • mutagenesis libraries are created and screened to further optimize the sequence of the best binders.
  • structural analysis of protein-protein interaction is used to suggest peptides that mimic the binding activity of the peptides described herein.
  • the crystal structure resulting from such an analysis suggests the identity and relative orientation of critical residues of the peptide, from which a peptide is designed.
  • compositions for modulating an inflammatory disorder comprising co-administering (a) a therapeutically-effective amount of a first active agent that targets PF4 and/or RANTES, and (b) a therapeutically-effective amount of a second active agent, wherein the second active agent is selected from (i) an agent that treats an inflammatory disorder through an alternative pathway (i.e., the "second anti-inflammatory agent"), or (ii) an agent that treats a cardiovascular disorder.
  • combining (a) the first active agent; and (b) the second active agent is synergistic and results in a more efficacious therapy.
  • the therapy is more efficacious as it treats inflammatory disorders by multiple pathways.
  • the therapy is more efficacious as it treats inflammatory disorders by multiple pathways and treats and/or ameliorates undesired inflammation resulting from the second agent.
  • the therapy is more efficacious as it allows (partially or fully) a medical professional to increase the prescribed dosage of the second active agent.
  • administering causes (either partially or fully)
  • administration of sulfasalazine results in (either partially or fully) pneumonitis with or without eosinophilia, vasculitis, pericarditis with or without tamponade, hepatitis, allergic myocarditis, pancreatitis, nephritis, exfoliative dermatitis, serum vasculitis, and/or pleuritis.
  • administration of mesalamine results in (either partially or fully) pericarditis, myocarditis, pancreatitis, hepatitis, interstitial pneumonitis, pleuritis, interstitial nephritis, and/or pneumonitis.
  • administration of olsalazine results in (either partially or fully) myocarditis, pericarditis, pancreatitis, interstitial nephritis and/or pneumonitis.
  • the first active agent and a 5-ASA treat an inflammatory disorder by (1) decreasing the chemotaxis of leukocytes, and (2) reducing the synthesis of eicosanoids and inflammatory cytokines.
  • the first active agent also decreases any undesired inflammation (e.g., pancreatitis) resulting from administration of the 5-ASA.
  • the first active agent and an anti-TNF agent treat an inflammatory disorder by (1) decreasing the chemotaxis of leukocytes, and (2) suppressing a TNF-induced cytokine cascade.
  • the first active agent also decreases any undesired inflammation (e.g., tuberculosis) resulting from administration of the anti-TNF agent.
  • the first active and a leukotriene inhibitor treat an inflammatory disorder by (1) decreasing the chemotaxis of leukocytes, and (2) antagonizing LTA4, LTB4, LTC4, LTD4, LTE4, LTF4, LTA4R, LTB4R, LTB4R1, LTB4R2, LTC4R, LTD4R, LTE4R, CYSLTRl, or CYSLTR2; or inhibiting the synthesis of a leukotriene via 5- LO, FLAP, LTA4H, LTA4S, or LTC4S.
  • the first active agent also decreases any undesired inflammation (e.g., tuberculosis) resulting from administration of the leukotriene inhibitor.
  • the first active agent and an IL-1 receptor antagonist treat an inflammatory disorder by (1) decreasing the chemotaxis of leukocytes, and (2) blocking the stimulation of T cell IL-1 receptor.
  • the first active agent also decreases any undesired inflammation (e.g., pneumonia, and bone and joint infections) resulting from
  • the first active agent and an IL-2 receptor antagonist treat an inflammatory disorder by (1) decreasing the chemotaxis of leukocytes, and (2) blocking the stimulation of T cell IL-2 receptor.
  • the first active agent also decreases any undesired inflammation (e.g., gastrointestinal disorders) resulting from administration of the IL-2 receptor antagonist.
  • the first active agent and a cytotoxic agent treat an inflammatory disorder by (1) decreasing the chemotaxis of leukocytes, and (2) treating neoplastic disease.
  • the first active agent also decreases any undesired inflammation (e.g., neutropenia) resulting from administration of the cytotoxic agent.
  • the first active agent and an immunomodulatory agent treat an inflammatory disorder by (1) decreasing the chemotaxis of leukocytes, and (2) enhancing, or suppressing the immune system.
  • the first active agent also decreases any undesired inflammation (e.g., hematologic side effects) resulting from administration of the immunomodulatory agent.
  • the first active agent and an antibiotic treat an inflammatory disorder by (1) decreasing the chemotaxis of leukocytes, and (2) by blocking cell and/or microbial growth by disrupting the cell cycle, or by blocking histone deacetylase.
  • the first active agent also decreases any undesired inflammation (e.g., cardiotoxicity) resulting from administration of the antibiotic.
  • the first active agent and a T-cell co-stimulatory blocker treat an inflammatory disorder by (1) decreasing the chemotaxis of leukocytes, and (2) modulating a co- stimulatory signal which is required for full T-cell activation.
  • the first active agent also decreases any undesired inflammation (e.g., neutropenia) resulting from administration of the T-cell co-stimulatory blocker.
  • the first active agent and a B cell depleting agent treat an inflammatory disorder by (1) decreasing the chemotaxis of leukocytes, and (2) inhibiting B-cell activity.
  • the first active agent also decreases any undesired inflammation (e.g., Progressive Multifocal Leukoencephalopathy) resulting from administration of the B-cell depleting agent.
  • the first active agent and an immunosuppressive agent treat an inflammatory disorder by (1) decreasing the chemotaxis of leukocytes, and (2) selectively or non- selectively inhibiting or preventing activity of the immune system.
  • the first active agent also decreases any undesired inflammation (e.g., lymphoma) resulting from
  • the first active agent and an alkylating agent treat an inflammatory disorder by (1) decreasing the chemotaxis of leukocytes, and (2) inducing covalent binding of alkyl groups to cellular molecules.
  • the first active agent also decreases any undesired inflammation (e.g., immune suppression) resulting from administration of the alkylating agent.
  • the first active agent and an anti-metabolite treat an inflammatory disorder by (1) decreasing the chemotaxis of leukocytes, and (2) preventing the biosynthesis or use of normal cellular metabolites.
  • the first active agent also decreases any undesired inflammation (e.g., mutagenesis) resulting from administration of the anti-metabolite.
  • the first active agent and a plant alkaloid treat an inflammatory disorder by (1) decreasing the chemotaxis of leukocytes, and (2) interfering with normal microtubule breakdown during cell division.
  • the first active agent also decreases any undesired inflammation (e.g., leukopenia) resulting from administration of the plant alkaloid.
  • the first active agent and a terpenoid treat an inflammatory disorder by (1) decreasing the chemotaxis of leukocytes, and (2) treating neoplastic disease or microbial infections. In some embodiments, the first active agent also decreases any undesired inflammation resulting from administration of the terpenoid agent.
  • the first active agent and a topoisomerase inhibitor treat an inflammatory disorder by (1) decreasing the chemotaxis of leukocytes, and (2) modulating the action of cellular topoisomerase enzymes.
  • the first active agent also decreases any undesired inflammation (e.g., gastrointestinal effects) resulting from administration of the topoisomerase inhibitor.
  • the first active agent and an antibody treat an inflammatory disorder by (1) decreasing the chemotaxis of leukocytes, and (2) neutralizing inflammatory cytokines such as, for example, TNF alpha.
  • the first active agent also decreases any undesired inflammation (e.g., tuberculosis) resulting from administration of the antibody.
  • the first active agent and a hormonal therapy treat an inflammatory disorder by (1) decreasing the chemotaxis of leukocytes, and (2) suppressing cytokine release.
  • the first active agent also decreases any undesired inflammation (e.g., cancer) resulting from administration of the hormone.
  • the first active agent and an anti-diabetes therapy treat an inflammatory disorder by (1) decreasing the chemotaxis of leukocytes, and (2) improving sensitivity to insulin in muscle and adipose tissue.
  • the first active agent also decreases any undesired inflammation (e.g., liver inflammation, pancreatitis) resulting from administration of the anti-diabetes agent.
  • the first active agent and a statin synergistically treat a CVD by (1) decreasing the chemotaxis of leukocytes, (2) decreasing the synthesis of cholesterol, and (3) decreasing any undesired inflammation resulting from administration of the statin.
  • statins induce inflammation.
  • administration of a statin results (partially or fully) in myositis.
  • statin-induced myositis is dose-dependent.
  • prescribing the first active agent allows (partially or fully) a medical professional to increase the prescribed dosage of statin.
  • the first active agent and a fibrate synergistically treat a CVD by (1) decreasing the chemotaxis of leukocytes, and (2) increasing the concentration of HDL. In some embodiments, the first active agent also decreases any undesired inflammation resulting from administration of the fibrate.
  • the first active agent and an ApoAl modulator synergistically treat a CVD by (1) decreasing the chemotaxis of leukocytes, and (2) increasing the concentration of HDL. In some embodiments, the first active agent also decreases any undesired inflammation resulting from administration of the ApoAl modulator.
  • the first active agent and an ACAT modulator synergistically treat a CVD by (1) decreasing the chemotaxis of leukocytes, and (2) decreasing (a) the production and release of apoB-containing lipoproteins and (b) foam cell formation.
  • the first active agent also decreases any undesired inflammation resulting from administration of the ACAT inhibitor.
  • the first active agent and a CETP modulator synergistically treat a CVD by (1) decreasing the chemotaxis of leukocytes, and (2) decreasing the transfer cholesterol from HDL cholesterol to LDL. In some embodiments, the first active agent also decreases any undesired inflammation resulting from administration of the CETP inhibitor.
  • the first active agent and a GP Ilb/IIIa receptor antagonist synergistically treat a CVD by (1) decreasing the chemotaxis of leukocytes, and (2) inhibiting platelet aggregation. In some embodiments, the first active agent also decreases any undesired inflammation resulting from administration of the GP Ilb/IIIa receptor antagonist.
  • the first active agent and the P2Y12 receptor antagonist are identical to the first active agent and the P2Y12 receptor antagonist
  • the first active agent also decreases any undesired inflammation resulting from administration of the P2Y12 receptor antagonist.
  • the first active agent and an Lp-PLA2 antagonist synergistically treat a CVD by (1) decreasing the chemotaxis of leukocytes, and (2) inhibiting the formation of biologically active products from oxidized LDL.
  • the first active agent also decreases any undesired inflammation resulting from administration of the Lp-PLA2 antagonist.
  • the first active agent and a leukotriene antagonist synergistically treat a CVD by (1) decreasing the chemotaxis of leukocytes and (2) inhibiting the adhesion and activation of leukocytes on the endothelium.
  • the first active agent also decreases any undesired inflammation resulting from administration of the leukotriene antagonist.
  • the second anti-inflammatory agent is: cyclosporine A, alefacept, efalizumab, methotrexate, acitretin, isotretinoin, hydroxyurea, mycophenolate mofetil (MMF), sulfasalazine, 6-Thioguanine, Dovonex, Taclonex, betamethasone, tazarotene, hydroxychloroquine, etanercept, adalimumab, infliximab, abatacept, rituximab, trastuzumab, Anti-CD45 monoclonal antibody AHN- 12 (NCI), Iodine-131 Anti-Bl Antibody (Corixa Corp.), anti-CD66 monoclonal antibody BW 250/183 (NCI,shire General Hospital), anti-CD45 monoclonal antibody (NCI, Baylor College of Medicine), antibody anti-anb3 integrin (NCI),
  • cyclophosphamide leflunomide, d-penicillamine, amitriptyline, or nortriptyline, chlorambucil, nitrogen mustard, prasterone, LJP 394 (abetimus sodium), LJP 1082 (La Jolla Pharmaceutical), eculizumab, belibumab, rhuCD40L (NIAID), epratuzumab, sirolimus, tacrolimus, pimecrolimus, thalidomide, antithymocyte globulin-equine (Atgam, Pharmacia Upjohn), antithymocyte globulin- rabbit (Thymoglobulin, Genzyme), Muromonab-CD3 (FDA Office of Orphan Products
  • Glucotrol® (Glipizide), Glynasec (glyburide), Micronase® (glyburide), Orinase® (Tolbutamide), Tolinase® (Tolazamide), Glucophage, Riomet® (Metformin), Glucovance® (glyburide + metformin), Avandamet® (Rosiglitazone + metformin), Avandaryl® (Rosiglitazone+glimepiride), Byetta® (Exenatide), Insulins, Januvia® (Sitagliptin), Metaglip® (glipizide and metformin), Prandin® (Repaglinide), Precose® (Acarbose), Starlix® (Nateglinide), Xenical® (Orlistat), cisplatin; carboplatin; oxaliplatin; mechlorethamine; cyclophosphamide; chlorambucil; vincristine; vinblastine; vin
  • cladribine 5-fluorouracil
  • FUDR floxuridine
  • cytosine arabinoside methotrexate
  • trimethoprim pyrimethamine; pemetrexed; paclitaxel; docetaxel; etoposide; teniposide; irinotecan; topotecan; amsacrine; etoposide; etoposide phosphate; teniposide; dactinomycin; doxorubicin; daunorubicin; valrubicine; idarubicine; epirubicin; bleomycin; plicamycin; mitomycin; trastuzumab; cetuximab; rituximab; bevacizumab; finasteride; goserelin; aminoglutethimide; anastrozole;
  • letrozole vorozole; exemestane; 4-androstene-3,6,17-trione ("6-OXO"; l,4,6-androstatrien-3,17- dione (ATD); formestane; testolactone; fadrozole; A-81834 (3-(3-(l,l-dimethylethylthio-5- (quinoline-2- ylmethoxy)- 1 -(4-chloromethylphenyl)indole-2-yl)-2,2-dimethylpropionaldehyde oxime-O-2-acetic acid; AM103 (Amira); AM803 (Amira); atreleuton; BAY-x-1005 ((R)-(+)-alpha- cyclopentyl-4-(2-quinolinylmethoxy)-Benzeneacetic acid); CJ-13610 (4-(3-(4-(2-Methyl-imidazol-l- yl)-phen
  • WY-47,288 (2-[(l-naphthalenyloxy)methyl]quinoline); zileuton; ZD-2138 (6-((3- fluoro-5- (tetrahydro-4-methoxy-2H-pyran-4yl)phenoxy)methyl)-l-methyl-2(lH)-quinlolinone); busulphan; alemtuzumab; belatacept (LEA29Y); posaconazole; fingolimod (FTY720); an anti-CD40 ligand antibody (e.g., BG 9588); CTLA4Ig (BMS 188667); abetimus (UP 394); an anti-ILlO antibody; an anti-CD20 antibody (e.g. rituximab); an anti-C5 antibody (e.g., eculizumab); or combinations thereof.
  • ZD-2138 (6-((3- fluoro-5- (tetrahydro-4-methoxy-2H-pyran-4y
  • the second active agent is selected from: a niacin; a fibrate; a statin; an Apolipoprotein A-l modulator; an AC AT modulator; a CETP modulator; a glycoprotein Ilb/IIIa modulator; a P2Y12 modulator; an Lp-PLA2 modulator; or combinations thereof.
  • the second active agent is a statin.
  • the second active agent is atorvastatin; cerivastatin; fluvastatin; lovastatin; mevastatin; pitavastatin; pravastatin; rosuvastatin; simvastatin; simvastatin and ezetimibe; lovastatin and niacin, extended-release;

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Abstract

Disclosed herein, in certain embodiments, are peptides for use in inhibiting the interactions of PF4 and RANTES. Further disclosed herein, are methods for treating an inflammatory disease, disorder, condition, or symptom. In some embodiments, the method comprises co-administering an agent that inhibits the interactions of PF4 and RANTES and a second active agent.

Description

METHODS OF TREATING INFLAMMATION
CROSS-REFERENCE
[0001] This application claims priority to US Provisional Application 61/315,754, filed March 19, 2010; US Provisional Application 61/321,384, filed April 6, 2010; and to US Provisional
Application 61/345,511, filed May 17, 2010; all of which are incorporated herein by reference in their entirety.
BACKGROUND OF THE INVENTION
[0002] Inflammatory diseases, disorders, conditions and symptoms are characterized, in part, by the migration of lymphocytes and monocytes into the affected tissue. The migration of lymphocytes and monocytes induces tissue damage and exacerbates inflammatory diseases, disorders, conditions and symptoms.
[0003] RANTES (also known as CCL5) and PF4 are pro-inflammatory chemokines. In certain instances, they are secreted by an activated platelet in response to an inflammation or tissue injury. In certain instances, RANTES and PF4 induce chemotaxis in nearby leukocytes (e.g. monocytes) along their gradients.
SUMMARY OF THE INVENTION
[0004] There is a need for new methods of treating inflammatory diseases, disorders, conditions (e.g., atherosclerosis) and symptoms that do not interfere with (a) non-inflammatory processes or (b) desired-inflammatory processes. The inventors have discovered that undesired and harmful inflammation can be treated by inhibiting the interactions of PF4 and RANTES. Further, the inventors have discovered that targeting precise regions of PF4 and RANTES will inhibit the ability of the ligands to bind to each other and their receptors (thus, preventing undesired inflammation) without affecting other (e.g., desired and beneficial) interactions of PF4 and RANTES.
[0005] Disclosed herein, in certain embodiments, is a peptide that (a) mimics the RANTES binding domain of PF4 and (b) inhibits the formation of a PF4 and RANTES heterodimer. In some embodiments, the peptide comprises (a) 15-25 amino acids and (b) an amino acid sequence that is 85% homologous to an amino acid sequence selected from:
Figure imgf000002_0001
PRHITSLEVIKAGPHC (SEQ ID NO. 51) SSKCSNLAWFVTRR (SEQ ID NO. 74)
PTAQLIATLKNGRKIC (SEQ ID NO. 52) RRNRQVCANPEKKWV (SEQ ID NO. 75)
LDLQAPLYKKIIKKLLES (SEQ ID NO. EKKWVQEYINYLEMS (SEQ ID NO. 76) 53)
MSVAAVFRGLRPSPE (SEQ ID NO. 54) S SKS SNLA WFVTRCCKEYFYT (SEQ ID
NO. 77)
SPELLLLGLLFLPAV (SEQ ID NO. 55) SKS SNLA WFVTRCCKEYFYTS (SEQ ID
NO. 78)
PAVVAVTSAGPEESD (SEQ ID NO. 56) KSSNLAWFVTRCCKEYFYTSS (SEQ ID
NO. 79);
ESDGDLSCVCVKTIS (SEQ ID NO. 57) S SNLA WFVTRCCKEYFYT S SK (SEQ ID
NO. 80);
TISSGIHLKHITSLE (SEQ ID NO. 58) SNLA WFVTRCCKEYFYTS SKS (SEQ ID
NO. 81);
VIKAGRHCAVPQLIA (SEQ ID NO. 59) NLAVVFVTRCCKEYFYTSSKSS (SEQ ID
NO. 82);
LIATLKNGRKICLDR (SEQ ID NO. 60) SFKGTTVYALSNVRSYSFVKCC (SEQ
ID NO. 83);
LDRQAPLYKKVIKKI (SEQ ID NO. 61) FKGTTVYALSNVRSYSFVKCCS (SEQ
ID NO. 84);
QAPLYKKVIKKILES (SEQ ID NO. 62) SNVRSYSFVKCCSFKGTTVYAL (SEQ
ID NO. 85);
MKVSAAALAVILIAT (SEQ ID NO. 63) NVRSYSFVKCCSFKGTTVYALS (SEQ
ID NO. 86);
ATALCAPASASPYSS (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ
ID NO. 87);
SDTTPCCFAYIARPL (SEQ ID NO. 65) YSFVKCCSFKGTTVYALSNVRS (SEQ
ID NO. 88);
PLPRAHIKEYFYTSG (SEQ ID NO. 66) SFVKCCSFKGTTVYALSNVRSY (SEQ
ID NO. 89);
EYFYTSGKCSNPAW (SEQ ID NO. 67) FVKCCSFKGTTVYALSNVRSYS (SEQ
ID NO. 90)
VFVTRKNRQVCANPE (SEQ ID NO. 66)
In some embodiments, the peptide comprises an amino acid sequence is at least 90% homologous to an amino acid sequence selected from: MSSAAGFCASRP (SEQ ID NO. 46) EKKWVREYINSLEMS (SEQ ID NO. 69)
RPGLLFLGLLLLPL (SEQ ID NO. 47) MKISAAALTIILTAA (SEQ ID NO. 70)
PLWAFASAEA (SEQ ID NO. 48) AALCTPAPASPYGSD (SEQ ID NO. 71)
SAEAEEDGDLQC (SEQ ID NO. 49) SDTTPCCFAYLSLAL (SEQ ID NO. 72)
QCLCVKTTSQVRPRH (SEQ ID NO. 50) ALPRAHVKEYFYTSS (SEQ ID NO. 73)
PRHITSLEVIKAGPHC (SEQ ID NO. 51) SSKCSNLAWFVTRR (SEQ ID NO. 74)
PTAQLIATLKNGRKIC (SEQ ID NO. 52) RRNRQVCANPEKKWV (SEQ ID NO. 75)
LDLQAPLYKKIIKKLLES (SEQ ID NO. EKKWVQEYINYLEMS (SEQ ID NO. 76) 53)
MSVAAVFRGLRPSPE (SEQ ID NO. 54) S SKS SNLA WFVTRCCKEYFYT (SEQ ID
NO. 77)
SPELLLLGLLFLPAV (SEQ ID NO. 55) SKS SNLA WFVTRCCKEYFYTS (SEQ ID
NO. 78)
PAVVAVTSAGPEESD (SEQ ID NO. 56) KSSNLAWFVTRCCKEYFYTSS (SEQ ID
NO. 79);
ESDGDLSCVCVKTIS (SEQ ID NO. 57) SSNLAVVFVTRCCKEYFYTSSK (SEQ ID
NO. 80);
TISSGIHLKHITSLE (SEQ ID NO. 58) SNLAWFVTRCCKEYFYTSSKS (SEQ ID
NO. 81);
VIKAGRHCAVPQLIA (SEQ ID NO. 59) NLAVVFVTRCCKEYFYTSSKSS (SEQ ID
NO. 82);
LIATLKNGRKICLDR (SEQ ID NO. 60) SFKGTTVYALSNVRSYSFVKCC (SEQ
ID NO. 83);
LDRQAPLYKKVIKKI (SEQ ID NO. 61) FKGTTVYALSNVRSYSFVKCCS (SEQ
ID NO. 84);
QAPLYKKVIKKILES (SEQ ID NO. 62) SNVRSYSFVKCCSFKGTTVYAL (SEQ
ID NO. 85);
MKVSAAALAVILIAT (SEQ ID NO. 63) NVRSYSFVKCCSFKGTTVYALS (SEQ
ID NO. 86);
ATALCAPASASPYSS (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ
ID NO. 87);
SDTTPCCFAYIARPL (SEQ ID NO. 65) YSFVKCCSFKGTTVYALSNVRS (SEQ
ID NO. 88);
PLPRAHIKEYFYTSG (SEQ ID NO. 66) SFVKCCSFKGTTVYALSNVRSY (SEQ
ID NO. 89); EYFYTSGKCSNPAW (SEQ ID NO. 67) FVKCCSFKGTTVYALSNVRSYS (SEQ
ID NO. 90)
VFVTRKNRQVCANPE (SEQ ID NO. 66)
In some embodiments, the peptide comprises an amino acid sequence is at least 95% homologous to an amino acid sequence selected from:
MSSAAGFCASRP (SEQ ID NO. 46) EKKWVREYINSLEMS (SEQ ID NO. 69)
RPGLLFLGLLLLPL (SEQ ID NO. 47) MKISAAALTIILTAA (SEQ ID NO. 70)
PLWAFASAEA (SEQ ID NO. 48) AALCTPAPASPYGSD (SEQ ID NO. 71)
SAEAEEDGDLQC (SEQ ID NO. 49) SDTTPCCFAYLSLAL (SEQ ID NO. 72)
QCLCVKTTSQVRPRH (SEQ ID NO. 50) ALPRAHVKEYFYTSS (SEQ ID NO. 73)
PRHITSLEVIKAGPHC (SEQ ID NO. 51) SSKCSNLAWFVTRR (SEQ ID NO. 74)
PTAQLIATLKNGRKIC (SEQ ID NO. 52) RRNRQVCANPEKKWV (SEQ ID NO. 75)
LDLQAPLYKKIIKKLLES (SEQ ID NO. EKKWVQEYINYLEMS (SEQ ID NO. 76) 53)
MSVAAVFRGLRPSPE (SEQ ID NO. 54) S SKS SNLA WFVTRCCKEYFYT (SEQ ID
NO. 77)
SPELLLLGLLFLPAV (SEQ ID NO. 55) SKS SNLA WFVTRCCKEYFYTS (SEQ ID
NO. 78)
PAVVAVTSAGPEESD (SEQ ID NO. 56) KS SNLA WFVTRCCKEYFYT S S (SEQ ID
NO. 79);
ESDGDLSCVCVKTIS (SEQ ID NO. 57) S SNLA WFVTRCCKEYFYT S SK (SEQ ID
NO. 80);
TISSGIHLKHITSLE (SEQ ID NO. 58) SNLA WFVTRCCKEYFYTS SKS (SEQ ID
NO. 81);
VIKAGRHCAVPQLIA (SEQ ID NO. 59) NLAVVFVTRCCKEYFYTSSKSS (SEQ ID
NO. 82);
LIATLKNGRKICLDR (SEQ ID NO. 60) SFKGTTVYALSNVRSYSFVKCC (SEQ
ID NO. 83);
LDRQAPLYKKVIKKI (SEQ ID NO. 61) FKGTTVYALSNVRSYSFVKCCS (SEQ
ID NO. 84);
QAPLYKKVIKKILES (SEQ ID NO. 62) SNVRSYSFVKCCSFKGTTVYAL (SEQ
ID NO. 85);
MKVSAAALAVILIAT (SEQ ID NO. 63) NVRSYSFVKCCSFKGTTVYALS (SEQ
ID NO. 86);
ATALCAPASASPYSS (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 87);
SDTTPCCFAYIARPL (SEQ ID NO. 65) YSFVKCCSFKGTTVYALSNVRS (SEQ
ID NO. 88);
PLPRAHIKEYFYTSG (SEQ ID NO. 66) SFVKCCSFKGTTVYALSNVRSY (SEQ
ID NO. 89);
EYFYTSGKCSNPAW (SEQ ID NO. 67) FVKCCSFKGTTVYALSNVRSYS (SEQ
ID NO. 90)
VFVTRKNPvQVCANPE (SEQ ID NO. 66)
me embodiments, the peptide comprises an amino acid sequence selected from:
MSSAAGFCASRP (SEQ ID NO. 46) EKKWVREYINSLEMS (SEQ ID NO. 69)
RPGLLFLGLLLLPL (SEQ ID NO. 47) MKISAAALTIILTAA (SEQ ID NO. 70)
PLWAFASAEA (SEQ ID NO. 48) AALCTPAPASPYGSD (SEQ ID NO. 71)
SAEAEEDGDLQC (SEQ ID NO. 49) SDTTPCCFAYLSLAL (SEQ ID NO. 72)
QCLCVKTTSQVRPRH (SEQ ID NO. 50) ALPRAHVKEYFYTSS (SEQ ID NO. 73)
PRHITSLEVIKAGPHC (SEQ ID NO. 51) SSKCSNLAWFVTRR (SEQ ID NO. 74)
PTAQLIATLKNGRKIC (SEQ ID NO. 52) RRNRQVCANPEKKWV (SEQ ID NO. 75)
LDLQAPLYKKIIKKLLES (SEQ ID NO. EKKWVQEYINYLEMS (SEQ ID NO. 76) 53)
MSVAAVFRGLRPSPE (SEQ ID NO. 54) S SKS SNLA WFVTRCCKEYFYT (SEQ ID
NO. 77)
SPELLLLGLLFLPAV (SEQ ID NO. 55) SKS SNLA WFVTRCCKEYFYTS (SEQ ID
NO. 78)
PAVVAVTSAGPEESD (SEQ ID NO. 56) KSSNLAWFVTRCCKEYFYTSS (SEQ ID
NO. 79);
ESDGDLSCVCVKTIS (SEQ ID NO. 57) S SNLA WFVTRCCKEYFYT S SK (SEQ ID
NO. 80);
TISSGIHLKHITSLE (SEQ ID NO. 58) SNLA WFVTRCCKEYFYTS SKS (SEQ ID
NO. 81);
VIKAGRHCAVPQLIA (SEQ ID NO. 59) NLAVVFVTRCCKEYFYTSSKSS (SEQ ID
NO. 82);
LIATLKNGRKICLDR (SEQ ID NO. 60) SFKGTTVYALSNVRSYSFVKCC (SEQ
ID NO. 83);
LDRQAPLYKKVIKKI (SEQ ID NO. 61) FKGTTVYALSNVRSYSFVKCCS (SEQ
ID NO. 84);
QAPLYKKVIKKILES (SEQ ID NO. 62) SNVRSYSFVKCCSFKGTTVYAL (SEQ ID NO. 85);
MKVSAAALAVILIAT (SEQ ID NO. 63) NVRSYSFVKCCSFKGTTVYALS (SEQ
ID NO. 86);
ATALCAPASASPYSS (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ
ID NO. 87);
SDTTPCCFAYIARPL (SEQ ID NO. 65) YSFVKCCSFKGTTVYALSNVRS (SEQ
ID NO. 88);
PLPRAHIKEYFYTSG (SEQ ID NO. 66) SFVKCCSFKGTTVYALSNVRSY (SEQ
ID NO. 89);
EYFYTSGKCSNPAW (SEQ ID NO. 67) FVKCCSFKGTTVYALSNVRSYS (SEQ
ID NO. 90)
VFVTRKNRQVCANPE (SEQ ID NO. 66)
In some embodiments, the peptide comprises (a) N- and/or C-terminal chemical modifications to improve ADME-PK or (b) non-natural amino acids. In some embodiments, the peptide is a cyclical variant. In some embodiments, the peptide is used in the treatment of a disease characterized by inflammation mediated by the binding of PF4 and RANTES. In some embodiments, the peptide is used in the treatment of a disease characterized by inflammation mediated by the binding of PF4 and RANTES, wherein the disease is atherosclerosis, cystic fibrosis, AAA, RSV infection, emphysema, moderate asthma, severe asthma, or any combination thereof. In some embodiments, the use further comprises the co-administration of an active agent selected from: niacin, a fibrate, a statin, an Apo- Al mimetic peptide, an ApoA-I transcriptional up-regulator, an ACAT inhibitor, a CETP modulator, Glycoprotein (GP) Ilb/IIIa receptor antagonists, P2Y12 receptor antagonists, Lp-PLA2-inhibitors, an anti-TNF agent, an IL-1 receptor antagonist, an IL-2 receptor antagonist, a cytotoxic agent, an immunomodulatory agent, an antibiotic, a T-cell co-stimulatory blocker, a disorder-modifying antirheumatic agent, a B cell depleting agent, an immunosuppressive agent, an anti-lymphocyte antibody, an alkylating agent, an anti-metabolite, a plant alkaloid, a terpenoid, a topoisomerase inhibitor, an antitumor antibiotic, a monoclonal antibody, a hormone, or combinations thereof. In some embodiments, a pharmaceutical composition comprises any of the aforementioned peptides.
[0006] Disclosed herein, in certain embodiments, is a method of diagnosing an abdominal aortic aneurysm in an individual in need thereof, comprising (a) administering to the individual an agent that (i) specifically binds to all or a portion of PF4, RANTES, or a combination thereof, and (ii) is labeled with an imaging agent; (b) exposing the individual to an imaging device for a period of time sufficient to generate an image of the agent; and (c) diagnosing the individual with an abdominal aortic aneurysm if the image of the agent shows a concentration of the agent in the abdominal aorta that exceeds the concentration in a control. In some embodiments, the agent localizes to the site of an abdominal aortic aneurysm. In some embodiments, the agent is an antibody, a peptibody, a small molecule, or a peptide. In some embodiments, the agent is a peptide that (a) mimics all or a portion of PF4, or (b) all or a portion of RANTES. In some embodiments, the agent is a peptide that (a) mimics all or a portion of the RANTES binding domain of PF4, or (b) all or a portion of PF4 binding domain of RANTES. In some embodiments, the agent is a peptide that comprises an amino acid sequence that is at least 85% homologous to the sequence: CKEYFYTSSKSSNLAWFVTRC (SEQ ID NO: 13). In some embodiments, the agent is a peptide that comprises an amino acid sequence that is at least 85% homologous to the sequence: CKEYFYTSGKSSNPGTVFITRC (SEQ ID NO: 16). In some embodiments, the peptide is: CKEYFYTSSKSSNLAWFVTRC (SEQ ID NO: 13) or CKEYFYTSGKSSNPGTVFITRC (SEQ ID NO: 16). In some embodiments, the agent is an anti-PF4 antibody, an anti-RANTES antibody, or a combination thereof. In some embodiments, the agent is labeled for radio-imaging, PET imaging, MRI imaging, or fluorescent imaging. In some embodiments, the agent is imaged by use of a gamma camera, MRI, PET scanner, x-ray computed tomography (CT), functional magnetic resonance imaging (fMRI), or single photon emission computed tomography (SPECT). In some embodiments, the imaging agent is a radiolabel. In some embodiments, the imaging agent is a fluorescent label. In some embodiments, the imaging agent is magnetic, paramagnetic or superparamagnetic. In some embodiments, the imaging agent is 17F, 18F, 117I, U8I, U9I, 120I, 121I, 122I, 124I, 126I, 1281 131I, 10C, UC, 130, 140, 150, 74Br, 75Br, 76Br, 77Br, 78Br, 82Br, "'In, 225Ac, 13N, 82Rb, 32C1, 33C1, 34C1, 64Cu, monobromobimane (mBBr), dibromobimane, monobromotrimethylammoniobimane, indocyanine green acylthiazolidinethione (ICG-ATT), 7- amino -4 -methyl coumarin-3-acetic acid (AMCA), guanidinoethanethiol (GET), ferucarbotran, UC- dimethylamine, perfluoro-15-crown-5-ether (PFCE), perfluorooctylbromide (PFOB), Rhodamine 6G, Rhodamine B, Rhodamine 123, N-[2-(4-18F-fluorobenzamido)ethyl]maleimide, colloidal gold particles, colloidal silver particles, colloidal iron particles, colloidal gadolinium particles, iron dextran particles, gold dextran particles, silver dextran particles, or a combination thereof.
[0007] Disclosed herein, in certain embodiments, is a method of monitoring an abdominal aortic aneurysm in an individual in need thereof, comprising (a) administering to the individual an agent that (i) specifically binds to all or a portion of PF4, RANTES, or a combination thereof, and (ii) is labeled with an imaging agent; (b) exposing the individual to an imaging device for a period of time sufficient to generate an image of the agent; wherein (i) a decrease in the concentration of the agent in the abdominal aorta as compared to an earlier image of the agent indicates that the Abdominal Aortic Aneurysm (AAA) is regressing, (ii) an increase in the concentration of the agent in the abdominal aorta as compared to an earlier image of the agent indicates that the Abdominal Aortic Aneurysm (AAA) is progressing, or (iii) no change in the concentration of the agent in the abdominal aorta as compared to an earlier image of the agent indicates that the Abdominal Aortic Aneurysm (AAA) is neither regressing or progressing. In some embodiments, the agent localizes to the site of an abdominal aortic aneurysm. In some embodiments, the agent is an antibody, a peptibody, a small molecule, or a peptide. In some embodiments, the agent is a peptide that (a) mimics all or a portion of PF4, or (b) all or a portion of PvANTES. In some embodiments, the agent is a peptide that (a) mimics all or a portion of the PvANTES binding domain of PF4, or (b) all or a portion of PF4 binding domain of PvANTES. In some embodiments, the agent is a peptide that comprises an amino acid sequence that is at least 85% homologous to the sequence:
CKEYFYTSSKSSNLAVVFVTRC (SEQ ID NO: 13). In some embodiments, the agent is a peptide that comprises an amino acid sequence that is at least 85% homologous to the sequence:
CKEYFYTSGKSSNPGIVFITRC (SEQ ID NO: 16). In some embodiments, the peptide is:
CKEYFYTSSKSSNLAVVFVTRC (SEQ ID NO: 13) or CKEYFYTSGKSSNPGIVFITRC (SEQ ID NO: 16). In some embodiments, the agent is an anti-PF4 antibody, an anti-RANTES antibody, or a combination thereof. In some embodiments, the agent is labeled for radio-imaging, PET imaging, MRI imaging, or fluorescent imaging. In some embodiments, the agent is imaged by use of a gamma camera, MRI, PET scanner, x-ray computed tomography (CT), functional magnetic resonance imaging (fMRI), or single photon emission computed tomography (SPECT). In some embodiments, the imaging agent is a radiolabel. In some embodiments, the imaging agent is a fluorescent label. In some embodiments, the imaging agent is magnetic, paramagnetic or superparamagnetic. In some embodiments, the imaging agent is 17F, 18F, U7I, U8I, U9I, 120I, 121I, 122I, 124I, 126L 1281 131I, 10C, UC, 130, 140, 150, 74Br, 75Br, 76Br, 77Br, 78Br, 82Br, mIn, 225Ac, 13N, 82Rb, 32C1, 33C1, 34C1, 64Cu, monobromobimane (mBBr), dibromobimane, monobromotrimethylammoniobimane, indocyanine green acylthiazolidinethione (ICG- ATT), 7-amino-4-methyl coumarin-3 -acetic acid (AMCA), guanidinoethanethiol (GET), ferucarbotran, uC-dimethylamine, perfluoro-15-crown-5-ether (PFCE), perfluorooctylbromide (PFOB), Rhodamine 6G, Rhodamine B, Rhodamine 123, N-[2-(4- 18F-fluorobenzamido)ethyl]maleimide, colloidal gold particles, colloidal silver particles, colloidal iron particles, colloidal gadolinium particles, iron dextran particles, gold dextran particles, silver dextran particles, or a combination thereof.
[0008] Disclosed herein, in certain embodiments, is a stent comprising a peptide comprising an amino acid sequence that is at least 85%> homologous to the amino acid sequence
CKEYFYTSSKSSNLAVVFVTRC (SEQ ID NO: 13), or CKEYFYTSGKSSNPGIVFITRC (SEQ ID NO: 16). In some embodiments, the peptide is CKEYFYTSSKSSNLAVVFVTRC (SEQ ID NO: 13), or CKEYFYTSGKSSNPGIVFITRC (SEQ ID NO: 16).
[0009] Disclosed herein, in certain embodiments, is a method of treating RSV infection, comprising administering an agent that specifically binds to all or a portion of PF4, RANTES, or a combination thereof. In some embodiments, the agent is an antibody, a peptibody, a small molecule, or a peptide. In some embodiments, the agent (a) mimics all or a portion of PF4, or (b) all or a portion of RANTES. In some embodiments, the agent (a) mimics all or a portion of the RANTES binding domain of PF4, or (b) all or a portion of PF4 binding domain of RANTES. In some embodiments, the agent is a peptide comprising an amino acid sequence that is at least 85% homologous to the amino acid sequence: CKEYFYTSSKSSNLAWFVTRC (SEQ ID NO: 13). In some embodiments, the agent is a peptide comprising an amino acid sequence that is at least 85% homologous to the amino acid sequence: CKEYFYT SGKS SNPGIVFITRC (SEQ ID NO: 16). In some embodiments, the agent is CKEYFYTSSKSSNLAWFVTRC (SEQ ID NO: 13), or
CKEYFYTSGKS SNPGIVFITRC (SEQ ID NO: 16).
[0010] Disclosed herein, in certain embodiments, is a method of treating emphysema, comprising administering an agent that specifically binds to all or a portion of PF4, RANTES, or a combination thereof. In some embodiments, the agent is an antibody, a peptibody, a small molecule, or a peptide. In some embodiments, the agent (a) mimics all or a portion of PF4, or (b) all or a portion of RANTES. In some embodiments, the agent (a) mimics all or a portion of the RANTES binding domain of PF4, or (b) all or a portion of PF4 binding domain of RANTES. In some embodiments, the agent is a peptide comprising an amino acid sequence that is at least 85% homologous to the amino acid sequence: CKEYFYTSSKSSNLAWFVTRC (SEQ ID NO: 13).
In some embodiments, the agent is a peptide comprising an amino acid sequence that is at least 85% homologous to the amino acid sequence: CKEYFYT SGKS SNPGIVFITRC (SEQ ID NO: 16). In some embodiments, the agent is CKEYFYTSSKSSNLAWFVTRC (SEQ ID NO: 13), or
CKEYFYTSGKS SNPGIVFITRC (SEQ ID NO: 16).
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:
[0012] Figure 1. The peptide of SEQ ID NO: 13 showed a dose-dependent inhibition of the increase in aorta diameter caused by PPE infusion in a porcine pancreatic elastase (PPE) infusion mouse model of human AAA. An aneurysm is defined as a 50% increase in aortic diameter or the presence of aortic dissection.
[0013] Figure 2. AAA developed in all porcine pancreatic elastase (PPE) infusion mice treated with the vehicle within 7 days. In contrast, AAA developed within 14 days in 3 porcine pancreatic elastase (PPE) infusion mice treated with 10 mg /kg/day of peptide of SEQ ID NO: 13, and 1 porcine pancreatic elastase (PPE) infusion mouse treated with 20 mg /kg/day of peptide of SEQ ID NO: 13. [0014] Figure 3. The peptide of SEQ ID NO: 13 showed a dose-dependent inhibition of the increase in aorta diameter caused by PPE infusion in a porcine pancreatic elastase (PPE) infusion mouse model of human AAA. *p<0.05 and **p<0.01 AAA between two groups, n=5-8 mice in each group.
[0015] Figure 4. The elastic Masson staining and the anti-smooth muscle cell alpha actin antibody immunostaining revealed that medial elastin fibers and smooth muscle cell layers were well preserved in porcine pancreatic elastase (PPE) infusion mice treated with 10 mg /kg/day of peptide of SEQ ID NO: 13 or 20 mg /kg/day of peptide of SEQ ID NO: 13, as compared to the vehicle- treated porcine pancreatic elastase (PPE) infusion mice.
[0016] Figure 5. Treatment of porcine pancreatic elastase (PPE) infusion mice with 10 mg /kg/day of peptide of SEQ ID NO: 13 or 20 mg /kg/day of peptide of SEQ ID NO: 13 significantly reduced the numbers of macrophages, CD4+ T cells and neutrophils and newly formed blood vessels in the media and adventitia by immunostaining against leukocyte subsets and endothelial cells. Data are one of two independent experiments. *p<0.05 and **p<0.01 AAA compared to vehicle group, n=2-3 mice in each group.
[0017] Figure 6. The elastic Masson staining and the anti-smooth muscle cell alpha actin antibody immunostaining revealed that medial elastin fibers and smooth muscle cell layers were well preserved in the PPE infusion mice treated with 10 mg /kg/day of peptide of SEQ ID NO: 13 or 20 mg /kg/day of peptide of SEQ ID NO: 13, as compared to the vehicle-treated mice.
[0018] Figure 7. Treatment of mice infected with RSV. Peptide of SEQ ID NO: 13 given by IV injection on Day 0-2 at 1 or 10 mg/kg. Given in a delayed administration at 10 mg/kg by IV injection on Day 5-7. All animals were harvested on day 8.
[0019] Figure 8. Treatment of mice to determine airway hyperreactivity after a methacholine challenge (0.6 mg/ml). Drug given by IV injection on Day 0-2 at 1 or 10 mg/kg. Given in a delayed administration at 10 mg/kg by IV injection on Day 5-7. All animals were harvested on day 8.
[0020] Figure 9. Histology results of mice infected with RSV. Control mice received vehicle. Experimental mice received the peptide of SEQ ID NO: 13 at the indicated dosage.
[0021] Figure 10. Treatment of mice infected with RSV. Animals were given peptide of SEQ ID NO: 13 by IP injection on day 0-7 at lOmg/kg dose and harvested on day 8 post-infection, (no change in AHR was observed).
DETAILED DESCRIPTION OF THE INVENTION
Certain Definitions
[0022] The terms "individual," "patient," or "subject" are used interchangeably. As used herein, they mean any mammal (i.e. species of any orders, families, and genus within the taxonomic classification animalia: chordata: vertebrata: mammalia). In some embodiments, the mammal is a human. In some embodiments, the mammal is a non-human. In some embodiments, the mammal is a member of the taxonomic orders: primates (e.g. lemurs, lorids, galagos, tarsiers, monkeys, apes, and humans); rodentia (e.g. mice, rats, squirrels, chipmunks, and gophers); lagomorpha (e.g. hares, rabbits, and pika); erinaceomorpha (e.g. hedgehogs and gymnures); soricomorpha (e.g. shrews, moles, and solenodons); chiroptera (e.g., bats); cetacea (e.g. whales, dolphins, and porpoises); carnivora (e.g. cats, lions, and other feliformia; dogs, bears, weasels, and seals); perissodactyla (e.g. horse, zebra, tapir, and rhinoceros); artiodactyla (e.g. pigs, camels, cattle, and deer); proboscidea (e.g. elephants); sirenia (e.g. manatees, dugong, and sea cows); cingulata (e.g. armadillos); pilosa (e.g. anteaters and sloths); didelphimorphia (e.g. american opossums); paucituberculata (e.g. shrew opossums); microbiotheria (e.g. Monito del Monte); notoryctemorphia (e.g. marsupial moles); dasyuromorphia (e.g. marsupial carnivores); peramelemorphia (e.g. bandicoots and bilbies); or diprotodontia (e.g. wombats, koalas, possums, gliders, kangaroos, wallaroos, and wallabies). In some embodiments, the animal is a reptile (i.e. species of any orders, families, and genus within the taxonomic classification animalia: chordata: vertebrata: reptilia). In some embodiments, the animal is a bird (i.e. animalia: chordata: vertebrata: aves). None of the terms require or are limited to situation characterized by the supervision (e.g. constant or intermittent) of a health care worker (e.g. a doctor, a registered nurse, a nurse practitioner, a physician's assistant, an orderly, or a hospice worker).
[0023] The terms "treat," "treating" or "treatment," and other grammatical equivalents as used herein, include alleviating, inhibiting or reducing symptoms, reducing or inhibiting severity of, reducing incidence of, prophylactic treatment of, reducing or inhibiting recurrence of, preventing, delaying onset of, delaying recurrence of, abating or ameliorating a disease or condition symptoms, ameliorating the underlying metabolic causes of symptoms, inhibiting the disease or condition, e.g., arresting the development of the disease or condition, relieving the disease or condition, causing regression of the disease or condition, relieving a condition caused by the disease or condition, or stopping the symptoms of the disease or condition. The terms further include achieving a therapeutic benefit. By therapeutic benefit is meant eradication or amelioration of the underlying disorder being treated, and/or the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the individual.
[0024] The terms "prevent," "preventing" or "prevention," and other grammatical equivalents as used herein, include preventing additional symptoms, preventing the underlying metabolic causes of symptoms, inhibiting the disease or condition, e.g., arresting the development of the disease or condition and are intended to include prophylaxis. The terms further include achieving a prophylactic benefit. For prophylactic benefit, the compositions are optionally administered to an individual at risk of developing a particular disease, to an individual reporting one or more of the physiological symptoms of a disease, or to an individual at risk of reoccurrence of the disease. [0025] Where combination treatments or prevention methods are contemplated, it is not intended that the agents described herein be limited by the particular nature of the combination. For example, the agents described herein are optionally administered in combination as simple mixtures as well as chemical hybrids. An example of the latter is where the agent is covalently linked to a targeting carrier or to an active pharmaceutical. Covalent binding can be accomplished in many ways, such as, though not limited to, the use of a commercially available cross-linking agent. Furthermore, combination treatments are optionally administered separately or concomitantly.
[0026] As used herein, the terms "pharmaceutical combination", "administering an additional therapy", "administering an additional therapeutic agent" and the like refer to a pharmaceutical therapy resulting from the mixing or combining of more than one active ingredient and includes both fixed and non-fixed combinations of the active ingredients. The term "fixed combination" means that at least one of the agents described herein, and at least one co-agent, are both
administered to an individual simultaneously in the form of a single entity or dosage. The term "non- fixed combination" means that at least one of the agents described herein, and at least one co- agent, are administered to an individual as separate entities either simultaneously, concurrently or sequentially with variable intervening time limits, wherein such administration provides effective levels of the two or more agents in the body of the individual. In some instances, the co-agent is administered once or for a period of time, after which the agent is administered once or over a period of time. In other instances, the co-agent is administered for a period of time, after which, a therapy involving the administration of both the co-agent and the agent are administered. In still other embodiments, the agent is administered once or over a period of time, after which, the co- agent is administered once or over a period of time. These also apply to cocktail therapies, e.g. the administration of three or more active ingredients.
[0027] As used herein, the terms "co-administration", "administered in combination with" and their grammatical equivalents are meant to encompass administration of the selected therapeutic agents to a single individual, and are intended to include treatment regimens in which the agents are administered by the same or different route of administration or at the same or different times. In some embodiments, the agents described herein will be co-administered with other agents. These terms encompass administration of two or more agents to an animal so that both agents and/or their metabolites are present in the animal at the same time. They include simultaneous administration in separate compositions, administration at different times in separate compositions, and/or administration in a composition in which both agents are present. Thus, in some embodiments, the agents described herein and the other agent(s) are administered in a single composition. In some embodiments, the agents described herein and the other agent(s) are admixed in the composition.
[0028] The terms "effective amount" or "therapeutically effective amount" as used herein, refer to a sufficient amount of at least one agent being administered which achieve a desired result, e.g., to relieve to some extent one or more symptoms of a disease or condition being treated. In certain instances, the result is a reduction and/or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. In specific instances, the result is a decrease in the growth of, the killing of, or the inducing of apoptosis in at least one abnormally proliferating cell, e.g., a cancer stem cell. In certain instances, an "effective amount" for therapeutic uses is the amount of the composition comprising an agent as set forth herein required to provide a clinically significant decrease in a disease. An appropriate "effective" amount in any individual case is determined using any suitable technique, such as a dose escalation study.
[0029] The terms "administer," "administering", "administration," and the like, as used herein, refer to the methods that may be used to enable delivery of agents or compositions to the desired site of biological action. These methods include, but are not limited to oral routes, intraduodenal routes, parenteral injection (including intravenous, subcutaneous, intraperitoneal, intramuscular, intravascular or infusion), topical and rectal administration. Administration techniques that are optionally employed with the agents and methods described herein, include e.g., as discussed in Goodman and Gilman, The Pharmacological Basis of Therapeutics, current ed.; Pergamon; and Remington's, Pharmaceutical Sciences (current edition), Mack Publishing Co., Easton, Pa. In certain embodiments, the agents and compositions described herein are administered orally.
[0030] The term "pharmaceutically acceptable" as used herein, refers to a material that does not abrogate the biological activity or properties of the agents described herein, and is relatively nontoxic (i.e., the toxicity of the material significantly outweighs the benefit of the material). In some instances, a pharmaceutically acceptable material may be administered to an individual without causing significant undesirable biological effects or significantly interacting in a deleterious manner with any of the components of the composition in which it is contained.
[0031] The term "recruiting of monocytes" as described herein includes the migration of monocytes into or out of the endothelium, their attachment and propagation, for example, into endothelial fissures. The attachment of monocytes is also known as monocyte adhesion, or as monocyte arrest when the attachment occurs in shear flow as under physiological conditions, for example, in blood capillaries, microvascular or arterial streamlines.
[0032] The phrase "specifically binds" when referring to the interaction between a binding molecule (i.e., the agent; e.g., a peptide or peptide mimetic) and a protein or polypeptide or epitope, typically refers to a binding molecule that recognizes and specifically binds with high affinity to the target of interest (said binding is detectable). Preferably, under designated or physiological conditions, the specified antibodies or binding molecules bind to a particular polypeptide, protein or epitope yet does not bind in a significant or undesirable amount to other molecules present in a sample. In other words the specified antibody or binding molecule does not undesirably cross-react with non-target antigens and/or epitopes. A variety of immunoassay formats are used to select antibodies or other binding molecule that are immunoreactive with a particular polypeptide and have a desired specificity. For example, solid-phase ELISA immunoassays, BIAcore, flow cytometry and radioimmunoassays are used to select monoclonal antibodies having a desired immunoreactivity and specificity. See, Harlow, 1988, ANTIBODIES, A LABORATORY MANUAL, Cold Spring Harbor Publications, New York (hereinafter, "Harlow"), for a description of immunoassay formats and conditions that are used to determine or assess immunoreactivity and specificity.
[0033] "Selective binding," "selectivity," and the like refer the preference of agent to interact with one molecule as compared to another. Preferably, interactions between an agent disclosed herein and proteins are both specific and selective. Note that in some embodiments, an agent is designed to "specifically bind" and "selectively bind" two distinct, yet similar targets without binding to other undesirable targets.
[0034] The terms "polypeptide," "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to naturally occurring amino acid polymers as well as amino acid polymers in which one or more amino acid residues is a non-naturally occurring amino acid (e.g., an amino acid analog). The terms encompass amino acid chains of any length, including full length proteins (i.e., antigens), wherein the amino acid residues are linked by covalent peptide bonds.
[0035] The terms "motif and "domain" are used interchangeably. As used herein, they mean a discrete, contiguous or non-contiguous portion of a polypeptide that folds independently of the rest of the polypeptide and possesses its own function.
[0036] The term "disruption" means to interfere with the function of. For example, to disrupt a motif/domain means to interfere with the function of the motif/domain.
[0037] The term "antigen" refers to a substance that is capable of inducing the production of an antibody. In some embodiments, an antigen is a substance that specifically binds to an antibody variable region.
[0038] The terms "antibody" and "antibodies" refer to monoclonal antibodies, polyclonal antibodies, bi-specific antibodies, multispecific antibodies, grafted antibodies, human antibodies, humanized antibodies, synthetic antibodies, chimeric antibodies, camelized antibodies, single-chain Fvs (scFv), single chain antibodies, Fab fragments, F(ab') fragments, disulfide-linked Fvs (sdFv), intrabodies, and anti-idiotypic (anti-Id) antibodies and antigen-binding fragments of any of the above. In particular, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules that contain an antigen binding site.
Depending on the amino acid sequence of the constant motif/domain of their heavy chains, immunoglobulins can be assigned to different classes. The heavy-chain constant motif/domains (Fc) that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known. Immunoglobulin molecules are of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgGi; IgG2, IgG3; IgG4; IgAi and IgA2) or subclass. The terms "antibody" and
"immunoglobulin" are used interchangeably in the broadest sense. In some embodiments, an antibody is part of a larger molecule, formed by covalent or non-covalent association of the antibody with one or more other proteins or peptides.
[0039] The term "peptide mimetic", "mimetic peptide" and "analog" are used herein
interchangeably for the purposes of the specifications and claims, to mean a peptide that mimics part or all of the bioactivity of an endogenous protein ligand. In some embodiments, peptide mimetics are modeled after a specific peptide and they display an altered peptide backbone, altered amino acids and/or an altered primary amino acid sequence when compared to the peptide that it was designed to mimic.
[0040] The term "peptibody" refers to a molecule comprising peptide(s) fused either directly or indirectly to an antibody or one or more antibody motif/domains (e.g., an Fc motif/domain of an antibody), where the peptide moiety specifically binds to a desired target. The peptide(s) may be fused to either an Fc region or inserted into an Fc- Loop, a modified Fc molecule. The term
"peptibody" does not include Fc-fusion proteins (e.g., full length proteins fused to an Fc
motif/domain).
[0041] The terms "isolated" and "purified" refer to a material that is substantially or essentially removed from or concentrated in its natural environment. For example, an isolated nucleic acid is one that is separated from at least some of the nucleic acids that normally flank it or other nucleic acids or components (proteins, lipids, etc.) in a sample. In another example, a polypeptide is purified if it is substantially removed from or concentrated in its natural environment. Methods for purification and isolation of nucleic acids and proteins are documented methodologies.
Embodiments of "substantially" include at least 20%, at least 40%, at least 50%, at least 75%, at least 85%, at least 90%, at least 95%, or at least 99%.
[0042] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that are present in minor amounts. In some embodiments, monoclonal antibodies are made, for example, by the hybridoma method first described by Kohler and Milstein (1975) Nature 256:495. or are made by recombinant methods, e.g., as described in U.S. Pat. No. 4,816,567. In some embodiments, monoclonal antibodies are isolated from phage antibody libraries using the techniques described in Clackson et al, Nature 352:624-628 (1991), as well as in Marks et al, J. Mol. Biol. 222:581 -597 (1991).
RANTES and Platelet Factor 4 (PF4) RANTES
[0043] In some embodiments, the PF4/RANTES targeting agents, methods and compositions disclosed herein inhibit (partially or fully) the activity of RANTES.
[0044] RANTES (also known as CCL5) is a pro-inflammatory chemokine.
[0045] In certain instances, human RANTES is encoded by the following nucleic acid sequence:
ATG AAG GTC TCC GCG GCA GCC CTC GCT GTC ATC CTC
ATT GCT ACT GCC CTC TGC GCT CCT GCA TCT GCC TCC
CCA TAT TCC TCG GAC ACC ACA CCC TGC TGC TTT GCC
TAC ATT GCC CGC CCA CTG CCC CGT GCC CAC ATC AAG
GAG TAT TTC TAC ACC AGT GGC AAG TGC TCC AAC CCA
GCA GTC GTC TTT GTC ACC CGA AAG AAC CGC CAA GTG
TGT GCC AAC CCA GAG AAG AAA TGG GTT CGG GAG TAC
ATC AAC TCT TTG GAG ATG AGC TAG
[0046] In certain instances, mouse RANTES is encoded by the following nucleic acid sequence:
ATG AAG ATC TCT GCA GCT GCC CTC ACC ATC ATC CTC ACT GCA
GCC GCC CTC TGC ACC CCC GCA CCT GCC TCA CCA TAT GGC TCG
GAC ACC ACT CCC TGC TGC TTT GCC TAC CTC TCC CTC GCG CTG
CCT CGT GCC CAC GTC AAG GAG TAT TTC TAC ACC AGC AGC AAG
TGC TCC AAT CTT GCA GTC GTG TTT GTC ACT CGA AGG AAC CGC
CAA GTG TGT GCC AAC CCA GAG AAG AAG TGG GTT CAA GAA TAC
ATC AAC TAT TTG GAG ATG AGC TAG
[0047] In certain instances, human RANTES is encoded by the following peptide sequence:
MKVSAAALAVILIATALCAPASASPYSSDTTPCCFAYIARPLPRAHIKEYF
YTSGKCSNPAVVFVTRKNRQVCANPEKKWVREYINSLEMS
[0048] In certain instances, mouse RANTES is encoded by the following peptide sequence:
MKISAAALTIILTAAALCTPAPASPYGSDTTPCCFAYLSLALPRAHVKEYF
YTSSKCSNLAWFVTRRNRQVCANPEKKWVQEYINYLEMS
[0049] In certain instances, rat RANTES is encoded by the following peptide sequence:
MKISTAASLTVILVAAALCTPAPASPYGSDTTPCCFAYLSLALPRAHVKE
YF YT S S KC SNL A WF VTRRNRQ VC ANPEKKWVQE YINYLEM S
[0050] In certain instances, pig RANTES is encoded by the following peptide sequence:
MKVSTAALAVILAMAALCAPASASPYASDTTPCCFSYLSRPLPRAHLQEY
FYTSSKCSMAAWFITRKNRQVCANPEKKWVREYINSLELS
[0051] In certain instances, dog RANTES is encoded by the following peptide sequence:
MKVSAATFAILLATATFRAPASASPYASDTTPCCFAYISGRLPFTHVQEYF
YTSSKCSMPAVVFVTRKHRQVCANPQKKWVREYINSLEMS [0052] In certain instances, RANTES is secreted by an activated platelet in response to an inflammation or tissue injury. In certain instances, RANTES is a ligand for a CCR5 receptor found on the plasma membrane of a target leukocyte (e.g. monocyte). In certain instances, RANTES induces chemotaxis in nearby leukocytes (e.g. monocytes) along a RANTES gradient. In certain instances, RANTES induces the chemotaxis of a leukocyte to the site of an inflammation or tissue injury. In certain instances, the chemotaxis of monocytes along a RANTES gradient results in monocyte arrest (i.e., the deposition of monocytes on epithelium) at the site of injury or inflammation.
PF4
[0053] In some embodiments, the PF4/RANTES targeting agents, methods and compositions disclosed herein inhibit (partially or fully) the activity of Platelet Factor 4 (PF4).
[0054] PF4 (also known as CXCL4) is a chemokine.
[0055] In certain instances, human PF4 is encoded by the following nucleic acid sequence:
ATG AGC TCC GCA GCC GGG TTC TGC GCC TCA CGC CCC GGG CTG
CTG TTC CTG GGG TTG CTG CTC CTG CCA CTT GTG GTC GCC TTC
GCC AGC GCT GAA GCT GAA GAA GAT GGG GAC CTG CAG TGC CTG
TGT GTG AAG ACC ACC TCC CAG GTC CGT CCC AGG CAC ATC ACC
AGC CTG GAG GTG ATC AAG GCC GGA CCC CAC TGC CCC ACT GCC
CAA CTG ATA GCC ACG CTG AAG AAT GGA AGG AAA ATT TGC TTG
GAC CTG CAA GCC CCG CTG TAC AAG AAA ATA ATT AAG AAA CTT
TTG GAG AGT
[0056] In certain instances, mouse PF4 is encoded by the following nucleic acid sequence:
ATG AGC GTC GCT GCG GTG TTT CGA GGC CTC CGG CCC AGT CCT
GAG CTG CTG CTT CTG GGC CTG TTG TTT CTG CCA GCG GTG GTT
GCT GTC ACC AGC GCT GGT CCC GAA GAA AGC GAT GGA GAT CTT
AGC TGT GTG TGT GTG AAG ACC ATC TCC TCT GGG ATC CAT CTT
AAG CAC ATC ACC AGC CTG GAG GTG ATC AAG GCA GGA CGC CAC
TGT GCG GTT CCC CAG CTC ATA GCC ACC CTG AAG AAT GGG AGG
AAA ATT TGC CTG GAC CGG CAA GCA CCC CTA TAT AAG AAA GTA
ATC GAG AAA ATC CTG GAG AGT
[0057] In certain instances, human PF4 is encoded by the following peptide sequence:
MSSAAGFCASRPGLLFLGLLLLPLVVAFASAEAEEDGDLQCLCVKTTSQV RPRHITSLEVIKAGPHCPTAQLIATLKNGRKICLDLQAPLYKKIIKKLLES
[0058] In certain instances, mouse PF4 is encoded by the following peptide sequence: MSVAAVFRGLRPSPELLLLGLLFLPAVVAVTSAGPEESDGDLSCVCVKTI
S S GIHLKHIT SLEVIKAGRHCAVPQLIATLKNGRKICLDRQ APLYKKVIKKI LES
[0059] In certain instances, PF4 is secreted by the alpha granules of an activated platelet during platelet aggregation in response to tissue injury and/or inflammation. In certain instances, PF4 is a ligand for a CXC3 receptor (i.e., CXC3RB). In certain instances, it induces directed chemotaxis in nearby leukocytes (e.g. monocytes). In certain instances, PF4 induces the chemotaxis of a leukocyte to the site of an inflammation or tissue injury.
PF4/RANTES
[0060] In certain instances, RANTES and PF4 form a heteromultimer (e.g., a heterodimer). In certain instances, a RANTES and PF4 heteromultimer (e.g., a heterodimer) amplifies the effects of RANTES-induced monocyte arrest.
Disorders
[0061] Disclosed herein, in certain embodiments, are methods of treating diseases, disorders or conditions involving inflammation (e.g., acute or chronic) comprising administering an agent disclosed herein (e.g., an agent that inhibits the formation of a PF4/RANTES heterodimer, an agent that binds to the RANTES binding domain of PF4, an agent that binds to the PF4 binding domain of RANTES). In certain instances, inflammation results from (either partially or fully) an infection. In certain instances, inflammation results from (either partially or fully) damage to a tissue (e.g., by a burn, by frostbite, by exposure to a cytotoxic agent, or by trauma). In certain instances, inflammation results from (either partially or fully) an autoimmune disorder. In certain instances, inflammation results from (either partially or fully) the presence of a foreign body (e.g., a splinter). In certain instances, inflammation results from exposure to a toxin and/or chemical irritant.
[0062] Disclosed herein, in certain embodiments, is a method of treating an inflammatory disorder comprising administering to an individual in need thereof (a) a peptide that mimics the RANTES binding domain of PF4, and inhibits the formation of a PF4 and RANTES heterodimer, or (b) a peptide that comprises an amino acid sequence that is at least 85% homologous (i.e., is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homologous, including any amount of homology between 85% and 100%) to an amino acid sequence selected from:
Figure imgf000019_0001
PRHITSLEVIKAGPHC (SEQ ID NO. 51) SSKCSNLAWFVTRR (SEQ ID NO. 74)
PTAQLIATLKNGRKIC (SEQ ID NO. 52) RRNRQVCANPEKKWV (SEQ ID NO. 75)
LDLQAPLYKKIIKKLLES (SEQ ID NO. 53) EKKWVQEYINYLEMS (SEQ ID NO. 76)
MSVAAVFRGLRPSPE (SEQ ID NO. 54) S SKS SNLA WFVTRCCKEYFYT (SEQ ID
NO. 77)
SPELLLLGLLFLPAV (SEQ ID NO. 55) SKS SNLA WFVTRCCKEYFYTS (SEQ ID
NO. 78)
PAVVAVTSAGPEESD (SEQ ID NO. 56) KS SNLA WFVTRCCKEYFYT S S (SEQ ID
NO. 79);
ESDGDLSCVCVKTIS (SEQ ID NO. 57) S SNLA WFVTRCCKEYFYT S SK (SEQ ID
NO. 80);
TISSGIHLKHITSLE (SEQ ID NO. 58) SNLA WFVTRCCKEYFYTS SKS (SEQ ID
NO. 81);
VIKAGRHCAVPQLIA (SEQ ID NO. 59) NLA WFVTRCCKEYFYT S SKS S (SEQ ID
NO. 82);
LIATLKNGRKICLDR (SEQ ID NO. 60) SFKGTTVYALSNVRSYSFVKCC (SEQ ID
NO. 83);
LDRQAPLYKKVIKKI (SEQ ID NO. 61) FKGTTVYALSNVRSYSFVKCCS (SEQ ID
NO. 84);
QAPLYKKVIKKILES (SEQ ID NO. 62) SNVRSYSFVKCCSFKGTTVYAL (SEQ ID
NO. 85);
MKVSAAALAVILIAT (SEQ ID NO. 63) NVRSYSFVKCCSFKGTTVYALS (SEQ ID
NO. 86);
ATALCAPASASPYSS (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID
NO. 87);
SDTTPCCFAYIARPL (SEQ ID NO. 65) YSFVKCCSFKGTTVYALSNVRS (SEQ ID
NO. 88);
PLPRAHIKEYFYTSG (SEQ ID NO. 66) SFVKCCSFKGTTVYALSNVRSY (SEQ ID
NO. 89);
EYFYTSGKCSNPAW (SEQ ID NO. 67) FVKCCSFKGTTVYALSNVRSYS (SEQ ID
NO. 90)
VFVTRKNRQVCANPE (SEQ ID NO. 66)
[0063] Disclosed herein, in certain embodiments, is the use of (a) a peptide that mimics the RANTES binding domain of PF4, and inhibits the formation of a PF4 and RANTES heterodimer, or (b) a peptide that comprises an amino acid that is 85% homologous (i.e., is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homologous, including any amount of homology between 85% and 100%) to an amino acid sequence selected from:
Figure imgf000021_0001
NO. 89);
EYFYTSGKCSNPAW (SEQ ID NO. 67) FVKCCSFKGTTVYALSNVRSYS (SEQ ID
NO. 90)
VFVTRKNRQVCANPE (SEQ ID NO. 66)
for the treatment of inflammation mediated by the binding of PF4 and RANTES.
[0064] As used herein, "acute inflammation" refers to inflammation characterized in that it develops over the course of a few minutes to a few hours, and ceases once the stimulus has been removed (e.g., an infectious agent has been killed by an immune response or administration of a therapeutic agent, a foreign body has been removed by an immune response or extraction, or damaged tissue has healed). The short duration of acute inflammation results from the short half- lives of most inflammatory mediators.
[0065] In certain instances, acute inflammation begins with the activation of leukocytes (e.g., monocytes, macrophages, neutrophils, basophils, eosinophils, lymphocytes, dendritic cells, endothelial cells and mastocytes). In certain instances, the leukocytes release inflammatory mediators (e.g., histamines, proteoglycans, serine proteases, eicosanoids, and cytokines). In certain instances, inflammatory mediators result in (either partially or fully) the symptoms associated with inflammation. For example, in certain instances an inflammatory mediator dilates post capillary venules, and increases blood vessel permeability. In certain instances, the increased blood flow that follows vasodilation results in (either partially or fully) rubor and calor. In certain instances, increased permeability of the blood vessels results in an exudation of plasma into the tissue leading to edema. In certain instances, the latter allows leukocytes to migrate along a chemotactic gradient to the site of the inflammatory stimulant. Further, in certain instances, structural changes to blood vessels (e.g., capillaries and venules) occur. In certain instances, the structural changes are induced (either partially or fully) by monocytes and/or macrophages. In certain instances, the structural changes include, but are not limited to, remodeling of vessels, and angiogenesis. In certain instances, angiogenesis contributes to the maintenance of chronic inflammation by allowing for increased transport of leukocytes. Additionally, in certain instances, histamines and bradykinin irritate nerve endings leading to itching and/or pain.
[0066] In certain instances, chronic inflammation results from the presence of a persistent stimulant (e.g., persistent acute inflammation, bacterial infection (e.g., by Mycobacterium tuberculosis), prolonged exposure to chemical agents (e.g., silica, or tobacco smoke) and autoimmune reactions (e.g., rheumatoid arthritis)). In certain instances, the persistent stimulant results in continuous inflammation (e.g., due to the continuous recruitment of monocytes, and the proliferation of macrophages). In certain instances, the continuous inflammation further damages tissues which results in the additional recruitment of mononuclear cells thus maintaining and exacerbating the inflammation. In certain instances, physiological responses to inflammation further include angiogenesis and fibrosis.
[0067] Multiple disorders are associated with inflammation (i.e., inflammatory disorders).
Inflammatory disorders include, but are not limited to, Acute disseminated encephalomyelitis; Addison's disease; Ankylosing spondylitis; Antiphospholipid antibody syndrome; Autoimmune hemolytic anemia; Autoimmune hepatitis; Autoimmune inner ear disease; Bullous pemphigoid; Chagas disease; Chronic obstructive pulmonary disease; Coeliac disease; Dermatomyositis;
Diabetes mellitus type 1 ; Diabetes mellitus type 2; Endometriosis; Goodpasture's syndrome; Graves' disease; Guillain-Barre syndrome; Hashimoto's disease; Idiopathic thrombocytopenic purpura; Interstitial cystitis; Systemic lupus erythematosus (SLE); Metabolic syndrome, Multiple sclerosis; Myasthenia gravis; Myocarditis, Narcolepsy; Obesity; Pemphigus Vulgaris; Pernicious anaemia; Polymyositis; Primary biliary cirrhosis; Rheumatoid arthritis; Schizophrenia; Scleroderma; Sjogren's syndrome; Vasculitis; Vitiligo; Wegener's granulomatosis; Allergic rhinitis; Prostate cancer; Non- small cell lung carcinoma; Ovarian cancer; Breast cancer; Melanoma; Gastric cancer; Colorectal cancer; Brain cancer; Metastatic bone disorder; Pancreatic cancer; a Lymphoma; Nasal polyps; Gastrointestinal cancer; Ulcerative colitis; Crohn's disorder; Collagenous colitis; Lymphocytic colitis; Ischaemic colitis; Diversion colitis; Behcet's syndrome; Infective colitis; Indeterminate colitis; Inflammatory liver disorder, Endotoxin shock, Rheumatoid spondylitis, Ankylosing spondylitis, Gouty arthritis, Polymyalgia rheumatica, Alzheimer's disorder, Parkinson's disorder, Epilepsy, AIDS dementia, Asthma, Adult respiratory distress syndrome, Bronchitis, Cystic fibrosis, Acute leukocyte-mediated lung injury, Distal proctitis, Wegener's granulomatosis, Fibromyalgia, Bronchitis, Cystic fibrosis, Uveitis, Conjunctivitis, Psoriasis, Eczema, Dermatitis, Smooth muscle proliferation disorders, Meningitis, Shingles, Encephalitis, Nephritis, Tuberculosis, Retinitis, Atopic dermatitis, Pancreatitis, Periodontal gingivitis, Coagulative Necrosis, Liquefactive Necrosis, Fibrinoid Necrosis, Hyperacute transplant rejection, Acute transplant rejection, Chronic transplant rejection, Acute graft- versus-host disease, Chronic graft- versus-host disease, or combinations thereof.
[0068] Disclosed herein, in certain embodiments, is a method of treating cystic fibrosis comprising administering to an individual in need thereof (a) a peptide that mimics the RANTES binding domain of PF4, and inhibits the formation of a PF4 and RANTES heterodimer, or (b) a peptide that comprises an amino acid that is 85% homologous (i.e., is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homologous, including any amount of homology between 85% and 100%) to an amino acid sequence selected from:
Figure imgf000023_0001
SAEAEEDGDLQC (SEQ ID NO. 49) SDTTPCCFAYLSLAL (SEQ ID NO. 72)
QCLCVKTTSQVRPRH (SEQ ID NO. 50) ALPRAHVKEYFYTSS (SEQ ID NO. 73)
PRHITSLEVIKAGPHC (SEQ ID NO. 51) SSKCSNLAWFVTRR (SEQ ID NO. 74)
PTAQLIATLKNGRKIC (SEQ ID NO. 52) RRNRQVCANPEKKWV (SEQ ID NO. 75)
LDLQAPLYKKIIKKLLES (SEQ ID NO. 53) EKKWVQEYINYLEMS (SEQ ID NO. 76)
MSVAAVFRGLRPSPE (SEQ ID NO. 54) S SKS SNLA WFVTRCCKEYFYT (SEQ ID
NO. 77)
SPELLLLGLLFLPAV (SEQ ID NO. 55) SKS SNLA WFVTRCCKEYFYTS (SEQ ID
NO. 78)
PAVVAVTSAGPEESD (SEQ ID NO. 56) KS SNLA WFVTRCCKEYFYT S S (SEQ ID
NO. 79);
ESDGDLSCVCVKTIS (SEQ ID NO. 57) S SNLA WFVTRCCKEYFYT S SK (SEQ ID
NO. 80);
TISSGIHLKHITSLE (SEQ ID NO. 58) SNLA WFVTRCCKEYFYTS SKS (SEQ ID
NO. 81);
VIKAGRHCAVPQLIA (SEQ ID NO. 59) NLA WFVTRCCKEYFYT S SKS S (SEQ ID
NO. 82);
LIATLKNGRKICLDR (SEQ ID NO. 60) SFKGTTVYALSNVRSYSFVKCC (SEQ ID
NO. 83);
LDRQAPLYKKVIKKI (SEQ ID NO. 61) FKGTTVYALSNVRSYSFVKCCS (SEQ ID
NO. 84);
QAPLYKKVIKKILES (SEQ ID NO. 62) SNVRSYSFVKCCSFKGTTVYAL (SEQ ID
NO. 85);
MKVSAAALAVILIAT (SEQ ID NO. 63) NVRSYSFVKCCSFKGTTVYALS (SEQ ID
NO. 86);
ATALCAPASASPYSS (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID
NO. 87);
SDTTPCCFAYIARPL (SEQ ID NO. 65) YSFVKCCSFKGTTVYALSNVRS (SEQ ID
NO. 88);
PLPRAHIKEYFYTSG (SEQ ID NO. 66) SFVKCCSFKGTTVYALSNVRSY (SEQ ID
NO. 89);
EYFYTSGKCSNPAW (SEQ ID NO. 67) FVKCCSFKGTTVYALSNVRSYS (SEQ ID
NO. 90)
VFVTRKNRQVCANPE (SEQ ID NO. 66)
[0069] Disclosed herein, in certain embodiments, is the use of (a) a peptide that mimics the RANTES binding domain of PF4, and inhibits the formation of a PF4 and RANTES heterodimer, or (b) a peptide that comprises an amino acid that is 85% homologous (i.e., is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homologous, including any amount of homology between 85% and 100%) to an amino acid sequence selected from:
Figure imgf000025_0001
PLPRAHIKEYFYTSG (SEQ ID NO. 66) SFVKCCSFKGTTVYALSNVRSY (SEQ ID
NO. 89);
EYFYTSGKCSNPAW (SEQ ID NO. 67) FVKCCSFKGTTVYALSNVPvSYS (SEQ ID
NO. 90)
VFVTRKNPvQVCANPE (SEQ ID NO. 66)
for the treatment of cystic fibrosis.
[0070] Disclosed herein, in certain embodiments, is a method of treating atherosclerosis comprising administering to an individual in need thereof (a) a peptide that mimics the RANTES binding domain of PF4, and inhibits the formation of a PF4 and RANTES heterodimer, or (b) a peptide that comprises an amino acid that is 85% homologous (i.e., is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homologous, including any amount of homology between 85% and 100%) to an amino acid sequence selected from:
Figure imgf000026_0001
NO. 84);
QAPLYKKVIKKILES (SEQ ID NO. 62) SNVRSYSFVKCCSFKGTTVYAL (SEQ ID
NO. 85);
MKVSAAALAVILIAT (SEQ ID NO. 63) NVRSYSFVKCCSFKGTTVYALS (SEQ ID
NO. 86);
ATALCAPASASPYSS (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID
NO. 87);
SDTTPCCFAYIARPL (SEQ ID NO. 65) YSFVKCCSFKGTTVYALSNVRS (SEQ ID
NO. 88);
PLPRAHIKEYFYTSG (SEQ ID NO. 66) SFVKCCSFKGTTVYALSNVRSY (SEQ ID
NO. 89);
EYFYTSGKCSNPAW (SEQ ID NO. 67) FVKCCSFKGTTVYALSNVRSYS (SEQ ID
NO. 90)
VFVTRKNPvQVCANPE (SEQ ID NO. 66)
[0071] Disclosed herein, in certain embodiments, is the use of (a) a peptide that mimics the RANTES binding domain of PF4, and inhibits the formation of a PF4 and RANTES heterodimer, or (b) a peptide that comprises an amino acid that is 85% homologous (i.e., is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homologous, including any amount of homology between 85% and 100%) to an amino acid sequence selected from:
MSSAAGFCASRP (SEQ ID NO. 46) EKKWVREYINSLEMS (SEQ ID NO. 69)
RPGLLFLGLLLLPL (SEQ ID NO. 47) MKISAAALTIILTAA (SEQ ID NO. 70)
PLWAFASAEA (SEQ ID NO. 48) AALCTPAPASPYGSD (SEQ ID NO. 71)
SAEAEEDGDLQC (SEQ ID NO. 49) SDTTPCCFAYLSLAL (SEQ ID NO. 72)
QCLCVKTTSQVRPRH (SEQ ID NO. 50) ALPRAHVKEYFYTSS (SEQ ID NO. 73)
PRHITSLEVIKAGPHC (SEQ ID NO. 51) SSKCSNLAWFVTRR (SEQ ID NO. 74)
PTAQLIATLKNGRKIC (SEQ ID NO. 52) RRNRQVCANPEKKWV (SEQ ID NO. 75)
LDLQAPLYKKIIKKLLES (SEQ ID NO. 53) EKKWVQEYINYLEMS (SEQ ID NO. 76)
MSVAAVFRGLRPSPE (SEQ ID NO. 54) S SKS SNLA WFVTRCCKEYFYT (SEQ ID
NO. 77)
SPELLLLGLLFLPAV (SEQ ID NO. 55) SKS SNLA WFVTRCCKEYFYTS (SEQ ID
NO. 78)
PAVVAVTSAGPEESD (SEQ ID NO. 56) KS SNLA WFVTRCCKEYFYT S S (SEQ ID
NO. 79);
ESDGDLSCVCVKTIS (SEQ ID NO. 57) S SNLA WFVTRCCKEYFYT S SK (SEQ ID
NO. 80); TISSGIHLKHITSLE (SEQ ID NO. 58) SNLAWFVTRCCKEYFYTS SKS (SEQ ID
NO. 81);
VIKAGRHCAVPQLIA (SEQ ID NO. 59) NLAVVFVTRCCKEYF YT S SKS S (SEQ ID
NO. 82);
LIATLKNGRKICLDPv (SEQ ID NO. 60) SFKGTTVYALSNVRSYSFVKCC (SEQ ID
NO. 83);
LDRQAPLYKKVIKKI (SEQ ID NO. 61) FKGTTVYALSNVRSYSFVKCCS (SEQ ID
NO. 84);
QAPLYKKVIKKILES (SEQ ID NO. 62) SNVRSYSFVKCCSFKGTTVYAL (SEQ ID
NO. 85);
MKVSAAALAVILIAT (SEQ ID NO. 63) NVRSYSFVKCCSFKGTTVYALS (SEQ ID
NO. 86);
ATALCAPASASPYSS (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID
NO. 87);
SDTTPCCFAYIARPL (SEQ ID NO. 65) YSFVKCCSFKGTTVYALSNVRS (SEQ ID
NO. 88);
PLPRAHIKEYFYTSG (SEQ ID NO. 66) SFVKCCSFKGTTVYALSNVRSY (SEQ ID
NO. 89);
EYFYTSGKCSNPAW (SEQ ID NO. 67) FVKCCSFKGTTVYALSNVRSYS (SEQ ID
NO. 90)
VFVTRKNRQVCANPE (SEQ ID NO. 66)
for the treatment of atherosclerosis.
[0072] Disclosed herein, in certain embodiments, is a method of treating asthma comprising administering to an individual in need thereof (a) a peptide that mimics the RANTES binding domain of PF4, and inhibits the formation of a PF4 and RANTES heterodimer, or (b) a peptide that comprises an amino acid that is 85% homologous (i.e., is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homologous, including any amount of homology between 85% and 100%) to an amino acid sequence selected from:
Figure imgf000028_0001
LDLQAPLYKKIIKKLLES (SEQ ID NO. 53) EKKWVQEYINYLEMS (SEQ ID NO. 76)
MSVAAVFPvGLRPSPE (SEQ ID NO. 54) S SKS SNLA WFVTRCCKEYFYT (SEQ ID
NO. 77)
SPELLLLGLLFLPAV (SEQ ID NO. 55) SKS SNLA WFVTRCCKEYFYTS (SEQ ID
NO. 78)
PAVVAVTSAGPEESD (SEQ ID NO. 56) KS SNLA WFVTRCCKEYFYT S S (SEQ ID
NO. 79);
ESDGDLSCVCVKTIS (SEQ ID NO. 57) S SNLA WFVTRCCKEYFYT S SK (SEQ ID
NO. 80);
TISSGIHLKHITSLE (SEQ ID NO. 58) SNLA WFVTRCCKEYFYTS SKS (SEQ ID
NO. 81);
VIKAGRHCAVPQLIA (SEQ ID NO. 59) NLA WFVTRCCKEYFYT S SKS S (SEQ ID
NO. 82);
LIATLKNGRKICLDPv (SEQ ID NO. 60) SFKGTTVYALSNVRSYSFVKCC (SEQ ID
NO. 83);
LDRQAPLYKKVIKKI (SEQ ID NO. 61) FKGTTVYALSNVRSYSFVKCCS (SEQ ID
NO. 84);
QAPLYKKVIKKILES (SEQ ID NO. 62) SNVRSYSFVKCCSFKGTTVYAL (SEQ ID
NO. 85);
MKVSAAALAVILIAT (SEQ ID NO. 63) NVRSYSFVKCCSFKGTTVYALS (SEQ ID
NO. 86);
ATALCAPASASPYSS (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID
NO. 87);
SDTTPCCFAYIARPL (SEQ ID NO. 65) YSFVKCCSFKGTTVYALSNVRS (SEQ ID
NO. 88);
PLPRAHIKEYFYTSG (SEQ ID NO. 66) SFVKCCSFKGTTVYALSNVRSY (SEQ ID
NO. 89);
EYFYTSGKCSNPAW (SEQ ID NO. 67) FVKCCSFKGTTVYALSNVRSYS (SEQ ID
NO. 90)
VFVTRKNRQVCANPE (SEQ ID NO. 66)
In some embodiments, the asthma is moderate asthma. In some embodiments, the asthma is sever asthma.
[0073] Disclosed herein, in certain embodiments, is the use of (a) a peptide that mimics the
RANTES binding domain of PF4, and inhibits the formation of a PF4 and RANTES heterodimer, or (b) a peptide that comprises an amino acid that is 85% homologous (i.e., is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homologous, including any amount of homology between 85% and 100%) to an amino acid sequence selected from:
Figure imgf000030_0001
NO. 89);
EYFYTSGKCSNPAW (SEQ ID NO. 67) FVKCCSFKGTTVYALSNVPvSYS (SEQ ID
NO. 90)
VFVTRKNPvQVCANPE (SEQ ID NO. 66)
for the treatment of asthma. In some embodiments, the asthma is moderate asthma. In some embodiments, the asthma is severe asthma.
[0074] Disclosed herein, in certain embodiments, is a method of treating respiratory syncytial virus (RSV), comprising administering to an individual in need thereof a peptide disclosed herein. In some embodiments, the peptide is (a) a peptide that mimics the RANTES binding domain of PF4, and inhibits the formation of a PF4 and RANTES heterodimer, or (b) a peptide that mimics the PF4 binding domain of RANTES, and inhibits the formation of a PF4 and RANTES heterodimer. In some embodiments, the peptide comprises an amino acid that is 85% homologous (i.e., is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homologous, including any amount of homology between 85% and 100%) to an amino acid sequence CKEYFYTSSKSSNLAVVFVTRC (SEQ ID NO: 13). In some embodiments, the peptide comprises an amino acid that is 85% homologous (i.e., is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homologous, including any amount of homology between 85% and 100%) to an amino acid sequence CKEYFYTSGKSSNPGIVFITRC (SEQ ID NO: 16). In some embodiments, the peptide is CKEYFYTSSKSSNLAVVFVTRC (SEQ ID NO: 13) or CKEYFYTSGKSSNPGIVFITRC (SEQ ID NO: 16).
[0075] Disclosed herein, in certain embodiments, is a peptide disclosed herein for use in the treatment of respiratory syncytial virus (RSV), wherein the peptide is (a) a peptide that mimics the RANTES binding domain of PF4, and inhibits the formation of a PF4 and RANTES heterodimer, or (b) a peptide that mimics the PF4 binding domain of RANTES, and inhibits the formation of a PF4 and RANTES heterodimer . In some embodiments, the peptide comprises an amino acid that is 85% homologous (i.e., is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%), 99%, or 100% homologous, including any amount of homology between 85% and 100%) to an amino acid sequence CKEYFYTSSKSSNLAVVFVTRC (SEQ ID NO: 13). In some embodiments, the peptide comprises an amino acid that is 85% homologous (i.e., is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homologous, including any amount of homology between 85% and 100%) to an amino acid sequence
CKEYFYTSGKSSNPGIVFITRC (SEQ ID NO: 16). In some embodiments, the peptide is
CKEYFYTSSKSSNLAVVFVTRC (SEQ ID NO: 13) or CKEYFYTSGKSSNPGIVFITRC (SEQ ID NO: 16).
[0076] Disclosed herein, in certain embodiments, is a method of treating emphysema, comprising administering to an individual in need thereof a peptide disclosed herein. In some embodiments, the peptide is (a) a peptide that mimics the RANTES binding domain of PF4, and inhibits the formation of a PF4 and RANTES heterodimer, or (b) a peptide that mimics the PF4 binding domain of RANTES, and inhibits the formation of a PF4 and RANTES heterodimer. In some embodiments, the peptide comprises an amino acid that is 85% homologous (i.e., is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homologous, including any amount of homology between 85% and 100%) to an amino acid sequence CKEYFYTSSKSSNLAVVFVTRC (SEQ ID NO: 13). In some embodiments, the peptide comprises an amino acid that is 85% homologous (i.e., is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%), 99%, or 100% homologous, including any amount of homology between 85% and 100%) to an amino acid sequence CKEYFYTSGKSSNPGIVFITRC (SEQ ID NO: 16). In some embodiments, the peptide is CKEYFYTSSKSSNLAVVFVTRC (SEQ ID NO: 13) or
CKEYFYTSGKSSNPGIVFITRC (SEQ ID NO: 16).
[0077] Disclosed herein, in certain embodiments, is a peptide disclosed herein for use in the treatment of emphysema, wherein the peptide is (a) a peptide that mimics the RANTES binding domain of PF4, and inhibits the formation of a PF4 and RANTES heterodimer, or (b) a peptide that mimics the PF4 binding domain of RANTES, and inhibits the formation of a PF4 and RANTES heterodimer . In some embodiments, the peptide for use is CKEYFYTSSKSSNLAVVFVTRC (SEQ ID NO: 13). In some embodiments, the peptide comprises an amino acid that is 85% homologous (i.e., is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) homologous, including any amount of homology between 85% and 100%) to an amino acid sequence CKEYFYTSGKSSNPGIVFITRC (SEQ ID NO: 16). In some embodiments, the peptide is CKEYFYTSSKSSNLAVVFVTRC (SEQ ID NO: 13) or CKEYFYTSGKSSNPGIVFITRC (SEQ ID NO: 16).
[0078] In some embodiments, the PF4/RANTES targeting agents, methods and compositions described herein treat a T-cell mediated autoimmune disorder. In certain instances, a T-cell mediated autoimmune disorder is characterized by a T-cell mediated immune response against self (e.g., native cells and tissues).
[0079] Examples of T-cell mediated autoimmune disorders include, but are not limited to colitis, multiple sclerosis, arthritis, rheumatoid arthritis, osteoarthritis, juvenile arthritis, psoriatic arthritis, acute pancreatitis, chronic pancreatitis, diabetes, insulin-dependent diabetes mellitus (IDDM or type I diabetes), insulitis, inflammatory bowel disease, Crohn's disease, ulcerative colitis, autoimmune hemolytic syndromes, autoimmune hepatitis, autoimmune neuropathy, autoimmune ovarian failure, autoimmune orchitis, autoimmune thrombocytopenia, reactive arthritis, ankylosing spondylitis, silicone implant associated autoimmune disease, Sjogren's syndrome, systemic lupus erythematosus (SLE), vasculitis syndromes (e.g., giant cell arteritis, Behcet's disease & Wegener's granulomatosis), vitiligo, secondary hematologic manifestation of autoimmune diseases (e.g., anemias), drug-induced autoimmunity, Hashimoto's thyroiditis, hypophysitis, idiopathic thrombocytic pupura, metal-induced autoimmunity, myasthenia gravis, pemphigus, autoimmune deafness (e.g., Meniere's disease), Goodpasture's syndrome, Graves' disease, HlV-related autoimmune syndromes and Gullain-Barre disease.
[0080] In some embodiments, the PF4/RANTES targeting agents, methods and compositions described herein treat pain. Pain includes, but is not limited to acute pain, acute inflammatory pain, chronic inflammatory pain and neuropathic pain.
[0081] In some embodiments, the PF4/RANTES targeting agents, methods and compositions described herein treat hypersensitivity. As used herein, "hypersensitivity" refers to an undesirable immune system response. Hypersensitivity is divided into four categories. Type I hypersensitivity includes allergies (e.g., Atopy, Anaphylaxis, or Asthma). Type II hypersensitivity is
cytotoxic/antibody mediated (e.g., Autoimmune hemolytic anemia, Thrombocytopenia,
Erythroblastosis fetalis, or Goodpasture's syndrome). Type III is immune complex diseases (e.g., Serum sickness, Arthus reaction, or SLE). Type IV is delayed-type hypersensitivity (DTH), Cell- mediated immune memory response, and antibody-independent (e.g., Contact dermatitis, Tuberculin skin test, or Chronic transplant rejection).
[0082] As used herein, "allergy" means a disorder characterized by excessive activation of mast cells and basophils by IgE. In certain instances, the excessive activation of mast cells and basophils by IgE results (either partially or fully) in an inflammatory response. In certain instances, the inflammatory response is local. In certain instances, the inflammatory response results in the narrowing of airways (i.e., bronchoconstriction). In certain instances, the inflammatory response results in inflammation of the nose (i.e., rhinitis). In certain instances, the inflammatory response is systemic (i.e., anaphylaxis).
[0083] In some embodiments, the PF4/RANTES targeting agents, methods and compositions described herein treat angiogenesis. As used herein, "angiogenesis" refers to the formations of new blood vessels. In certain instances, angiogenesis occurs with chronic inflammation. In certain instances, angiogenesis is induced by monocytes and/or macrophages.
[0084] In some embodiments, the present invention comprises a method of treating a neoplasia. In certain instances, a neoplastic cell induces an inflammatory response. In certain instances, part of the inflammatory response to a neoplastic cell is angiogenesis. In certain instances, angiogenesis facilitates the development of a neoplasia. In some embodiments, the neoplasia is: angiosarcoma, Ewing sarcoma, osteosarcoma, and other sarcomas, cecum carcinoma, colon carcinoma, lung carcinoma, ovarian carcinoma, pharyngeal carcinoma, rectosigmoid carcinoma, pancreatic carcinoma, renal carcinoma, endometrial carcinoma, gastric carcinoma, liver carcinoma, head and neck carcinoma, breast carcinoma and other carcinomas, Hodgkins lymphoma and other lymphomas, malignant and other melanomas, parotid tumor, chronic lymphocytic leukemia and other leukemias, astrocytomas, gliomas, hemangiomas, retinoblastoma, neuroblastoma, acoustic neuroma, neurofibroma, trachoma and pyogenic granulomas.
[0085] In some embodiments, the PF4/RANTES targeting agents, methods and compositions described herein treat obesity. As used herein, "obesity" means an accumulation of adipose tissue with a BMI of greater than or equal to 30kg/m2. In certain instances, obesity is characterized a proinflammatory state, increasing the risk of thrombosis. In certain instances, obesity is associated with a low-grade inflammation of white adipose tissue (WAT). In certain instances, WAT associated with obesity is characterized by an increased production and secretion of a wide range of inflammatory molecules including TNF-alpha and interleukin-6 (IL-6). In certain instances, WAT is infiltrated by macrophages, which produce pro-inflammatory cytokines. In certain instances, TNF- alpha is overproduced in adipose tissue. In certain instances, IL-6 production increases during obesity.
[0086] In some embodiments, the PF4/RANTES targeting agents, methods and compositions described herein treat metabolic syndrome. In certain instances, metabolic syndrome is associated with fasting hyperglycemia; high blood pressure; central obesity; decreased HDL levels; elevated triglyceride levels; systemic inflammation; or combinations thereof. In certain instances, metabolic syndrome is characterized by an increase in the levels of C-reactive protein, fibrinogen, IL-6, and TNFa.
[0087] In some embodiments, the PF4/RANTES targeting agents, methods and compositions described herein treat a cardiovascular disorder involving inflammation. As used herein, the term "cardiovascular disease" (CVD) refers to a disease or disorder characterized by impairment or dysfunction of the heart, an artery, and/or vein. In some embodiments, the disorder is a
dyslipidemia. In some embodiments, the disorder is hyperlipidemia; hypercholesterolemia;
hyperglyceridemia; combined hyperlipidemia; hypolipoproteinemia; hypocholesterolemia;
abetlipoproteinemia; Tangier disease; or a combination thereof. In some embodiments, the disorder is acute coronary syndrome; unstable angina; non-ST segment elevation myocardial infarction; ST segment elevation myocardial infarction; stable angina; Prinzmetal's angina; arteriosclerosis;
atherosclerosis; stenosis; restenosis; venous thrombosis; arterial thrombosis; stroke; transient ischemic attack; peripheral vascular disease; coronary artery disease; obesity; diabetes; metabolic syndrome; or combinations thereof.
[0088] In some embodiments, the PF4/RANTES targeting agents, methods and compositions described herein treat dyslipidemia. As used herein, the term "dyslipidemia" means a disruption (i.e., variation from a normal range) in the concentration of a lipid in the blood. In certain instances, a dyslipidemia is an increase in lipid (e.g. cholesterol, glycerides, or triglyceride) concentrations over a normal range (i.e., a hyperlipidemia). In certain instances, a hyperlipidemia involves an increase in the concentration of cholesterol (i.e., hypercholesterolemia); glycerides (i.e., hyperglyceridemia); triglycerides (i.e., hypertriglyceridemia); lipoproteins (i.e.,
hyperlipoproteinemia); chylomicrons (i.e., hyperchylomicronemia); or combinations thereof (e.g., combined hyperlipidemia). In certain instances, a dyslipidemia is a decrease in lipid concentrations below a normal range (i.e., a hypolipidemia). In certain instances, a hypolipidemia involves a decrease in the concentration of lipoproteins (i.e., hypolipoproteinemia); cholesterol (i.e., hypocholesterolemia); beta lipoproteins (i.e., abetalipoproteinemia); HDL (i.e., Tangier disease); or combinations thereof. In certain instances, a dyslipidemia results from environmental factors (e.g., lack of exercise or food intake). In certain instances, a dyslipidemia results from genetic factors (e.g., aberrant expression of ApoAl, Apo B, ApoC2, LPL, or LDL receptor).
[0089] In certain instances, blood comprises lipoproteins. In certain instances, a lipoprotein is a complex of proteins (e.g., ApoAl, ApoA2, ApoA4, ApoA5, ApoCl, ApoC2, ApoC3, ApoD, ApoE, LCAT, PAF-AH, PON1, GPX, serum amyloid A, a-1 antitrypsin, and amyloid-β) and lipids. In certain instances, a lipoprotein is a high density lipoprotein (HDL). In certain instances, a lipoprotein is a low density lipoprotein (LDL).
[0090] HDL is a type of lipoprotein that transports cholesterol and triglycerides to the liver. In certain instances, HDL comprises ApoAl and ApoA2. In certain instances, ApoAl and ApoA2 are expressed in the liver. In certain instances, the liver synthesized HDL. In certain instances, HDL transport cholesterol from cells to the liver, adrenals, ovary and/or testes. In certain instances, cholesterol transported to the liver is excreted as bile. In certain instances, cholesterol transported to adrenals, ovaries and/or testes are used to synthesize steroid hormones. HDL comprises multiple subclasses of lipoprotein. In certain instances, the subclasses of HDL differ in size, density, protein and lipid composition. In certain instances, some HDL are protective, anti-oxidative, anti-inflammatory and/or anti-atherogenic. In certain instances, some HDL are neutral. In certain instances, some HDL enhance oxidation, increase inflammation and/or are pro-atherogenic. In certain instances, increasing the concentration of HDL across all or most sub-classes results in the production of reactive oxygen species (ROS). In certain instances, an enzyme associated with HDL modifies a phospholipid into an oxidized phospholipid. In certain instances, an enzyme associated with HDL modifies cholesterol into an oxidized sterol. In certain instances, an oxidized sterol and/or an oxidized phospholipid results in pro-inflammatory and/or pro-atherogenic HDL.
[0091] In certain instances, cholesteryl ester transfer protein (CETP) exchanges triglycerides transported by VLDL (very low density lipoprotein) for cholesteryl esters transported by HDL. In certain instances, the exchange of triglycerides for cholesteryl esters results in VLDL being processed into LDL. In certain instances, LDL is removed from circulation by the LDL receptor pathway. In certain instances, the triglycerides are degraded by hepatic lipase. In certain instances, delipidified HDL recirculate in the blood and transport additional lipids to the liver. [0092] In certain instances, inhibiting CETP disrupts the metabolism of HDL. In certain instances, inhibiting CETP prevents transfer of HDL-cholesterol and increases circulating levels of cholesteryl-ester enriched (larger) HDL subfractions. In some embodiments, inhibiting (partially or fully) CETP treat CVD. In certain instances, slowing the catabolism of HDL increases total circulating HDL levels. In certain instances, increasing total circulating HDL levels treats atherogenesis. In some embodiments, inhibiting (partially or fully) CETP results (partially or fully) in inflammation and/or worsening of CVD. In certain instances, increasing total circulating HDL levels generates a lipid pool with reduced clearance (kinetics). In certain instances, reduced clearance of lipids increases HDL capacity to harbor oxidizable and potentially inflammatory lipid stores.
[0093] Low-density lipoprotein (LDL) is a type of lipoprotein that transports cholesterol and triglycerides from the liver to peripheral tissues. In certain instances, LDL comprises an
Apolipoprotein B (ApoB). In certain instances, ApoB is expressed as two isoforms, ApoB48 and ApoBlOO. In certain instances, ApoB48 is synthesized by intestinal cells. In certain instances, ApoB 100 is synthesized in the liver. In certain instances, Hspl 10 stabilizes ApoB.
[0094] In some embodiments, the PF4/RANTES targeting agents, methods and compositions described herein treat atherosclerosis. As used herein, "atherosclerosis" means inflammation of an arterial wall. In certain instances, the inflammation results from (partially or fully) the accumulation of macrophage white blood cells. In certain instances, the inflammation results from (partially or fully) the presence of oxidized LDL. In certain instances, oxidized LDL damages an arterial wall. In certain instances, monocytes respond to (i.e., follow a chemotactic gradient to) the damaged arterial wall. In certain instances, the monocytes differentiate into macrophages. In certain instances, macrophages endocytose the oxidized-LDL (cells such as macrophages with endocytosed LDL are called "foam cells"). In certain instances, a foam cell dies. In certain instances, the rupture of a foam cell deposits oxidized cholesterol into the artery wall. In certain instances, the arterial wall becomes inflamed due to the damaged caused by the oxidized LDL. In certain instances, cells form a hard covering over the inflamed area. In certain instances, the cellular covering narrows an artery.
[0095] In certain instances, an atheromatous plaque is divided into three distinct components: (a) the atheroma (i.e., a nodular accumulation of a soft, flaky, yellowish material comprised of macrophages nearest the lumen of the artery; (b) areas of cholesterol crystals; and (c) calcification at the outer base.
[0096] In certain instances, an atherosclerotic plaque results (partially or fully) in stenosis (i.e., the narrowing of blood vessel). In certain instances, stenosis results (partially or fully) in decreased blood flow. In some embodiments, the PF4/RANTES targeting agents, methods and compositions described herein treat stenosis and/or restenosis. In certain instances, an atherosclerotic plaque results (partially or fully) in the development of an aneurysm. In some embodiments, the PF4/RANTES targeting agents, methods and compositions described herein treat an aneurysm. In certain instances, the rupture of an atherosclerotic plaque results (partially or fully) in an infarction (i.e., the deprivation of oxygen) to a tissue. In some embodiments, the PF4/RANTES targeting agents, methods and compositions described herein treat an infarction.
[0097] In some embodiments, the PF4/RANTES targeting agents, methods and compositions described herein treat a myocardial infarction. "Myocardial infarction" and "heart attack" are used interchangeably. As used herein, both terms refer to an interruption in the blood supply to the heart. In certain instances, an interruption in the blood supply to the heart results from (partially or fully) the occlusion of a coronary artery by a ruptured atherosclerotic plaque. In certain instances, occlusion of an artery results in the infarction of myocardium. In certain instances, the infarction of myocardium results in the scarring of myocardial tissue. In certain instances, scarred of myocardial tissue conducts electrical impulses more slowly than unscarred tissue. In certain instances, the difference in conduction velocity between scarred and unscarred tissue results (partially or fully) in ventricular fibrillation or ventricular tachycardia.
[0098] In some embodiments, the PF4/RANTES targeting agents, methods and compositions described herein treat an angina (e.g., stable or unstable). As used herein, "angina pectoris" refers chest pain resulting from (partially or fully) ischemia of the heart.
[0099] In some embodiments, the PF4/RANTES targeting agents, methods and compositions described herein treat a thrombosis (venous or arterial). As used herein, "thrombosis" refers to the formation of a blood clot. In certain instances, the blood clot forms in a vein (i.e., venous thrombosis). In certain instances, the blood clot forms in an artery (i.e., arterial thrombosis). In certain instances, a piece of or the entire blood clot is transported (i.e., an embolism) to the lungs (i.e., a pulmonary embolism). In some embodiments, the PF4/RANTES targeting agents, methods and compositions described herein treat an embolism.
[00100] In some embodiments, the PF4/RANTES targeting agents, methods and compositions described herein treat a stroke. As used herein, "stroke" refers to a loss of brain function (e.g., necrosis of brain tissue) resulting from (partially or fully) a disturbance in blood supply (e.g., ischemia). In certain instances, a stroke results from (partially or fully) a thrombosis or an embolism.
[00101] In certain instances, an atherosclerotic plaque results (partially or fully) in the development of an aneurysm. In some embodiments, the PF4/RANTES targeting agents, methods and compositions described herein treat an aneurysm. In some embodiments, the PF4/RANTES targeting agents, methods and compositions described herein treat an abdominal aortic aneurysm ("AAA"). As used herein, an "abdominal aortic aneurysm" is a localized dilatation of the abdominal aorta. In certain instances, the rupture of an AAA results in bleeding, leading to hypovolemic shock with hypotension, tachycardia, cyanosis, and altered mental status.
Treatment of AAA [00102] In some embodiments, the PF4/RANTES targeting agents, methods and compositions described herein treat abdominal aortic aneurysms ("AAA").
[00103] Disclosed herein, in certain embodiments, is a method of treating AAA comprising administering to an individual in need thereof (a) a peptide that mimics the RANTES binding domain of PF4, and inhibits the formation of a PF4 and RANTES heterodimer, or (b) a peptide that comprises an amino acid that is 85% homologous (i.e., is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homologous, including any amount of homology between 85% and 100%) to an amino acid sequence selected from:
Figure imgf000038_0001
NO. 86);
ATALCAPASASPYSS (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID
NO. 87);
SDTTPCCFAYIARPL (SEQ ID NO. 65) YSFVKCCSFKGTTVYALSNVRS (SEQ ID
NO. 88);
PLPRAHIKEYFYTSG (SEQ ID NO. 66) SFVKCCSFKGTTVYALSNVRSY (SEQ ID
NO. 89);
EYFYTSGKCSNPAW (SEQ ID NO. 67) FVKCCSFKGTTVYALSNVRSYS (SEQ ID
NO. 90)
VFVTRKNPvQVCANPE (SEQ ID NO. 66)
[00104] Disclosed herein, in certain embodiments, is the use of (a) a peptide that mimics the
RANTES binding domain of PF4, and inhibits the formation of a PF4 and RANTES heterodimer, or (b) a peptide that comprises an amino acid that is 85% homologous (i.e., is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homologous, including any amount of homology between 85% and 100%) to an amino acid sequence selected from:
MSSAAGFCASRP (SEQ ID NO. 46) EKKWVREYINSLEMS (SEQ ID NO. 69)
RPGLLFLGLLLLPL (SEQ ID NO. 47) MKISAAALTIILTAA (SEQ ID NO. 70)
PLWAFASAEA (SEQ ID NO. 48) AALCTPAPASPYGSD (SEQ ID NO. 71)
SAEAEEDGDLQC (SEQ ID NO. 49) SDTTPCCFAYLSLAL (SEQ ID NO. 72)
QCLCVKTTSQVRPRH (SEQ ID NO. 50) ALPRAHVKEYFYTSS (SEQ ID NO. 73)
PRHITSLEVIKAGPHC (SEQ ID NO. 51) SSKCSNLAWFVTRR (SEQ ID NO. 74)
PTAQLIATLKNGRKIC (SEQ ID NO. 52) RRNRQVCANPEKKWV (SEQ ID NO. 75)
LDLQAPLYKKIIKKLLES (SEQ ID NO. 53) EKKWVQEYINYLEMS (SEQ ID NO. 76)
MSVAAVFRGLRPSPE (SEQ ID NO. 54) S SKS SNLA WFVTRCCKEYFYT (SEQ ID
NO. 77)
SPELLLLGLLFLPAV (SEQ ID NO. 55) SKS SNLA WFVTRCCKEYFYTS (SEQ ID
NO. 78)
PAVVAVTSAGPEESD (SEQ ID NO. 56) KS SNLA WFVTRCCKEYFYT S S (SEQ ID
NO. 79);
ESDGDLSCVCVKTIS (SEQ ID NO. 57) S SNLA WFVTRCCKEYFYT S SK (SEQ ID
NO. 80);
TISSGIHLKHITSLE (SEQ ID NO. 58) SNLA WFVTRCCKEYFYTS SKS (SEQ ID
NO. 81);
VIKAGRHCAVPQLIA (SEQ ID NO. 59) NLA WFVTRCCKEYFYT S SKS S (SEQ ID
NO. 82); LIATLKNGRKICLDR (SEQ ID NO. 60) SFKGTTVYALSNVRSYSFVKCC (SEQ ID
NO. 83);
LDRQAPLYKKVIKKI (SEQ ID NO. 61) FKGTTVYALSNVRSYSFVKCCS (SEQ ID
NO. 84);
QAPLYKKVIKKILES (SEQ ID NO. 62) SNVRSYSFVKCCSFKGTTVYAL (SEQ ID
NO. 85);
MKVSAAALAVILIAT (SEQ ID NO. 63) NVRSYSFVKCCSFKGTTVYALS (SEQ ID
NO. 86);
ATALCAPASASPYSS (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID
NO. 87);
SDTTPCCFAYIARPL (SEQ ID NO. 65) YSFVKCCSFKGTTVYALSNVRS (SEQ ID
NO. 88);
PLPRAHIKEYFYTSG (SEQ ID NO. 66) SFVKCCSFKGTTVYALSNVRSY (SEQ ID
NO. 89);
EYFYTSGKCSNPAW (SEQ ID NO. 67) FVKCCSFKGTTVYALSNVRSYS (SEQ ID
NO. 90)
VFVTRKNRQVCANPE (SEQ ID NO. 66)
for the treatment of AAA.
[00105] In some embodiments, the PF4/RANTES targeting agents, methods and compositions described herein prevents (partially or fully) or reduces inflammation at the site of an AAA. In some embodiments, the PF4/RANTES targeting agents, methods and compositions described herein decreases the dilation of the abdominal aorta. In some embodiments, the PF4/RANTES targeting agents, methods and compositions described herein partially or fully inhibit the breakdown of a structural protein (e.g., elastin and collagen) in an AAA. In some embodiments, the PF4/RANTES targeting agents, methods and compositions described herein facilitate the regeneration of a structural protein (e.g., elastin and collagen) in an AAA. In some embodiments, the PF4/RANTES targeting agents, methods and compositions described herein partially or fully inhibit the activation of an MMP in an AAA. In some embodiments, the PF4/RANTES targeting agents, methods and compositions described herein inhibit the upregulation of MMP-1, MMP-9 or MMP-12 in an AAA.
Use as an Imaging and/or Diagnostic Agent
[00106] Disclosed herein, in certain embodiments, is a method of monitoring a disease, disorder or condition characterized by inflammation mediated by the binding of PF4 and RANTES. In some embodiments, the method comprises (a) administering to the individual an agent that (i) specifically binds to all or a portion of PF4, RANTES, or a combination thereof, and (ii) is labeled with an imaging agent; (b) exposing the individual to an imaging device for a period of time sufficient to generate an image of the agent. In some embodiments, the agent localizes to the site of an abdominal aortic aneurysm. In some embodiments, the agent is an antibody, a peptibody, a small molecule, or a peptide. In some embodiments, the agent is a peptide that (a) mimics all or a portion of PF4, or (b) all or a portion of PvANTES. In some embodiments, the agent is a peptide that (a) mimics all or a portion of the PvANTES binding domain of PF4, or (b) all or a portion of PF4 binding domain of PvANTES. In some embodiments, the peptide comprises an amino acid that is 85% homologous (i.e., is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homologous, including any amount of homology between 85% and 100%) to an amino acid sequence: CKEYFYTSSKSSNLAVVFVTRC (SEQ ID NO: 13). In some embodiments, the peptide comprises an amino acid that is 85% homologous (i.e., is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homologous, including any amount of homology between 85% and 100%) to an amino acid sequence
CKEYFYTSGKSSNPGIVFITRC (SEQ ID NO: 16). In some embodiments, the peptide is
CKEYFYTSSKSSNLAVVFVTRC (SEQ ID NO: 13) or CKEYFYTSGKSSNPGIVFITRC (SEQ ID NO: 16). In some embodiments, the agent is an anti-PF4 antibody, an anti-RANTES antibody, or a combination thereof.
[00107] Disclosed herein, in certain embodiments, is a method of diagnosing an abdominal aortic aneurysm in an individual in need thereof, comprising (a) administering to the individual an agent that (i) specifically binds to all or a portion of PF4, RANTES, or a combination thereof, and (ii) is labeled with an imaging agent; (b) exposing the individual to an imaging device for a period of time sufficient to generate an image of the agent; and (c) diagnosing the individual with an abdominal aortic aneurysm if the image of the agent shows a concentration of the agent in the abdominal aorta that exceeds the concentration in a control. In some embodiments, the agent localizes to the site of an abdominal aortic aneurysm. In some embodiments, the agent is an antibody, a peptibody, a small molecule, or a peptide. In some embodiments, the agent is a peptide that (a) mimics all or a portion of PF4, or (b) all or a portion of RANTES. In some embodiments, the agent is a peptide that (a) mimics all or a portion of the RANTES binding domain of PF4, or (b) all or a portion of PF4 binding domain of RANTES. In some embodiments, the peptide comprises an amino acid that is 85% homologous (i.e., is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%), 99%, or 100% homologous, including any amount of homology between 85% and 100%) to an amino acid sequence: CKEYFYTSSKSSNLAVVFVTRC (SEQ ID NO: 13). In some embodiments, the peptide comprises an amino acid that is 85% homologous (i.e., is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homologous, including any amount of homology between 85% and 100%) to an amino acid sequence
CKEYFYTSGKSSNPGIVFITRC (SEQ ID NO: 16). In some embodiments, the peptide is
CKEYFYTSSKSSNLAVVFVTRC (SEQ ID NO: 13) or CKEYFYTSGKSSNPGIVFITRC (SEQ ID NO: 16). In some embodiments, the agent is an anti-PF4 antibody, an anti-PvANTES antibody, or a combination thereof.
[00108] Disclosed herein, in certain embodiments, is a method of monitoring an abdominal aortic aneurysm in an individual in need thereof, comprising (a) administering to the individual an agent that (i) specifically binds to all or a portion of PF4, PvANTES, or a combination thereof, and (ii) is labeled with an imaging agent; (b) exposing the individual to an imaging device for a period of time sufficient to generate an image of the agent; wherein a decrease in the concentration of the agent in the abdominal aorta as compared to an earlier image of the agent indicates that the Abdominal Aortic Aneurysm (AAA) is regressing, (ii) an increase in the concentration of the agent in the abdominal aorta as compared to an earlier image of the agent indicates that the Abdominal Aortic Aneurysm (AAA) is progressing, or (iii) no change in the concentration of the agent in the abdominal aorta as compared to an earlier image of the agent indicates that the Abdominal Aortic Aneurysm (AAA) is neither regressing or progressing. In some embodiments, the agent localizes to the site of an abdominal aortic aneurysm. In some embodiments, the agent is an antibody, a peptibody, a small molecule, or a peptide. In some embodiments, the agent is a peptide that (a) mimics all or a portion of PF4, or (b) all or a portion of PvANTES. In some embodiments, the agent is a peptide that (a) mimics all or a portion of the PvANTES binding domain of PF4, or (b) all or a portion of PF4 binding domain of RANTES. In some embodiments, the peptide comprises an amino acid that is 85% homologous (i.e., is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%o, 97%o, 98%o, 99%o, or 100% homologous, including any amount of homology between 85% and 100%) to an amino acid sequence: CKEYFYTSSKSSNLAVVFVTRC (SEQ ID NO: 13). In some embodiments, the peptide comprises an amino acid that is 85% homologous (i.e., is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homologous, including any amount of homology between 85% and 100%) to an amino acid sequence
CKEYFYTSGKSSNPGIVFITRC (SEQ ID NO: 16). In some embodiments, the peptide is
CKEYFYTSSKSSNLAVVFVTRC (SEQ ID NO: 13) or CKEYFYTSGKSSNPGIVFITRC (SEQ ID NO: 16).
[00109] In some embodiments, the agent that specifically binds to all or a portion of PF4, RANTES, or a combination thereof is administered to identify and/or locate a site of inflammation. In some embodiments, the agent that specifically binds to all or a portion of PF4, RANTES, or a combination thereof disclosed herein is administered to identify and/or locate a site of chronic inflammation. In some embodiments, the agent that specifically binds to all or a portion of PF4, RANTES, or a combination thereof disclosed herein is administered to identify and/or locate a site of acute inflammation.
[00110] In some embodiments, the agent that specifically binds to all or a portion of PF4, RANTES, or a combination thereof disclosed herein is administered to identify and/or locate an atherosclerotic plaque. In some embodiments, the agent that specifically binds to all or a portion of PF4, RANTES, or a combination thereof disclosed herein is administered to identify and/or locate an AAA. In some embodiments, the agent that specifically binds to all or a portion of PF4, RANTES, or a combination thereof disclosed herein is administered to identify and/or locate an occlusion of an artery. In some embodiments, the agent that specifically binds to all or a portion of PF4, RANTES, or a combination thereof disclosed herein is administered to identify and/or locate a thrombus. In some embodiments, the agent that specifically binds to all or a portion of PF4, RANTES, or a combination thereof disclosed herein is administered to identify and/or locate an embolism.
[00111] In some embodiments, the agent that specifically binds to all or a portion of PF4, RANTES, or a combination thereof disclosed herein localizes to areas of a body with high concentrations of leukocytes. In some embodiments, the agent that specifically binds to all or a portion of PF4, RANTES, or a combination thereof disclosed herein localizes to areas of a circulatory system with high concentrations of leukocytes. In some embodiments, an area of the circulatory system with high concentrations of leukocytes is an atherosclerotic plaque. In some embodiments, an area of the circulatory system with high concentrations of leukocytes is an AAA. In some embodiments, an area of the circulatory system with high concentrations of leukocytes is an occlusion of an artery. In some embodiments, an area of the circulatory system with high concentrations of leukocytes is a thrombus. In some embodiments, an area of the circulatory system with high concentrations of leukocytes is an embolism.
[00112] In some embodiments, the agent that specifically binds to all or a portion of PF4, RANTES, or a combination thereof disclosed herein is labeled for imaging (e.g., molecular imaging). In some embodiments, the agent that specifically binds to all or a portion of PF4, RANTES, or a combination thereof disclosed herein is labeled for medical imaging. In some embodiments, the agent that specifically binds to all or a portion of PF4, RANTES, or a combination thereof disclosed herein is labeled for radio-imaging, PET imaging, SPECT imaging, MRI imaging, ultrasound imaging, optical imaging modalities (e.g., near-infrared, fluorescent, optical coherence tomography (OCT)), and combination imaging modalities (e.g., CT/SPECT, CT/PET, MR/optical, PET/optical).
[00113] In some embodiments, the label is a radiolabel. In some embodiments, the label is a fluorescent label. In some embodiments, the label is magnetic, paramagnetic or superparamagnetic. In some embodiments, the agent is labeled with 17F, 18F, 117I, U8I, U9I, 120I, 121I, 122I, 124I, 126L 1281 131I, 10C, UC, 130, 140, 150, 74Br, 75Br, 76Br, 77Br, 78Br, 82Br, mIn, 225Ac, 13N, 82Rb, 32C1, 33C1, 34C1, MCu, monobromobimane (mBBr), dibromobimane, monobromotrimethylammoniobimane, indocyanine green acylthiazolidinethione (ICG- ATT), 7-amino-4-methyl coumarin-3 -acetic acid (AMCA), guanidinoethanethiol (GET), ferucarbotran, uC-dimethylamine, perfluoro-15-crown-5-ether (PFCE), perfluorooctylbromide (PFOB), Rhodamine 6G, Rhodamine B, Rhodamine 123, N-[2-(4- 18F-fluorobenzamido)ethyl]maleimide, colloidal gold particles, colloidal silver particles, colloidal iron particles, colloidal gadolinium particles, iron dextran particles, gold dextran particles, silver dextran particles, or a combination thereof.
[00114] In some embodiments, a labeled agent disclosed herein is detected by any suitable method (e.g., by use of a gamma camera, MRI, PET scanner, x-ray computed tomography (CT), functional magnetic resonance imaging (fJVIRI), and single photon emission computed tomography (SPECT)).
[00115] In some embodiments, the agent that specifically binds to all or a portion of PF4, RANTES, or a combination thereof disclosed herein is labeled by conjugating the imaging agent to a portion of the PF4/RANTES targeting agent (e.g., a peptide residue) that has minimal effect on the binding affinity of the PF4/RANTES targeting agent.
AAA
[00116] In some embodiments, an Abdominal Aortic Aneurysm (AAA) is diagnosed by (a) administering to an individual the agent that specifically binds to all or a portion of PF4, RANTES, or a combination thereof that has been labeled (the "labeled agent"), (b) exposing the individual to an imaging device for a period of time sufficient to generate an image of the labeled agent, and (c) indicating that the individual should be selected for treatment of a Abdominal Aortic Aneurysm (AAA) if the image of the labeled agent shows a concentration of the labeled agent in the abdominal aorta that exceeds the level in a control. In some embodiments, the control is the image produced from a healthy individual that (a) was administered the agent that specifically binds to all or a portion of PF4, RANTES, or a combination thereof that had been labeled; and (b) exposed to an imaging device for a period of time sufficient to generate an image of the labeled agent. In some embodiments, wherein the image of the labeled agent shows a concentration of the labeled agent in the abdominal aorta that exceeds the level in a control, the individual is administered an agent disclosed herein. In some embodiments, the PF4/RANTES targeting agent is
CKEYFYT S SKS SNLAVVFVTRC (SEQ ID NO: 13). In some embodiments, the PF4/RANTES targeting agent is CKEYF YTS GKS SNPGIVFITRC (SEQ ID NO: 16). In some embodiments, the peptide is CKEYF YTSSKSSNLAVVFVTRC (SEQ ID NO: 13) or
CKEYFYTSGKS SNPGIVFITRC (SEQ ID NO: 16).
[00117] In some embodiments, high concentrations of PF4 or RANTES are found at the site of an AAA. In some embodiments, the PF4/RANTES targeting agent localizes to areas of the circulatory system with high concentrations of PF4 or RANTES. In some embodiments, the PF4/RANTES targeting agent localizes to an AAA.
[00118] In some embodiments, the progression or regression of an Abdominal Aortic Aneurysm (AAA) is determined by (a) administering to an individual the agent that specifically binds to all or a portion of PF4, RANTES, or a combination thereof that has been labeled (the "labeled agent"), and (b) exposing the individual to an imaging device for a period of time sufficient to generate an image of the labeled agent. [00119] In some embodiments, a decrease in the concentration of the labeled agent in the abdominal aorta as compared to an earlier image of the label agent indicates that the Abdominal Aortic Aneurysm (AAA) is regressing. In some embodiments, where an Abdominal Aortic Aneurysm (AAA) is regressing, the dose of the agent administered to the individual is maintained. In some embodiments, where an Abdominal Aortic Aneurysm (AAA) is regressing, the dose of the agent administered to the individual is decreased.
[00120] In some embodiments, an increase in the concentration of the labeled agent in the abdominal aorta as compared to an earlier image of the labeled agent indicates that the Abdominal Aortic Aneurysm (AAA) is progressing. In some embodiments, where an Abdominal Aortic Aneurysm (AAA) is progressing, the dose of the agent administered to the individual is increased. In some embodiments, where an Abdominal Aortic Aneurysm (AAA) is progressing, an alternative agent is administered to the individual. In some embodiments, the alternative agent is administered in combination with the agent administered previously.
[00121] In some embodiments, no change in the concentration of the labeled agent in the abdominal aorta as compared to an earlier image of the labeled agent indicates that the Abdominal Aortic Aneurysm (AAA) is neither regressing nor progressing. In some embodiments, where an Abdominal Aortic Aneurysm (AAA) is neither progressing nor regressing, the dose of the agent administered to the individual is increased. In some embodiments, where an Abdominal Aortic Aneurysm (AAA) is neither progressing nor regressing, an alternative agent is administered to the individual. In some embodiments, the alternative agent is administered in combination with the agent administered previously.
[00122] In some embodiments, the agent that specifically binds to all or a portion of PF4, RANTES, or a combination thereof disclosed herein is labeled for imaging (e.g., molecular imaging). In some embodiments, the agent that specifically binds to all or a portion of PF4, RANTES, or a combination thereof disclosed herein is labeled for medical imaging. In some embodiments, the agent that specifically binds to all or a portion of PF4, RANTES, or a combination thereof disclosed herein is labeled for radio-imaging, PET imaging, SPECT imaging, MRI imaging, ultrasound imaging, optical imaging modalities (e.g., near-infrared, fluorescent, optical coherence tomography (OCT)), and combination imaging modalities (e.g., CT/SPECT, CT/PET, MR/optical, PET/optical).
[00123] In some embodiments, the label is a radiolabel. In some embodiments, the label is a fluorescent label. In some embodiments, the label is magnetic, paramagnetic or superparamagnetic. In some embodiments, the agent is labeled with 17F, 18F, 117I, U8I, U9I, 120I, 121I, 122I, 124I, 126I, 1281 131I, 10C, UC, 130, 140, 150, 74Br, 75Br, 76Br, 77Br, 78Br, 82Br, mIn, 225Ac, 13N, 82Rb, 32C1, 33C1, 34C1, MCu, monobromobimane (mBBr), dibromobimane, monobromotrimethylammoniobimane, indocyanine green acylthiazolidinethione (ICG- ATT), 7-amino-4-methyl coumarin-3 -acetic acid (AMCA), guanidinoethanethiol (GET), ferucarbotran, uC-dimethylamine, perfluoro-15-crown-5-ether (PFCE), perfluorooctylbromide (PFOB), Rhodamine 6G, Rhodamine B, Rhodamine 123, N-[2-(4- 18F-fluorobenzamido)ethyl]maleimide, colloidal gold particles, colloidal silver particles, colloidal iron particles, colloidal gadolinium particles, iron dextran particles, gold dextran particles, silver dextran particles, or a combination thereof.
[00124] In some embodiments, a labeled agent disclosed herein is detected by any suitable method (e.g., by use of a gamma camera, MRI, PET scanner, x-ray computed tomography (CT), functional magnetic resonance imaging (fJVIRI), and single photon emission computed tomography (SPECT)).
[00125] In some embodiments, the agent that specifically binds to all or a portion of PF4, RANTES, or a combination thereof disclosed herein is labeled by conjugating the imaging agent to a portion of the PF4/RANTES targeting agent (e.g., a peptide residue) that has minimal effect on the binding affinity of the PF4/RANTES targeting agent.
[00126] In some embodiments, an individual in need thereof displays one or more risk factors for developing an Abdominal Aortic Aneurysm (AAA) (e.g., 60 years of age or older; male; cigarette smoking; high blood pressure; high serum cholesterol; diabetes mellitus; atherosclerosis).
Small Molecules as PF4/RANTES Targeting Agents
[00127] In some embodiments, the ability of RANTES and PF4 to bind together is disrupted by use of a small molecule that binds to the PF4 binding domain of RANTES. In some embodiments, the ability of PF4 and RANTES to bind together is disrupted by use of a small molecule that binds to the RANTES binding domain of PF4.
[00128] In some embodiments, the function of a RANTES/PF4 heteromultimer (e.g., a heterodimer) is disrupted by use of a small molecule that binds to a RANTES/PF4 heterodimer.
Antibodies as PF4/RANTES Targeting Agents
[00129] In some embodiments, an inflammatory disease, disorder, condition, or symptom is treated by disrupting the ability of PF4 and RANTES to bind each other.
[00130] In some embodiments, an inflammatory disease, disorder, condition, or symptom is treated by administering to an individual in need an antibody that binds to PF4, one or more PF4 motifs. In some embodiments, an inflammatory disease, disorder, condition, or symptom is treated by administering to an individual in need an antibody that binds to the RANTES binding domain of PF4.
[00131] In some embodiments, an inflammatory disease, disorder, condition, or symptom is treated by administering to an individual in need an antibody that binds to RANTES, one or more RANTES motifs. In some embodiments, an inflammatory disease, disorder, condition, or symptom is treated by administering to an individual in need an antibody that binds to the PF4 binding domain of RANTES. [00132] In some embodiments, an inflammatory disease, disorder, condition, or symptom is treated by administering to an individual in need an antibody that binds to a PF4/RANTES multimer, or one or more motifs on a PF4/RANTES multimer.
[00133] In some embodiments, the antibody is a human antibody or a humanized antibody. In some embodiments, the antibody is a human IgG. In some embodiments, the antibody is or comprises one or more polypeptides derived from a human IgGl, IgG4, IgG2, IgD, IgA or IgM. An antibody disclosed herein is generated by any suitable method.
Antigen-Based Antibody Development
[00134] In some embodiments, an antibody disclosed herein is generated by contacting a host (e.g., a mouse or rabbit) with an antigen. In some embodiments, the antigen is PF4. In some embodiments, the antigen is RANTES. In some embodiments, the antigen is a PF4/RANTES multimer.
[00135] In some embodiments, the antigen is a fragment of a full-length RANTES polypeptide. In some embodiments, the antigen is a polypeptide that encompasses all or part of a RANTES alpha- helix (e.g., human RANTES58.67) or a peptide that is at least 85% homologous thereto. In some embodiments, the antigen is a polypeptide that encompasses all or part of a RANTES beta-strand (e.g., human RANTES25.30, human RANTES38.44, human RANTES47.51) or a peptide that is at least 85% homologous thereto. In some embodiments, the antigen is a polypeptide that encompasses all or part of a RANTES beta-sheet (e.g., human RANTES 25-51) or a peptide that is at least 85% homologous thereto. In some embodiments, the antigen is a polypeptide that encompasses all or part of human RANTES25.44 (e.g., KEYFYTSGKCSNPAWFVTR) or a peptide that is at least 85% homologous thereto. In some embodiments, the antigen is a polypeptide that encompasses all or part of mouse RANTES25-44 (e.g., KEYFYTSSKCSNLAWFVTR) or a peptide that is at least 85% homologous thereto. In some embodiments, the antigen is a polypeptide that encompasses all or part of rat RANTES25-44 (e.g., KEYFYTSSKCSNLAWFVTR) or a peptide that is at least 85% homologous thereto. In some embodiments, the antigen is a polypeptide that encompasses all or part of pig RANTES25-44 (e.g., QEYFYTS SKCSMAAWFITR) or a peptide that is at least 85% homologous thereto. In some embodiments, the antigen is a polypeptide that encompasses all or part of dog RANTES25-44 (e.g., QEYFYT S SKC SMPA WFVTR) or a peptide that is at least 85% homologous thereto.
[00136] In some embodiments, the antigen is a fragment of a full-length PF4 polypeptide. In some embodiments, the antigen is a polypeptide that encompasses all or part of the PF4 alpha-helix (e.g., human PF49i_i0i) or a peptide that is at least 85% homologous thereto. In some embodiments, the antigen is a polypeptide that encompasses all or part of a PF4 beta-strand (e.g., human PF455_62, human PF470_75, human PF480_84) or a peptide that is at least 85% homologous thereto. In some embodiments, the antigen is a polypeptide that encompasses all or part of the PF4 beta-sheet (e.g., human PF455_84) or a peptide that is at least 85% homologous thereto. In some embodiments, the antigen is a polypeptide that encompasses all or part of a PF4 DLQ motif (e.g., human PF438_4o, human PF485.87) or a peptide that is at least 85% homologous thereto. In some embodiments, the antigen is a polypeptide that encompasses all or part of the PF4 external loop (human PF451_54) or a peptide that is at least 85% homologous thereto.
[00137] In some embodiments, an antibody disclosed herein is generated by contacting a host (e.g., a mouse or rabbit) with at least two antigens selected from a peptide sequence that encompasses all or part of: human RANTES25-44 (i.e., KEYFYTSGKCSNPAVVFVTR), a sequence that is at least 85% homologous to human RANTES25-44, mouse RANTES25-44 (i.e., KEYFYTSSKCSNLAVVFVTR), a sequence that is at least 85% homologous to mouse RANTES25-44, rat RANTES25-44 (i.e.,
KEYFYTSSKCSNLAVVFVTR), a sequence that is at least 85% homologous to rat RANTES25-44, pig RANTES25-44 (i.e., QEYFYTSSKCSMAAWFITR), a sequence that is at least 85% homologous to pig RANTES25-44, dog RANTES25-44 (i.e., QEYFYTSSKCSMPAWFVTR), a sequence that is at least 85% homologous to dog RANTES25-44, a RANTES alpha-helix (e.g., human RANTESsg-ev), a sequence that is at least 85% homologous to a RANTES alpha-helix, a RANTES beta-strand (e.g., human RANTES25.30, RANTES38.44, RANTES47.51), a sequence that is at least 85% homologous to a RANTES beta-strand, a RANTES beta-sheet (e.g., human RANTES 25-51), a sequence that is at least 85%) homologous to a RANTES beta-sheet, a PF4 alpha-helix (e.g., human PF491.10i), a sequence that is at least 85% homologous to a PF4 alpha-helix, a PF4 beta-strand (e.g., human PF455_62, human PF470_75, human PF480_84), a sequence that is at least 85% homologous to a PF4 beta-strand, a PF4 beta-sheet (e.g., human PF455_84), a sequence that is at least 85% homologous to a PF4 beta- sheet, a PF4 DLQ motif (e.g., human PF438.4o, human PF485.87), a sequence that is at least 85% homologous to a PF4 DLQ motif, a PF4 external loop (e.g., human PF451_54), and a sequence that is at least 85% homologous to a PF4 external loop.
[00138] In some embodiments, an antibody disclosed herein is generated by contacting a host (e.g., a mouse or rabbit) with at least three antigens selected from a peptide sequence that encompasses all or part of: human RANTES25-44 (i.e., KEYFYTSGKCSNPAVVFVTR), a sequence that is at least 85%) homologous to human RANTES25-44, mouse RANTES25-44 (i.e.,
KEYFYTSSKCSNLAVVFVTR), a sequence that is at least 85% homologous to mouse RANTES 25- 44, rat RANTES25-44 (i.e., KEYFYTSSKCSNLAVVFVTR), a sequence that is at least 85% homologous to rat RANTES25-44, pig RANTES25-44 (i.e., QEYFYTSSKCSMAAWFITR), a sequence that is at least 85% homologous to pig RANTES25-44, dog RANTES25-44 (i.e.,
QEYFYTSSKCSMPAWFVTR), a sequence that is at least 85% homologous to dog RANTES25-44, a RANTES alpha-helix (e.g., human RANTES58.67), a sequence that is at least 85% homologous to a RANTES alpha-helix, a RANTES beta-strand (e.g., human RANTES25.30, RANTES38.44, RANTES47. 51), a sequence that is at least 85% homologous to a RANTES beta-strand, a RANTES beta-sheet (e.g., human RANTES 25-51), a sequence that is at least 85% homologous to a RANTES beta-sheet, a PF4 alpha-helix (e.g., human PF491.10i), a sequence that is at least 85% homologous to a PF4 alpha- helix, a PF4 beta-strand (e.g., human PF455_62, human PF470_75, human PF480_84), a sequence that is at least 85% homologous to a PF4 beta-strand, a PF4 beta-sheet (e.g., human PF455_84), a sequence that is at least 85% homologous to a PF4 beta-sheet, a PF4 DLQ motif (e.g., human PF438_4o, human PF485.87), a sequence that is at least 85% homologous to a PF4 DLQ motif, a PF4 external loop (e.g., human PF45 i_54), and a sequence that is at least 85% homologous to a PF4 external loop.
DNA-Based Antibody Development
[00139] In some embodiments, an antibody disclosed herein is generated by contacting a host with a nucleic acid sequence encoding part or all of a RANTES polypeptide (alternatively, "RANTES nucleic acid sequence"). In some embodiments, an antibody disclosed herein is generated by contacting a host with a nucleic acid sequence encoding part or all of a PF4 polypeptide
(alternatively, "PF4 nucleic acid sequence").
[00140] In some embodiments, the nucleic acid sequence has been cloned into an expression vector (e.g., a plasmid).
[00141] In some embodiments, the host is a mammal. In some embodiments, the host is a mouse, a rabbit, or a rat. In some embodiments, the host is a mammalian cell. In some embodiments, the host is a bacterial cell.
[00142] In some embodiments, the nucleic acid sequence (i.e., PF4 or RANTES) is contacted with the host by injecting the nucleic acid sequence into the host intramuscularly or intradermally. In some embodiments, the contacting further comprises applying an electric current to the site of injection (i.e., electroporation). In some embodiments, the nucleic acid sequence (i.e., PF4 or RANTES) is contacted with the host by use of a gene gun.
[00143] In some embodiments, nucleic acid sequence (i.e., PF4 or RANTES) is expressed by a host cell (or a plurality of host cells) to generate an antigen. In some embodiments, the RANTES nucleic acid sequence is expressed by a host cell (or a plurality of host cells) to generate an expressed RANTES polypeptide. In some embodiments, the PF4 nucleic acid sequence is expressed by a host cell (or a plurality of host cells) to generate an expressed PF4 polypeptide.
[00144] In some embodiments, a method of generating an antibody disclosed herein further comprises contacting the host with an adjuvant. In some embodiments, the adjuvant is administered as a nucleic acid sequence. In some embodiments, the adjuvant is administered as a polypeptide or polysaccharide. In some embodiments, the adjuvant is a cytokine, a lymphokine, or a combination thereof. In some embodiments, the adjuvant is an interleukin, a tumor necrosis factor, GM-CSF, or a combination thereof. In some embodiments, the adjuvant is B7-1, B7-2, CD40L, or a combination thereof. In some embodiments, the expression vector containing the nucleic acid sequence (i.e., PF4 or RANTES) further comprises a nucleic acid sequence encoding an adjuvant. In some
embodiments, the host is contacted with a second expression vector encoding an adjuvant. [00145] In some embodiments, the nucleic acid sequence (a) encodes part or all of a PF4 alpha-helix (e.g., TAC AAG AAA ATA ATT AAG AAA CTT TTG GAG AGT), or (b) is a sequence that is at least 85% homologous to a PF4 alpha-helix. In some embodiments, the nucleic acid sequence (a) encodes part or all of a PF4 beta-strand (e.g., ATC ACC AGC CTG GAG GTG ATC AAG, GCC CAA CTG ATA GCC ACG, AGG AAA ATT TGC TTG), or (b) is a sequence that is at least 85% homologous to a PF4 beta-strand. In some embodiments, the nucleic acid sequence (a) encodes part or all of a PF4 beta-sheet (e.g., CTG GAG GTG ATC AAG GCC GGA CCC CAC TGC CCC ACT GCC CAA CTG ATA GCC ACG CTG AAG AAT GGA AGG AAA ATT TGC TTG), or (b) is a sequence that is at least 85% homologous to a PF4 beta-sheet. In some embodiments, the nucleic acid sequence (a) encodes part or all of a PF4 DLQ motif (e.g., GAC CTG CAG, GAC CTG CAA), or (b) is a sequence that is at least 85% homologous to a PF4 DLQ motif. In some embodiments, the nucleic acid sequence (a) encodes part or all of a PF4 external loop (e.g., CGT CCC AGG CAC), or (b) is a sequence that is at least 85% homologous to a PF4 external loop.
[00146] In some embodiments, the nucleic acid sequence (a) encodes part or all of the RANTES alpha-helix (e.g., TGG GTT CGG GAG TAC ATC AAC TCT TTG GAG), or (b) is a sequence that is at least 85% homologous to a RANTES alpha-helix. In some embodiments, the nucleic acid sequence (a) encodes part or all of a RANTES beta-strand (e.g., ATC AAG GAG TAT TTC TAC, CCA GCA GTC GTC TTT GTC ACC, AAC CGC CAA GTG TGT), or (b) is a sequence that is at least 85%) homologous to a RANTES beta-strand. In some embodiments, the nucleic acid sequence (a) encodes part or all of the RANTES beta-sheet (e.g., ATC AAG GAG TAT TTC TAC ACC AGT GGC AAG TGC TCC AAC CCA GCA GTC GTC TTT GTC ACC CGA AAG AAC CGC CAA GTG TGT), or (b) is a sequence that is at least 85% homologous to a RANTES beta-sheet.
[00147] In some embodiments, an antibody disclosed herein is generated by contacting a host with at least two nucleic acid sequences selected from sequences that encode part or all of: a PF4 alpha- helix (e.g., TAC AAG AAA ATA ATT AAG AAA CTT TTG GAG AGT), a sequence that is at least 85% homologous to a PF4 alpha-helix, a PF4 beta-strand (e.g., ATC ACC AGC CTG GAG GTG ATC AAG, GCC CAA CTG ATA GCC ACG, AGG AAA ATT TGC TTG), a sequence that is at least 85% homologous to a PF4 beta-strand, a PF4 beta-sheet (e.g., CTG GAG GTG ATC AAG GCC GGA CCC CAC TGC CCC ACT GCC CAA CTG ATA GCC ACG CTG AAG AAT GGA AGG AAA ATT TGC TTG), a sequence that is at least 85% homologous to a PF4 beta-sheet, a PF4 DLQ motif (e.g., GAC CTG CAG, GAC CTG CAA), a sequence that is at least 85% homologous to a PF4 DLQ motif, a PF4 external loop (CGT CCC AGG CAC), a sequence that is at least 85% homologous to a PF4 external loop, a RANTES alpha-helix (e.g., TGG GTT CGG GAG TAC ATC AAC TCT TTG GAG), a sequence that is at least 85% homologous to a RANTES alpha-helix, a RANTES beta-strand (e.g., ATC AAG GAG TAT TTC TAC, CCA GCA GTC GTC TTT GTC ACC, AAC CGC CAA GTG TGT), a sequence that is at least 85% homologous to a RANTES beta- strand, a RANTES beta-sheet (e.g., ATC AAG GAG TAT TTC TAC ACC AGT GGC AAG TGC TCC AAC CCA GCA GTC GTC TTT GTC ACC CGA AAG AAC CGC CAA GTG TGT), and a sequence that is at least 85% homologous to a RANTES beta-sheet.
[00148] In some embodiments, an antibody disclosed herein is generated by contacting a host with at least three nucleic acid sequences selected from sequences that encode part or all of: a PF4 alpha- helix (e.g., TAC AAG AAA ATA ATT AAG AAA CTT TTG GAG AGT), a sequence that is at least 85% homologous to a PF4 alpha-helix, a PF4 beta-strand (e.g., ATC ACC AGC CTG GAG GTG ATC AAG, GCC CAA CTG ATA GCC ACG, AGG AAA ATT TGC TTG), a sequence that is at least 85% homologous to a PF4 beta-strand, a PF4 beta-sheet (e.g., CTG GAG GTG ATC AAG GCC GGA CCC CAC TGC CCC ACT GCC CAA CTG ATA GCC ACG CTG AAG AAT GGA AGG AAA ATT TGC TTG), a sequence that is at least 85% homologous to a PF4 beta-sheet, a PF4 DLQ motif (e.g., GAC CTG CAG, GAC CTG CAA), a sequence that is at least 85% homologous to a PF4 DLQ motif, a PF4 external loop (CGT CCC AGG CAC), a sequence that is at least 85% homologous to a PF4 external loop, a RANTES alpha-helix (e.g., TGG GTT CGG GAG TAC ATC AAC TCT TTG GAG), a sequence that is at least 85% homologous to a RANTES alpha-helix, a RANTES beta-strand (e.g., ATC AAG GAG TAT TTC TAC, CCA GCA GTC GTC TTT GTC ACC, AAC CGC CAA GTG TGT), a sequence that is at least 85% homologous to a RANTES beta- strand, a RANTES beta-sheet (e.g., ATC AAG GAG TAT TTC TAC ACC AGT GGC AAG TGC TCC AAC CCA GCA GTC GTC TTT GTC ACC CGA AAG AAC CGC CAA GTG TGT), and a sequence that is at least 85% homologous to a RANTES beta-sheet.
Production of Antibodies
[00149] In some embodiments, an antibody disclosed herein is produced via the use of a hybridoma. As used herein, a "hybridoma" is an immortalized antibody producing cell. In some embodiments, a host (e.g., a mouse or a rabbit) is inoculated with an antigen or a nucleic acid. In some embodiments, B-cells from the host's spleen are extracted. In some embodiments, a hybridoma is generated by fusing (1) an extracted B-cell with (2) a myeloma cell (i.e., hypoxanthine -guanine -phosphoribosyl transferase negative, immortalized myeloma cells). In some embodiments, the B-cell and the myeloma cells are cultured together and exposed to an agent that renders their cell membranes more permeable (e.g., PEG).
[00150] In some embodiments, the culture comprises a plurality of hybridoma, a plurality of myeloma cells, and a plurality of B-cells. In some embodiments, the cells are individual to culturing conditions that select for hybridoma (e.g., culturing with HAT media).
[00151] In some embodiments, an individual hybridoma (i.e., the clone) is isolated and cultured. In some embodiments, the hybridoma are injected into a laboratory animal. In some embodiments, the hybridoma are cultured in a cell culture.
Humanized Antibodies [00152] In some embodiments, the methods described herein comprise a humanized monoclonal antibody. In some embodiments, a humanized monoclonal antibody comprises heavy and light chain constant regions from a human source and variable regions from a murine source.
[00153] In some embodiments, humanized immunoglobulins, including humanized antibodies, are constructed by genetic engineering. In some embodiments, humanized immunoglobulins comprise a framework that is identical to the framework of a particular human immunoglobulin chain (i.e., an acceptor or recipient), and three CDRs from a non-human (donor) immunoglobulin chain. In some embodiments, a limited number of amino acids in the framework of a humanized immunoglobulin chain are identified and chosen to be the same as the amino acids at those positions in the donor rather than in the acceptor.
[00154] In some embodiments, a framework is used from a particular human immunoglobulin that is homologous to the donor immunoglobulin to be humanized. For example, comparison of the sequence of a mouse heavy (or light) chain variable region against human heavy (or light) variable regions in a data bank (for example, the National Biomedical Research Foundation Protein
Identification Resource or the protein sequence database of the National Center for Biotechnology Information - NCBI) shows that the extent of homology to different human regions can vary greatly, for example from about 40% to about 60%, about 70%, about 80%, or higher. By choosing as the acceptor immunoglobulin one of the human heavy chain variable regions that is most homologous to the heavy chain variable region of the donor immunoglobulin, fewer amino acids will be changed in going from the donor immunoglobulin to the humanized immunoglobulin. By choosing as the acceptor immunoglobulin one of the human light chain variable regions that is most homologous to the light chain variable region of the donor immunoglobulin, fewer amino acids will be changed in going from the donor immunoglobulin to the humanized immunoglobulin.
[00155] In some embodiments, a humanized immunoglobulin comprises light and heavy chains from the same human antibody as acceptor sequences. In some embodiments, a humanized
immunoglobulin comprises light and heavy chains from different human antibody germline sequences as acceptor sequences; when such combinations are used, one can readily determine whether the VH and VL bind an epitope of interest using conventional assays (e.g., an ELISA). In some embodiments, the human antibody will be chosen in which the light and heavy chain variable regions sequences, taken together, are overall most homologous to the donor light and heavy chain variable region sequences. In some embodiments, higher affinity is achieved by selecting a small number of amino acids in the framework of the humanized immunoglobulin chain to be the same as the amino acids at those positions in the donor rather than in the acceptor.
[00156] Any suitable method of modifying a framework region is contemplated herein. In some embodiments, the relevant framework amino acids to change are selected based on differences in amino acid framework residues between the donor and acceptor molecules. In some embodiments, the amino acid positions to change are residues known to be important or to contribute to CDR conformation (e.g., canonical framework residues are important for CDR conformation and/or structure). In some embodiments, the relevant framework amino acids to change are selected based on frequency of an amino acid residue at a particular framework position (e.g., comparison of the selected framework with other framework sequences within its subfamily can reveal residues that occur at minor frequencies at a particular position or positions). In some embodiments, the relevant framework amino acids to change are selected based on proximity to a CDR. In some embodiments, the relevant framework amino acids to change are selected based on known or predicted proximity to the antigen-CDR interface or predicted to modulate CDR activity. In some embodiments, the relevant framework amino acids to change are framework residues that are known to, or predicted to, form contacts between the heavy (VH) and light (VL) chain variable region interface. In some embodiments, the relevant framework amino acids to change are framework residues that are inaccessible to solvent.
[00157] In some embodiments, amino acid changes at some or all of the selected positions are incorporated into encoding nucleic acids for the acceptor variable region framework and donor CDRs. In some embodiments, altered framework or CDR sequences are individually made and tested, or are sequentially or simultaneously combined and tested.
[00158] In some embodiments, the variability at any or all of the altered positions is from a few to a plurality of different amino acid residues, including all twenty naturally occurring amino acids or functional equivalents and analogues thereof. In some embodiments, non-naturally occurring amino acids are considered.
[00159] In some embodiments, the humanized antibody sequence is cloned into a vector. In some embodiments, any suitable vector is used. In some embodiments, the vector is a plasmid, viral e.g. 'phage, or phagemid, as appropriate. For further details see, for example, Molecular Cloning: a Laboratory Manual: 2nd edition, Sambrook et al., 1989, Cold Spring Harbor Laboratory Press. Many known techniques and protocols for manipulation of nucleic acid, for example in preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells and gene expression, and analysis of proteins, are described in detail in Short Protocols in Molecular Biology, Second Edition, Ausubel et al. eds., John Wiley & Sons, 1992. The disclosures of Sambrook et al. and Ausubel et al. are incorporated herein by reference for such disclosure.
[00160] In some embodiments, any suitable host cell is transformed with the vector expressing the humanized antibody sequence. In some embodiments, the host cell is bacteria, mammalian cells, yeast and baculovirus systems. The expression of antibodies and antibody fragments in prokaryotic cells such as E. coli is well established in the art. For a review, see for example Pluckthun, A.
Bio/Technology 9: 545-551 (1991). Expression in eukaryotic cells in culture is also available to those skilled in the art as an option for production of the antibodies and antigen-binding fragments described herein, see for recent reviews, for example Raff, M.E. (1993) Curr. Opinion Biotech. 4: 573-576; Trill J.J. et al. (1995) Curr. Opinion Biotech 6: 553-560, each of which is incorporated herein by reference for such disclosure.
[00161] In some embodiments, a mammalian expression system is used. In some embodiments, the mammalian expression system is dehydrofolate reductase deficient ("dhfr- ") Chinese hamster ovary cells. In some embodiments, dhfr- CHO cells are transfected with an expression vector containing a functional DHFR gene, together with a gene that encodes part or all of a desired humanized antibody.
[00162] In some embodiments, DNA is transformed by any suitable method. For eukaryotic cells, suitable techniques include, for example, calcium phosphate transfection, DEAE Dextran, electroporation, liposome-mediated transfection and transduction using retrovirus or other virus, e.g., vaccinia or, for insect cells, baculovirus. For bacterial cells, suitable techniques include, for example, calcium chloride transformation, electroporation and transfection using bacteriophage.
[00163] In some embodiments, a DNA sequence encoding an antibody or antigen-binding fragment thereof is prepared synthetically rather than cloned. In some embodiments, the DNA sequence is designed with the appropriate codons for the antibody or antigen-binding fragment amino acid sequence. In general, one will select preferred codons for the intended host if the sequence will be used for expression. In some embodiments, the complete sequence is assembled from overlapping oligonucleotides prepared by standard methods and assembled into a complete coding sequence. See, e.g., Edge, Nature, 292:756 (1981); Nambair et al., Science, 223: 1299 (1984); Jay et al., J. Biol. Chem., 259:6311 (1984), each of which is incorporated herein by reference for such disclosure.
Peptide Mimics as PF4/RANTES Targeting Agents
[00164] In some embodiments, the interaction of RANTES and PF4 is disrupted by use of a peptide that mimics all or part of RANTES. In some embodiments, the interaction of RANTES and PF4 is disrupted by use of a peptide that mimics the PF4 binding domain of RANTES. In certain instances, PF4 binds to the peptide mimic and thus does not bind to RANTES.
[00165] In some embodiments, the interaction of RANTES and PF4 is disrupted by use of a peptide that mimics all or part of PF4. In some embodiments, the interaction of RANTES and PF4 is disrupted by use of a peptide that mimics the RANTES binding domain of RANTES. In certain instances, RANTES binds to the peptide mimic and thus does not bind to PF4.
[00166] In some embodiments, a peptide mimic described herein is an isolated peptide,
pharmacologically acceptable salts, derivatives, and conjugates thereof.
[00167] In some embodiments, a peptide mimic described herein does not effect (or only partially effect) other functions of the RANTES and/or PF4. In some embodiments, a selective blocking of the recruiting of monocytes is achieved, for example, on endothelium. [00168] In some embodiments, a peptide mimic described herein provides a high specificity, and does not effect (or only partially effect) the many metabolic processes mediated by the chemokines RANTES and PF4, for example, the immune or clotting systems.
[00169] In some embodiments, a peptide mimic described herein comprises between 15 and 25 amino acids. In some embodiments, a peptide mimic described herein comprises between 19 and 25 amino acids. In some embodiments, a peptide mimic described herein comprises no more than 25 amino acids. In a further embodiment, a peptide mimic described herein comprises aboutl5 to about 25 amino acids. Ina further embodiment, a peptide mimic described herein comprises about 15 to about 22 amino acids. In further embodiments, a peptide mimic described herein comprises about 18 to about 23 amino acids, including in the range of about 18 to about 22 amino acids, and including, in the range of about 19 to about 22 amino acids, and also including in the range of about 20 to about 21 amino acids. In certain embodiments, a peptide mimic described herein comprises 22 amino acids.
[00170] In some embodiments, a peptide mimic described herein comprises a cysteine residue at each of the amino-terminal and carboxy-terminal ends. In some embodiments, the cysteine residue at the amino-terminus and the cysteine residue at the carboxy terminus bind together, yielding a ring. In some embodiments, a cyclical peptide mimic is more stable than a non-cyclical peptide mimic.
[00171] The peptide mimics described herein are prepared by any suitable manner (e.g., literature methods).
[00172] In some embodiments, a peptide mimic described herein comprises an amino acid sequence that is at least 85% homologous (i.e., is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%), 96%), 97%), 98%), 99%, or 100% homologous, including any amount of homology between 85%o and 100%) to the amino acid sequence SEQ ID NO: 1, as indicated below:
C-Xl -X2-YFYTS-X3-X4-X5-X6-X7-X8-X9-X10-Xl 1 -XI 2-X13-X14-X15-C, wherein
XI is chosen from the group containing lysine, glutamine, arginine, histidine and asparagine, or an amino acid deletion;
X2 is chosen from the group containing glutamic acid, aspartic acid and glutamine, or an amino acid deletion;
X3 is chosen from the group containing glycine, serine and alanine;
X4 is chosen from the group containing lysine, leucine and arginine;
X5 is chosen from the group containing serine, cysteine, glycine and threonine;
X6 is chosen from the group containing proline and alanine;
X7 is chosen from the group containing asparagine and glutamine;
X8 is chosen from the group containing proline, tyrosine and glycine; X9 is chosen from the group containing glycine, alanine and serine;
XI 0 is chosen from the group containing isoleucine, valine and asparagine;
XI 1 is chosen from the group containing valine, isoleucine and asparagine;
XI 2 is chosen from the group containing phenylalanine, tyrosine, isoleucine, valine, leucine and methionine;
XI 3 is chosen from the group containing isoleucine, valine, leucine, methionine and phenylalanine; XI 4 is chosen from the group containing threonine, glycine, alanine, serine and tyrosine;
XI 5 is chosen from the group containing arginine, lysine, alanine, glutamine, histidine and asparagine, or an amino acid deletion.
[00173] In some embodiments, a peptide mimic described herein is derived from a human RANTES amino acid sequence. In some embodiments, a peptide mimic described herein comprises a portion of a human RANTES amino acid sequence. In some embodiments, a peptide mimic described herein comprises an amino acid sequence that is at least 85% homologous (i.e., is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homologous, including any amount of homology between 85% and 100%) to the amino acid sequence SEQ ID NO: 2, as indicated below:
C-KEYFYTSGKCSNPAWFVTR-C.
[00174] In some embodiments, a peptide mimic described herein is derived from a mouse RANTES amino acid sequence. In some embodiments, a peptide mimic described herein comprises a portion of a mouse RANTES amino acid sequence. In some embodiments, a peptide mimic described herein comprises an amino acid sequence that is at least 85% homologous (i.e., is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homologous, including any amount of homology between 85% and 100%) to the amino acid sequence SEQ ID NO: 3, as indicated below:
C- KEYFYTSSKCSNLAWFVTR-C.
[00175] In some embodiments, a peptide mimic described herein is derived from a pig RANTES amino acid sequence. In some embodiments, a peptide mimic described herein comprises a portion of a pig RANTES amino acid sequence. In some embodiments, a peptide mimic described herein comprises an amino acid sequence that is at least 85% homologous (i.e., is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homologous, including any amount of homology between 85% and 100%) to the amino acid sequence SEQ ID NO: 4, as indicated below:
C- QEYFYTSSKCSMAAWFITR-C.
[00176] In some embodiments, a peptide mimic described herein comprises an amino acid sequence that is at least 85% homologous (i.e., is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%), 98%o, 99%, or 100% homologous, including any amount of homology between 85%o and 100%) to the amino acid sequence SEQ ID NO. 5, as indicated below:
C-Xl -X2-YFYTS-X3-X4-X5-X6-X7-X8-X9-X10-Xl 1 -XI 2-X13-X14-X15-C;
where:
XI is chosen from the group containing lysine, glutamine, arginine, histidine and asparagine, or an amino acid deletion;
X2 is chosen from the group containing glutamic acid, aspartic acid and glutamine, or an amino acid deletion;
X3 is chosen from the group containing glycine, serine and alanine;
X4 is chosen from the group containing lysine, leucine and arginine;
X5 is chosen from the group containing serine, cysteine, glycine and threonine;
X6 is chosen from the group containing proline and alanine;
X7 is chosen from the group containing asparagine and glutamine;
X8 is chosen from the group containing proline, tyrosine and glycine;
X9 is chosen from the group containing glycine, alanine and serine;
XI 0 is chosen from the group containing isoleucine, valine and asparagine;
XI I is chosen from the group containing valine, isoleucine and asparagine;
XI 2 is chosen from the group containing phenylalanine, tyrosine, isoleucine, valine, leucine and methionine;
XI 3 is chosen from the group containing isoleucine, valine, leucine, methionine and phenylalanine; XI 4 is chosen from the group containing threonine, glycine, alanine, serine and tyrosine; and XI 5 is chosen from the group containing arginine, lysine, alanine, glutamine, histidine and asparagine, or an amino acid deletion.
[00177] In some embodiments, a peptide mimic described herein comprises an amino acid sequence that is at least 85% homologous (i.e., is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%), 96%), 97%), 98%), 99%, or 100% homologous, including any amount of homology between 85%) and 100%) to the amino acid sequence SEQ ID NO: 6, as indicated below:
C-X1-X2-YFYTS-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-C (SEQ ID NO: 6) where:
XI is chosen from the group containing lysine, glutamine, arginine, histidine and asparagine, or an amino acid deletion;
X2 is chosen from the group containing glutaminic acid, asparaginic acid and glutamine, or an amino acid deletion; X3 is chosen from the group containing glycine, serine and alanine;
X4 is chosen from the group containing lysine, leucine and arginine;
X5 is chosen from the group containing serine, cysteine, glycine and threonine;
X6 is chosen from the group containing serine, glycine and threonine; X7 is chosen from the group containing methionine, isoleucine, leucine, and phenylalanine;
X8 is chosen from the group containing proline, tyrosine and glycine;
X9 is chosen from the group containing glycine, alanine and serine;
XI 0 is chosen from the group containing isoleucine, valine and asparagine;
XI 1 is chosen from the group containing valine, isoleucine and asparagine;
XI 2 is chosen from the group containing phenylalanine, tyrosine, isoleucine, valine, leucine and methionine;
XI 3 is chosen from the group containing isoleucine, valine, leucine, methionine and phenylalanine; XI 4 is chosen from the group containing threonine, glycine, alanine, serine and tyrosine; and XI 5 is chosen from the group containing arginine, alanine, lysine, glutamine, histidine and asparagine, or an amino acid deletion.
[00178] In some embodiments, a peptide mimic described herein comprises an amino acid sequence that is at least 85% homologous (i.e., is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%o, 97%), 98%), 99%, or 100% homologous, including any amount of homology between 85%) and 100%) to the amino acid sequence SEQ ID NO: 7, as indicated below:
C-X1 -X2-YFYTS-X3-X4-X5-X6-X7-X8-X9-X10-X1 1 -X12-X13-X14-X15-C (SEQ ID NO: 7) where:
XI is chosen from the ; group containing ; lysine, glutamine, arginine, histidine and asparagine, or an amino acid deletion;
X2 is chosen from the ; group containing ; glutaminic acid, asparaginic acid and glutamine, or an amino acid deletion;
X3 is chosen from the ; group containing ; glycine, serine and alanine;
X4 is chosen from the ; group containing ; lysine, leucine and arginine;
X5 is chosen from the ; group containing ; serine, cysteine, glycine and threonine;
X6 is chosen from the ; group containing ; serine, glycine and threonine;
X7 is chosen from the ; group containing ; asparagine and glutamine;
X8 is chosen from the ; group containing ; leucine, isoleucine, phenylalanine, alanine, valine, threonine and methionine;
X9 is chosen from the ; group containing ; glycine, alanine and serine;
XI 0 is chosen from the group containing isoleucine, valine and asparagine;
XI 1 is chosen from the group containing valine, isoleucine and asparagine;
XI 2 is chosen from the group containing phenylalanine, tyrosine, isoleucine, valine, leucine and methionine;
XI 3 is chosen from the group containing isoleucine, valine, leucine, methionine and phenylalanine; XI 4 is chosen from the group containing threonine, glycine, alanine, serine and tyrosine; and XI 5 is chosen from the group containing arginine, alanine, lysine, glutamine, histidine and asparagine, or an amino acid deletion.
[00179] In some embodiments, a peptide mimic described herein comprises an amino acid sequence that is at least 85% homologous (i.e., is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%), 96%), 97%), 98%), 99%, or 100% homologous, including any amount of homology between 85%) and 100%) to the amino acid sequence SEQ ID NO: 8, as indicated below:
C-X1-X2-YFYTS-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-C (SEQ ID NO: 8) where:
XI is chosen from the group containing lysine, glutamine, arginine, histidine and asparagine, or an amino acid deletion;
X2 is chosen from the group containing glutaminic acid, asparaginic acid and glutamine, or an amino acid deletion;
X3 is chosen from the group containing glycine, serine and alanine;
X4 is chosen from the group containing lysine, leucine and arginine;
X5 is chosen from the group containing serine, cysteine, glycine and threonine;
X6 is chosen from the group containing proline and alanine;
X7 is chosen from the group containing methionine, isoleucine, leucine, and phenylalanine;
X8 is chosen from the group containing proline, tyrosine and glycine;
X9 is chosen from the group containing glycine, alanine and serine;
XI 0 is chosen from the group containing isoleucine, valine and asparagine;
XI I is chosen from the group containing valine, isoleucine and asparagine;
XI 2 is chosen from the group containing phenylalanine, tyrosine, isoleucine, valine, leucine and methionine;
XI 3 is chosen from the group containing isoleucine, valine, leucine, methionine and phenylalanine; XI 4 is chosen from the group containing threonine, glycine, alanine, serine and tyrosine; and XI 5 is chosen from the group containing arginine, alanine, lysine, glutamine, histidine and asparagine, or an amino acid deletion.
[00180] In some embodiments, a peptide mimic described herein comprises an amino acid sequence that is at least 85% homologous (i.e., is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%), 96%), 97%), 98%), 99%, or 100% homologous, including any amount of homology between 85%) and 100%) to the amino acid sequence SEQ ID NO: 9, as indicated below:
C-X1-X2-YFYTS-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-C (SEQ ID NO: 9) where:
XI is chosen from the group containing lysine, glutamine, arginine, histidine and asparagine, or an amino acid deletion; X2 is chosen from the group containing glutaminic acid, asparaginic acid and glutamine, or an amino acid deletion;
X3 is chosen from the group containing glycine, serine and alanine;
X4 is chosen from the group containing lysine, leucine and arginine;
X5 is chosen from the group containing serine, cysteine, glycine and threonine;
X6 is chosen from the group containing serine, glycine and threonine;
X7 is chosen from the group containing methionine, isoleucine, leucine, and phenylalanine;
X8 is chosen from the group containing leucine, isoleucine, phenylalanine, alanine, valine, threonine and methionine;
X9 is chosen from the group containing glycine, alanine and serine;
XI 0 is chosen from the group containing isoleucine, valine and asparagine;
XI 1 is chosen from the group containing valine, isoleucine and asparagine;
XI 2 is chosen from the group containing phenylalanine, tyrosine, isoleucine, valine, leucine and methionine;
XI 3 is chosen from the group containing isoleucine, valine, leucine, methionine and phenylalanine; XI 4 is chosen from the group containing threonine, glycine, alanine, serine and tyrosine; and XI 5 is chosen from the group containing arginine, alanine, lysine, glutamine, histidine and asparagine, or an amino acid deletion.
[00181] In some embodiments, a peptide mimic described herein comprises an amino acid sequence that is at least 85% homologous (i.e., is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%o, 97%), 98%), 99%, or 100% homologous, including any amount of homology between 85%) and 100%) to the amino acid sequence SEQ ID NO: 10, as indicated below:
C-X1 -X2-YFYTS-X3-X4-X5-X6-X7-X8-X9-X10-X1 1 -X12-X13-X14-X15-C (SEQ ID NO: 10) where:
XI is chosen from the ; group containing ; lysine, glutamine, arginine, histidine and asparagine, or an amino acid deletion;
X2 is chosen from the ; group containing ; glutaminic acid, asparaginic acid and glutamine, or an amino acid deletion;
X3 is chosen from the ; group containing ; glycine, serine and alanine;
X4 is chosen from the ; group containing ; lysine, leucine and arginine;
X5 is chosen from the ; group containing ; serine, cysteine, glycine and threonine;
X6 is chosen from the ; group containing ; proline and alanine;
X7 is chosen from the ; group containing ; asparagine and glutamine;
X8 is chosen from the ; group containing ; leucine, isoleucine, phenylalanine, alanine, valine, threonine and methionine;
X9 is chosen from the ; group containing ; glycine, alanine and serine; XI 0 is chosen from the group containing isoleucine, valine and asparagine;
XI 1 is chosen from the group containing valine, isoleucine and asparagine;
XI 2 is chosen from the group containing phenylalanine, tyrosine, isoleucine, valine, leucine and methionine;
XI 3 is chosen from the group containing isoleucine, valine, leucine, methionine and phenylalanine; XI 4 is chosen from the group containing threonine, glycine, alanine, serine and tyrosine; and XI 5 is chosen from the group containing arginine, alanine, lysine, glutamine, histidine and asparagine, or an amino acid deletion.
[00182] In some embodiments, a peptide mimic described herein comprises an amino acid sequence that is at least 85% homologous (i.e., is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%o, 97%), 98%), 99%, or 100% homologous, including any amount of homology between 85%) and 100%) to the amino acid sequence SEQ ID NO: 1 1 , as indicated below:
C-X1 -X2-YFYTS-X3-X4-X5-X6-X7-X8-X9-X10-X1 1 -X12-X13-X14-X15-C (SEQ ID NO: 1 1) where:
XI is chosen from the ; group containing ; lysine, glutamine, arginine, histidine and asparagine, or an amino acid deletion;
X2 is chosen from the ; group containing ; glutaminic acid, asparaginic acid and glutamine, or an amino acid deletion;
X3 is chosen from the ; group containing ; glycine, serine and alanine;
X4 is chosen from the ; group containing ; lysine, leucine and arginine;
X5 is chosen from the ; group containing ; serine, cysteine, glycine and threonine;
X6 is chosen from the ; group containing ; proline and alanine;
X7 is chosen from the ; group containing ; methionine, isoleucine, leucine, and phenylalanine;
X8 is chosen from the ; group containing ; leucine, isoleucine, phenylalanine, alanine, valine, threonine and methionine;
X9 is chosen from the ; group containing ; glycine, alanine and serine;
XI 0 is chosen from the group containing isoleucine, valine and asparagine;
XI 1 is chosen from the group containing valine, isoleucine and asparagine;
XI 2 is chosen from the group containing phenylalanine, tyrosine, isoleucine, valine, leucine and methionine;
XI 3 is chosen from the group containing isoleucine, valine, leucine, methionine and phenylalanine; XI 4 is chosen from the group containing threonine, glycine, alanine, serine and tyrosine; and XI 5 is chosen from the group containing arginine, alanine, lysine, glutamine, histidine and asparagine, or an amino acid deletion.
[00183] In some embodiments, a peptide mimic described herein comprises an amino acid sequence that is at least 85% homologous (i.e., is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%), 98%o, 99%, or 100% homologous, including any amount of homology between 85%o and 100%) to the amino acid sequence SEQ ID NO: 12, as indicated below:
C-X1-X2-YFYTS-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-C (SEQ ID NO: 12) where:
XI is chosen from the group containing lysine, glutamine, arginine, histidine and/or asparagine, or an amino acid deletion;
X2 is chosen from the group containing glutaminic acid, asparaginic acid and/or glutamine, or an amino acid deletion;
X3 is chosen from the group containing glycine, serine and/or alanine;
X4 is chosen from the group containing lysine, leucine and/or arginine;
X5 is chosen from the group containing serine, cysteine, glycine and/or threonine;
X6 is chosen from the group containing serine, glycine and/or threonine;
X7 is chosen from the group containing asparagine and/or glutamine;
X8 is chosen from the group containing proline, tyrosine and/or glycine;
X9 is chosen from the group containing glycine, alanine and/or serine;
XI 0 is chosen from the group containing isoleucine, valine and/or asparagine;
XI I is chosen from the group containing valine, isoleucine and/or asparagine;
X12 is chosen from the group containing phenylalanine, tyrosine, isoleucine, valine, leucine and/or methionine;
XI 3 is chosen from the group containing isoleucine, valine, leucine, methionine and/or phenylalanine;
X14 is chosen from the group containing threonine, glycine, alanine, serine and/or tyrosine; and XI 5 is chosen from the group containing arginine, lysine, glutamine, histidine and/or asparagine, or an amino acid deletion.
[00184] In some embodiments, a peptide mimic described herein comprises an amino acid sequence that is at least 85% homologous (i.e., is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%), 96%), 97%), 98%), 99%, or 100% homologous, including any amount of homology between 85%) and 100%) to the amino acid sequence SEQ ID NO: 13, as indicated below:
CKEYFYT S SKS SNLAVVFVTRC (SEQ ID NO: 13).
[00185] In some embodiments, a peptide mimic described herein comprises an amino acid sequence that is at least 85% homologous (i.e., is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%), 96%), 97%), 98%), 99%, or 100% homologous, including any amount of homology between 85%) and 100%) to the amino acid sequence SEQ ID NO: 14, as indicated below:
CSFKGTTVYALSNVRSYSFVKC (SEQ ID NO. 14).
[00186] In some embodiments, a peptide mimic described herein comprises an amino acid sequence that is at least 85% homologous (i.e., is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%o, 97%o, 98%o, 99%, or 100% homologous, including any amount of homology between 85%o and 100%) to the amino acid sequence SEQ ID NO: 15, as indicated below:
CSFKGTNVYALTKVRSYSFVSC (SEQ ID NO. 15).
[00187] In some embodiments, a peptide mimic described herein comprises an amino acid sequence that is at least 85% homologous (i.e., is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%o, 96%o, 97%o, 98%o, 99%, or 100% homologous, including any amount of homology between 85%o and 100%) to the amino acid sequence SEQ ID NO: 16, as indicated below:
CKEYF YTS GKS SNPGIVFITRC (SEQ ID NO: 16).
[00188] In some embodiments, a peptide mimic described herein comprises an amino acid sequence that is at least 85% homologous (i.e., is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%o, 96%o, 97%o, 98%o, 99%, or 100% homologous, including any amount of homology between 85%o and 100%) to an amino acid sequence listed in Table 1.
Figure imgf000063_0001
Species Sequence
Felis catus QEYFYTSSKCSMPAWFVTR (SEQ ID NO. 31)
Canis lupus QEYFYTSSKCSMPAWFVTR (SEQ ID NO. 32)
familiaris
Sus scrofa QEYFYTSSKCSMAAWFITR (SEQ ID NO. 33)
Bos taurus QEYFYTSSKCSMAAWFITR (SEQ ID NO. 34)
Equus caballus QEYFYTSSKCSIPAWFVTR (SEQ ID NO. 35)
Monodelphis REYFYTS SRCGNLGWFITR (SEQ ID NO. 36)
domestica
Loxodonta KEYFYTSGKCSMPAV (SEQ ID NO. 37)
africana
Dasypus KEYFYTSGKCSNPAV (SEQ ID NO. 38)
novemcinctus
Echinops telfairi REYFYTSSKCTSPAVVFVTR (SEQ ID NO. 39)
Erinaceus QEYFYTSSKCSIPSAVVFVTR (SEQ ID NO. 40)
europaeus
Tupaia belangeri REYFYTSGKCSNPAWFITR (SEQ ID NO. 41)
Sorex araneus QDYFYTSSKCSMPAWFVTR (SEQ ID NO. 42)
Gallus gallus KDYFYTS SKCPQAAWFITR (SEQ ID NO. 43)
Anas KDYFYTSSKCPQPAWFITR (SEQ ID NO. 44)
platyrhynchos
Myotis lucifugus QEYFYTSSKCSMPAWLITR (SEQ ID NO. 45)
Table 1
In some embodiments, a peptide mimic described herein comprises an amino acid sequence that is at least 85% homologous (i.e., is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%), 98%), 99%), or 100% homologous, including any amount of homology between 85% and 100%) to disclosed in Table 2.
Figure imgf000064_0001
MSVAAVFRGLRPSPE (SEQ ID NO. 54) S SKS SNLA WFVTRCCKEYFYT (SEQ ID
NO. 77)
SPELLLLGLLFLPAV (SEQ ID NO. 55) SKS SNLA WFVTRCCKEYFYTS (SEQ ID
NO. 78)
PAVVAVTSAGPEESD (SEQ ID NO. 56) KSSNLAWFVTRCCKEYFYTSS (SEQ ID
NO. 79);
ESDGDLSCVCVKTIS (SEQ ID NO. 57) SSNLAWFVTRCCKEYFYTSSK (SEQ ID
NO. 80);
TISSGIHLKHITSLE (SEQ ID NO. 58) SNLA WFVTRCCKEYFYTS SKS (SEQ ID
NO. 81);
VIKAGRHCAVPQLIA (SEQ ID NO. 59) NLA WFVTRCCKEYFYT S SKS S (SEQ ID
NO. 82);
LIATLKNGRKICLDR (SEQ ID NO. 60) SFKGTTVYALSNVRSYSFVKCC (SEQ ID
NO. 83);
LDRQAPLYKKVIKKI (SEQ ID NO. 61) FKGTTVYALSNVRSYSFVKCCS (SEQ ID
NO. 84);
QAPLYKKVIKKILES (SEQ ID NO. 62) SNVRSYSFVKCCSFKGTTVYAL (SEQ ID
NO. 85);
MKVSAAALAVILIAT (SEQ ID NO. 63) NVRSYSFVKCCSFKGTTVYALS (SEQ ID
NO. 86);
ATALCAPASASPYSS (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID
NO. 87);
SDTTPCCFAYIARPL (SEQ ID NO. 65) YSFVKCCSFKGTTVYALSNVRS (SEQ ID
NO. 88);
PLPRAHIKEYFYTSG (SEQ ID NO. 66) SFVKCCSFKGTTVYALSNVRSY (SEQ ID
NO. 89);
EYFYTSGKCSNPAW (SEQ ID NO. 67) FVKCCSFKGTTVYALSNVRSYS (SEQ ID
NO. 90)
VFVTRKNRQVCANPE (SEQ ID NO. 66)
Table 2
[00189] In some embodiments, a peptide mimic described herein comprises an amino acid sequence that is at least 85% homologous (i.e., is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%), 96%), 97%), 98%), 99%, or 100% homologous, including any amount of homology between 85% and 100%) to an amino acid sequence selected from: SSKSSNLA WFVTRCCKEYFYT (SEQ ID NO. 91); SKS SNLAVVFVTRCCKEYFYTS (SEQ ID NO. 92);
KSSNLAWFVTRCCKEYFYTSS (SEQ ID NO. 93); SSNLAWFVTRCCKEYFYTSSK (SEQ ID NO. 94); SNLAWFVTRCCKEYFYTSSKS (SEQ ID NO. 95); NLAWFVTRCCKEYFYTSSKSS (SEQ ID NO. 96); SFKGTTVYALSNVRSYSFVKCC (SEQ ID NO. 97);
FKGTTVYALSNVRSYSFVKCCS (SEQ ID NO. 98); SNVRSYSFVKCCSFKGTTVYAL (SEQ ID NO. 99); NVRSYSFVKCCSFKGTTVYALS (SEQ ID NO. 100);
SYSFVKCCSFKGTTVYALSNVR (SEQ ID NO. 101); YSFVKCCSFKGTTVYALSNVRS (SEQ ID NO. 102); SFVKCCSFKGTTVYALSNVRSY (SEQ ID NO. 103); or
FVKCCSFKGTTVYALSNVRSYS (SEQ ID NO. 104).
[00190] Disclosed herein, in certain embodiments, is a peptide that (a) mimics the RANTES binding domain of PF4, and (b) inhibits the formation of a PF4 and RANTES heterodimer. In some embodiments, the peptide comprises an amino acid that is 85% homologous (i.e., is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homologous, including any amount of homology between 85% and 100%) to an amino acid sequence selected from:
Figure imgf000066_0001
LDRQAPLYKKVIKKI (SEQ ID NO. 61) FKGTTVYALSNVRSYSFVKCCS (SEQ ID
NO. 84);
QAPLYKKVIKKILES (SEQ ID NO. 62) SNVRSYSFVKCCSFKGTTVYAL (SEQ ID
NO. 85);
MKVSAAALAVILIAT (SEQ ID NO. 63) NVRSYSFVKCCSFKGTTVYALS (SEQ ID
NO. 86);
ATALCAPASASPYSS (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID
NO. 87);
SDTTPCCFAYIARPL (SEQ ID NO. 65) YSFVKCCSFKGTTVYALSNVRS (SEQ ID
NO. 88);
PLPRAHIKEYFYTSG (SEQ ID NO. 66) SFVKCCSFKGTTVYALSNVRSY (SEQ ID
NO. 89);
EYFYTSGKCSNPAW (SEQ ID NO. 67) FVKCCSFKGTTVYALSNVRSYS (SEQ ID
NO. 90)
VFVTRKNRQVCANPE (SEQ ID NO. 66)
In some embodiments, the peptide comprises an amino acid sequence that is at least 85% homologous (i.e., is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%o, 99%, or 100% homologous, including any amount of homology between 85% and 100%) to the sequence: CKEYFYTSGAAAAPGIVFITRC (SEQ ID NO: 91).
[00191] In some embodiments, the peptide comprises N- and/or C-terminal chemical modifications to improve ADME-PK; non-natural amino acids. In some embodiments, the peptide is a cyclical variant.
Metabolites
[00192] In some embodiments, the agent that inhibits the interaction of PF4/RANTES interaction is a fragment of any peptide sequence disclosed herein (hereinafter, "peptide fragment"). As used herein, "peptide fragment" means an amino acid polymer produced by cleaving any peptide of comprising an amino acid sequence that is at least 85% homologous to the sequences SEQ ID NO. 1 through SEQ ID NO. 90. In some embodiments, a peptide comprising an amino acid sequence that is at least 85% homologous to any of SEQ ID NO. 1 through SEQ ID NO. 90 is cleaved at one site (e.g., one peptide bond is broken). In some embodiments, a peptide comprising an amino acid sequence that is at least 85% homologous to any of SEQ ID NO. 1 through SEQ ID NO. 90 is cleaved at two sites (e.g., two peptide bonds are broken). In some embodiments, the peptide fragment is produced by the metabolism of a peptide comprising an amino acid sequence that is at least 85% homologous to any of SEQ ID NO. 1 through SEQ ID NO. 90. [00193] In some embodiments, the fragment has structural features similar to a peptide disclosed herein. In some embodiments, the fragment is linear.
[00194] In some embodiments, the fragment comprises between 5 and 10 amino acids. In some embodiments, the fragment comprises 5 amino acids. In some embodiments, the fragment comprises between 6 and 10 amino acids. In some embodiments, the fragment comprises 6 amino acids. In some embodiments, the fragment comprises between 7 and 10 amino acids. In some embodiments, the fragment comprises between 8 and 10 amino acids. In some embodiments, the fragment comprises between 9 and 10 amino acids.
[00195] In some embodiments, the metabolite comprises a formula selected from:
C-X1-X2-X3-X4-X5-T-X6-X7-X8-X9-S-N-X10-X11-X12-X13-X14-X15-X16
C-X1-X2-X3-X4-X5-T-X6-X7-X8-X9-S-N-X10-X11-X12-X13-X14-X15
C-X1-X2-X3-X4-X5-T-X6-X7-X8-X9-S-N-X10-X11-X12-X13-X14
C-X1-X2-X3-X4-X5-T-X6-X7-X8-X9-S-N-X10-X11-X12-X13
C-X 1 -X2-X3 -X4-X5-T-X6-X7-X8 -X9-S-N-X 10-Xl 1 -X 12
C-X1-X2-X3-X4-X5-T-X6-X7-X8-X9-S-N-X10-X11
C-X1-X2-X3-X4-X5-T-X6-X7-X8-X9-S-N-X10
C-X1-X2-X3-X4-X5-T-X6-X7-X8-X9-S-N
C-X 1 -X2-X3 -X4-X5-T-X6-X7-X8 -X9
C-X1-X2-X3-X4-X5-T-X6-X7-X8
C-X1-X2-X3-X4-X5-T-X6-X7
C-X1-X2-X3-X4-X5-T-X6
C-X1-X2-X3-X4-X5-T
X1-X2-X3-X4-X5-T-X6-X7-X8-X9-S-N-X10-X11-X12-X13-X14-X15-X16-K-C
X2-X3-X4-X5-T-X6-X7-X8-X9-S-N-X10-X11-X12-X13-X14-X15-X16-K-C
X3-X4-X5-T-X6-X7-X8-X9-S-N-X10-X11-X12-X13-X14-X15-X16-K-C
X4-X5-T-X6-X7-X8-X9-S-N-X10-X11-X12-X13-X14-X15-X16-K-C X5-T-X6-X7-X8-X9-S-N-X10-X11-X12-X13-X14-X15-X16-K-C
T-X6-X7-X8-X9-S-N-X10-X11-X12-X13-X14-X15-X16-K-C
X6-X7-X8-X9-S-N-X10-X11-X12-X13-X14-X15-X16-K-C
X7-X8-X9-S-N-X10-X11 -X12-X13-X14-X15-X16-K-C
X8-X9-S-N-X10-X11-X12-X13-X14-X15-X16-K-C
X9-S-N-X10-Xl 1 -XI 2-X13-X14-X15-X16-K-C
S-N-Xl 0-X 11 -X 12-X 13 -X 14-X15 -X 16-K-C
N-X10-X11-X12-X13-X14-X15-X16-K-C
C-X1-X2-X3-X4-X5-T-X6-X7-X8-X9-S-N
C-X1-X2-X3-X4-X5-T-X6-X7-X8-X9-S C-X 1 -X2-X3 -X4-X5-T-X6-X7-X8 -X9
C-X1-X2-X3-X4-X5-T-X6-X7-X8
C-X1-X2-X3-X4-X5-T-X6-X7
C-X1-X2-X3-X4-X5-T-X6
C-X1-X2-X3-X4-X5-T
T-X6-X7-X8-X9-S-N-X10-Xl 1 -XI 2-X13-X14-X15-X16-K-C
X6-X7-X8-X9-S-N-X10-X11-X12-X13-X14-X15-X16-K-C
X7-X8-X9-S-N-X10-X11-X12-X13-X14-X15-X16-K-C
X8-X9-S-N-X10-Xl 1 -XI 2-X13 -XI 4-X15-X16-K-C
X9-S-N-X10-Xl 1 -XI 2-X13 -XI 4-X15-X16-K-C
S-N-X10-X11-X12-X13-X14-X15-X16-K-C
N-X10-X11-X12-X13- X14-X15-X16-K-C
wherein:
XI is selected from serine and lysine;
X2 is selected from glutamic acid, phenylalanine and serine;
X3 is selected from lysine and tyrosine;
X4 is selected from phenylalanine and glycine;
X5 is selected from threonine and tyrosine;
X6 is selected from serine and valine;
X7 is selected from serine and tyrosine;
X8 is selected from alanine and lysine;
X9 is selected from leucine and serine;
XI 0 is selected from leucine and valine;
XI I is selected from alanine and arginine;
XI 2 is selected from serine and valine;
XI 3 is selected from valine and tyrosine;
XI 4 is selected from phenylalanine and serine;
XI 5 is selected from phenylalanine and valine; and
XI 6 is selected from threonine and valine.
SAR Chemistry
[00196] In some embodiments, any of the aforementioned peptides and/or peptide fragments is used as a "model" to do structure-activity relationship (SAR) chemistry. In some embodiments, the SAR chemistry yields smaller peptides. In some embodiments, the smaller peptides yield small molecules that disrupt the activity of RANTES and/or PF4 (e.g., by figuring out the amino acid residues involved in disrupting the activity of RANTES and/or PF4). Peptide Mimetics
[00197] In some embodiments, a peptide mimetic is used in place of the peptides described herein, including for use in the treatment or prevention of the diseases disclosed herein.
[00198] Peptide mimetics (and peptide-based inhibitors) are developed using, for example, computerized molecular modeling. Peptide mimetics are designed to include structures having one or more peptide linkages optionally replaced by a linkage selected from the group consisting of:— CH2NH— ,— CH2S— ,— CH2— CH2— ,— CH=CH-(cis and trans),— CH=CF-(trans),— CoCH2 — ,— CH(OH)CH2— , and— CH2SO— . In some embodiments, such peptide mimetics have greater chemical stability, enhanced pharmacological properties (half-life, absorption, potency, efficacy, etc.), altered specificity (e.g., a broad-spectrum of biological activities), reduced antigenicity, and are more economically prepared. In some embodiments, peptide mimetics include covalent attachment of one or more labels or conjugates, directly or through a spacer (e.g., an amide group), to non-interfering positions(s) on the analog that are predicted by quantitative structure-activity data and/or molecular modeling. Such non-interfering positions generally are positions that do not form direct contacts with the receptor(s) to which the peptide mimetic binds to produce the therapeutic effect. In some embodiments, systematic substitution of one or more amino acids of a consensus sequence with a D-amino acid of the same type (e.g., D-lysine in place of L-lysine) are used to generate more stable peptides with desired properties.
[00199] In some embodiments, a peptide mimetic is generated by use of phage display peptide libraries. For disclosure regarding the creation of a phage display peptide library see Scott, J. K. et al. (1990) Science 249:386; Devlin, J. J. et al. (1990) Science 249:404; US5,223,409, US5,733,731 ; US5,498,530; US5,432,018;US5,338,665;US5,922,545; WO 96/40987and WO 98/15833 each of which is incorporated by reference for such disclosure. In such libraries, random peptide sequences are displayed by fusion with coat proteins of filamentous phage. Typically, the displayed peptides are affinity-eluted against an antibody-immobilized extracellular domain (in this case PF4 or RANTES). In some embodiments, peptide mimetics are isolated by biopanning. In some embodiments, whole cells expressing PF4 or RANTES are used to screen the library utilizing FACs to isolate phage bound cells. The retained phages are enriched by successive rounds of biopanning and repropagation. The best binding peptides are sequenced to identify key residues within one or more structurally related families of peptides. The peptide sequences also suggest which residues to replace by alanine scanning or by mutagenesis at the DNA level. In some embodiments, mutagenesis libraries are created and screened to further optimize the sequence of the best binders.
[00200] In some embodiments, structural analysis of protein-protein interaction is used to suggest peptides that mimic the binding activity of the peptides described herein. In some embodiments, the crystal structure resulting from such an analysis suggests the identity and relative orientation of critical residues of the peptide, from which a peptide is designed.
[00201] For further disclosure re PF4/RANTES, methods of treatment comprising inhibiting the interactions between PF4 and RANTES, and pharmaceutical compositions comprising PF4 and RANTES antagonists see U.S. Provisional Application 61/103,1872, filed October 06, 2008; and PCT International Publication No. WO 2007/042263, which are incorporated by reference herein for such disclosures.
Combinations
[00202] Disclosed herein, in certain embodiments, are methods and pharmaceutical compositions for modulating an inflammatory disorder comprising co-administering (a) a therapeutically-effective amount of a first active agent that targets PF4 and/or RANTES, and (b) a therapeutically-effective amount of a second active agent, wherein the second active agent is selected from (i) an agent that treats an inflammatory disorder through an alternative pathway (i.e., the "second anti-inflammatory agent"), or (ii) an agent that treats a cardiovascular disorder.
[00203] In some embodiments, combining (a) the first active agent; and (b) the second active agent is synergistic and results in a more efficacious therapy. In some embodiments, the therapy is more efficacious as it treats inflammatory disorders by multiple pathways. In some embodiments, the therapy is more efficacious as it treats inflammatory disorders by multiple pathways and treats and/or ameliorates undesired inflammation resulting from the second agent. In some embodiments, the therapy is more efficacious as it allows (partially or fully) a medical professional to increase the prescribed dosage of the second active agent.
[00204] In certain instances, administration of a 5-ASA causes (either partially or fully)
inflammation. In certain instances, administration of sulfasalazine results in (either partially or fully) pneumonitis with or without eosinophilia, vasculitis, pericarditis with or without tamponade, hepatitis, allergic myocarditis, pancreatitis, nephritis, exfoliative dermatitis, serum vasculitis, and/or pleuritis. In certain instances, administration of mesalamine results in (either partially or fully) pericarditis, myocarditis, pancreatitis, hepatitis, interstitial pneumonitis, pleuritis, interstitial nephritis, and/or pneumonitis. In certain instances, administration of olsalazine results in (either partially or fully) myocarditis, pericarditis, pancreatitis, interstitial nephritis and/or pneumonitis.
[00205] In some embodiments, the first active agent and a 5-ASA treat an inflammatory disorder by (1) decreasing the chemotaxis of leukocytes, and (2) reducing the synthesis of eicosanoids and inflammatory cytokines. In some embodiments, the first active agent also decreases any undesired inflammation (e.g., pancreatitis) resulting from administration of the 5-ASA.
[00206] In some embodiments, the first active agent and an anti-TNF agent treat an inflammatory disorder by (1) decreasing the chemotaxis of leukocytes, and (2) suppressing a TNF-induced cytokine cascade. In some embodiments, the first active agent also decreases any undesired inflammation (e.g., tuberculosis) resulting from administration of the anti-TNF agent.
[00207] In some embodiments, the first active and a leukotriene inhibitor treat an inflammatory disorder by (1) decreasing the chemotaxis of leukocytes, and (2) antagonizing LTA4, LTB4, LTC4, LTD4, LTE4, LTF4, LTA4R, LTB4R, LTB4R1, LTB4R2, LTC4R, LTD4R, LTE4R, CYSLTRl, or CYSLTR2; or inhibiting the synthesis of a leukotriene via 5- LO, FLAP, LTA4H, LTA4S, or LTC4S. In some embodiments, the first active agent also decreases any undesired inflammation (e.g., tuberculosis) resulting from administration of the leukotriene inhibitor.
[00208] In some embodiments, the first active agent and an IL-1 receptor antagonist treat an inflammatory disorder by (1) decreasing the chemotaxis of leukocytes, and (2) blocking the stimulation of T cell IL-1 receptor. In some embodiments, the first active agent also decreases any undesired inflammation (e.g., pneumonia, and bone and joint infections) resulting from
administration of the IL-1 receptor antagonist.
[00209] In some embodiments, the first active agent and an IL-2 receptor antagonist treat an inflammatory disorder by (1) decreasing the chemotaxis of leukocytes, and (2) blocking the stimulation of T cell IL-2 receptor. In some embodiments, the first active agent also decreases any undesired inflammation (e.g., gastrointestinal disorders) resulting from administration of the IL-2 receptor antagonist.
[00210] In some embodiments, the first active agent and a cytotoxic agent treat an inflammatory disorder by (1) decreasing the chemotaxis of leukocytes, and (2) treating neoplastic disease. In some embodiments, the first active agent also decreases any undesired inflammation (e.g., neutropenia) resulting from administration of the cytotoxic agent.
[00211] In some embodiments, the first active agent and an immunomodulatory agent treat an inflammatory disorder by (1) decreasing the chemotaxis of leukocytes, and (2) enhancing, or suppressing the immune system. In some embodiments, the first active agent also decreases any undesired inflammation (e.g., hematologic side effects) resulting from administration of the immunomodulatory agent.
[00212] In some embodiments, the first active agent and an antibiotic treat an inflammatory disorder by (1) decreasing the chemotaxis of leukocytes, and (2) by blocking cell and/or microbial growth by disrupting the cell cycle, or by blocking histone deacetylase. In some embodiments, the first active agent also decreases any undesired inflammation (e.g., cardiotoxicity) resulting from administration of the antibiotic.
[00213] In some embodiments, the first active agent and a T-cell co-stimulatory blocker treat an inflammatory disorder by (1) decreasing the chemotaxis of leukocytes, and (2) modulating a co- stimulatory signal which is required for full T-cell activation. In some embodiments, the first active agent also decreases any undesired inflammation (e.g., neutropenia) resulting from administration of the T-cell co-stimulatory blocker.
[00214] In some embodiments, the first active agent and a B cell depleting agent treat an inflammatory disorder by (1) decreasing the chemotaxis of leukocytes, and (2) inhibiting B-cell activity. In some embodiments, the first active agent also decreases any undesired inflammation (e.g., Progressive Multifocal Leukoencephalopathy) resulting from administration of the B-cell depleting agent.
[00215] In some embodiments, the first active agent and an immunosuppressive agent treat an inflammatory disorder by (1) decreasing the chemotaxis of leukocytes, and (2) selectively or non- selectively inhibiting or preventing activity of the immune system. In some embodiments, the first active agent also decreases any undesired inflammation (e.g., lymphoma) resulting from
administration of immunosuppressive agent.
[00216] In some embodiments, the first active agent and an alkylating agent treat an inflammatory disorder by (1) decreasing the chemotaxis of leukocytes, and (2) inducing covalent binding of alkyl groups to cellular molecules. In some embodiments, the first active agent also decreases any undesired inflammation (e.g., immune suppression) resulting from administration of the alkylating agent.
[00217] In some embodiments, the first active agent and an anti-metabolite treat an inflammatory disorder by (1) decreasing the chemotaxis of leukocytes, and (2) preventing the biosynthesis or use of normal cellular metabolites. In some embodiments, the first active agent also decreases any undesired inflammation (e.g., mutagenesis) resulting from administration of the anti-metabolite.
[00218] In some embodiments, the first active agent and a plant alkaloid treat an inflammatory disorder by (1) decreasing the chemotaxis of leukocytes, and (2) interfering with normal microtubule breakdown during cell division. In some embodiments, the first active agent also decreases any undesired inflammation (e.g., leukopenia) resulting from administration of the plant alkaloid.
[00219] In some embodiments, the first active agent and a terpenoid treat an inflammatory disorder by (1) decreasing the chemotaxis of leukocytes, and (2) treating neoplastic disease or microbial infections. In some embodiments, the first active agent also decreases any undesired inflammation resulting from administration of the terpenoid agent.
[00220] In some embodiments, the first active agent and a topoisomerase inhibitor treat an inflammatory disorder by (1) decreasing the chemotaxis of leukocytes, and (2) modulating the action of cellular topoisomerase enzymes. In some embodiments, the first active agent also decreases any undesired inflammation (e.g., gastrointestinal effects) resulting from administration of the topoisomerase inhibitor.
[00221] In some embodiments, the first active agent and an antibody treat an inflammatory disorder by (1) decreasing the chemotaxis of leukocytes, and (2) neutralizing inflammatory cytokines such as, for example, TNF alpha. In some embodiments, the first active agent also decreases any undesired inflammation (e.g., tuberculosis) resulting from administration of the antibody.
[00222] In some embodiments, the first active agent and a hormonal therapy treat an inflammatory disorder by (1) decreasing the chemotaxis of leukocytes, and (2) suppressing cytokine release. In some embodiments, the first active agent also decreases any undesired inflammation (e.g., cancer) resulting from administration of the hormone.
[00223] In some embodiments, the first active agent and an anti-diabetes therapy treat an inflammatory disorder by (1) decreasing the chemotaxis of leukocytes, and (2) improving sensitivity to insulin in muscle and adipose tissue. In some embodiments, the first active agent also decreases any undesired inflammation (e.g., liver inflammation, pancreatitis) resulting from administration of the anti-diabetes agent.
[00224] In some embodiments, the first active agent and a statin synergistically treat a CVD by (1) decreasing the chemotaxis of leukocytes, (2) decreasing the synthesis of cholesterol, and (3) decreasing any undesired inflammation resulting from administration of the statin. In certain instances, statins induce inflammation. In certain instances, administration of a statin results (partially or fully) in myositis. In certain instances, statin-induced myositis is dose-dependent. In some embodiments, prescribing the first active agent allows (partially or fully) a medical professional to increase the prescribed dosage of statin.
[00225] In some embodiments, the first active agent and a fibrate synergistically treat a CVD by (1) decreasing the chemotaxis of leukocytes, and (2) increasing the concentration of HDL. In some embodiments, the first active agent also decreases any undesired inflammation resulting from administration of the fibrate.
[00226] In some embodiments, the first active agent and an ApoAl modulator synergistically treat a CVD by (1) decreasing the chemotaxis of leukocytes, and (2) increasing the concentration of HDL. In some embodiments, the first active agent also decreases any undesired inflammation resulting from administration of the ApoAl modulator.
[00227] In some embodiments, the first active agent and an ACAT modulator synergistically treat a CVD by (1) decreasing the chemotaxis of leukocytes, and (2) decreasing (a) the production and release of apoB-containing lipoproteins and (b) foam cell formation. In some embodiments, the first active agent also decreases any undesired inflammation resulting from administration of the ACAT inhibitor.
[00228] In some embodiments, the first active agent and a CETP modulator synergistically treat a CVD by (1) decreasing the chemotaxis of leukocytes, and (2) decreasing the transfer cholesterol from HDL cholesterol to LDL. In some embodiments, the first active agent also decreases any undesired inflammation resulting from administration of the CETP inhibitor. [00229] In some embodiments, the first active agent and a GP Ilb/IIIa receptor antagonist synergistically treat a CVD by (1) decreasing the chemotaxis of leukocytes, and (2) inhibiting platelet aggregation. In some embodiments, the first active agent also decreases any undesired inflammation resulting from administration of the GP Ilb/IIIa receptor antagonist.
[00230] In some embodiments, the first active agent and the P2Y12 receptor antagonist
synergistically treat a CVD by (1) decreasing the chemotaxis of leukocytes, and (2) inhibiting platelet aggregation. In some embodiments, the first active agent also decreases any undesired inflammation resulting from administration of the P2Y12 receptor antagonist.
[00231] In some embodiments, the first active agent and an Lp-PLA2 antagonist synergistically treat a CVD by (1) decreasing the chemotaxis of leukocytes, and (2) inhibiting the formation of biologically active products from oxidized LDL. In some embodiments, the first active agent also decreases any undesired inflammation resulting from administration of the Lp-PLA2 antagonist.
[00232] In some embodiments, the first active agent and a leukotriene antagonist synergistically treat a CVD by (1) decreasing the chemotaxis of leukocytes and (2) inhibiting the adhesion and activation of leukocytes on the endothelium. In some embodiments, the first active agent also decreases any undesired inflammation resulting from administration of the leukotriene antagonist.
[00233] In some embodiments, the second anti-inflammatory agent is: cyclosporine A, alefacept, efalizumab, methotrexate, acitretin, isotretinoin, hydroxyurea, mycophenolate mofetil (MMF), sulfasalazine, 6-Thioguanine, Dovonex, Taclonex, betamethasone, tazarotene, hydroxychloroquine, etanercept, adalimumab, infliximab, abatacept, rituximab, trastuzumab, Anti-CD45 monoclonal antibody AHN- 12 (NCI), Iodine-131 Anti-Bl Antibody (Corixa Corp.), anti-CD66 monoclonal antibody BW 250/183 (NCI, Southampton General Hospital), anti-CD45 monoclonal antibody (NCI, Baylor College of Medicine), antibody anti-anb3 integrin (NCI), BIW-8962 (BioWa Inc.), Antibody BC8 (NCI), antibody muJ591 (NCI), indium In 111 monoclonal antibody MN-14 (NCI), yttrium Y 90 monoclonal antibody MN-14 (NCI), F105 Monoclonal Antibody (NIAID),
Monoclonal Antibody RAV12 (Raven Biotechnologies), CAT-192 (Human Anti-TGF-Betal Monoclonal Antibody, Genzyme), antibody 3F8 (NCI), 177Lu-J591 (Weill Medical College of Cornell University), TB-403 (Biolnvent International AB), anakinra, azathioprine,
cyclophosphamide, leflunomide, d-penicillamine, amitriptyline, or nortriptyline, chlorambucil, nitrogen mustard, prasterone, LJP 394 (abetimus sodium), LJP 1082 (La Jolla Pharmaceutical), eculizumab, belibumab, rhuCD40L (NIAID), epratuzumab, sirolimus, tacrolimus, pimecrolimus, thalidomide, antithymocyte globulin-equine (Atgam, Pharmacia Upjohn), antithymocyte globulin- rabbit (Thymoglobulin, Genzyme), Muromonab-CD3 (FDA Office of Orphan Products
Development), basiliximab, daclizumab, riluzole, cladribine, natalizumab, interferon beta- lb, interferon beta- la, tizanidine, baclofen, mesalazine, asacol, pentasa, mesalamine, balsalazide, olsalazine, 6-mercaptopurine, ΑΓΝ457 (Anti IL-17 Monoclonal Antibody, Novartis), theophylline, D2E7 (a human anti-TNF mAb from Knoll Pharmaceuticals), Mepolizumab (Anti-IL-5 antibody, SB 240563), Canakinumab (Anti-IL-1 Beta Antibody, NIAMS), Anti-IL-2 Receptor Antibody (Daclizumab, NHLBI), CNTO 328 (Anti IL-6 Monoclonal Antibody, Centocor), ACZ885 (fully human anti-interleukin-lbeta monoclonal antibody, Novartis), CNTO 1275 (Fully Human Anti-IL- 12 Monoclonal Antibody, Centocor), (3S)-N-hydroxy-4-( {4-[(4-hydroxy-2- butynyl)oxy]phenyl}sulfonyl)-2,2-dimet- hyl-3-thiomorpholine carboxamide (apratastat), golimumab (CNTO 148), Onercept, BG9924 (Biogen Idee), Certolizumab Pegol (CDP870, UCB Pharma), AZD9056 (AstraZeneca), AZD5069 (AstraZeneca), AZD9668 (AstraZeneca), AZD7928 (AstraZeneca), AZD2914 (AstraZeneca), AZD6067 (AstraZeneca), AZD3342 (AstraZeneca), AZD8309 (AstraZeneca), ), [(lR)-3-methyl-l-({(2S)-3-phenyl-2-[(pyrazin-2- ylcarbonyl)amino]propanoyl}amino.)butyl]boronic acid (Bortezomib), AMG-714, (Anti-IL 15 Human Monoclonal Antibody, Amgen), ABT-874 (Anti IL-12 monoclonal antibody, Abbott Labs), MRA(Tocilizumab, an Anti IL-6 Receptor Monoclonal Antibody, Chugai Pharmaceutical), CAT- 354 (a human anti-interleukin-13 monoclonal antibody, Cambridge Antibody Technology,
Medlmmune), aspirin, salicylic acid, gentisic acid, choline magnesium, choline magnesium salicylate, choline salicylate, magnesium salicylate, sodium salicylate, diflunisal, carprofen, fenoprofen, fenoprofen calcium, flurobiprofen, ibuprofen, ketoprofen, nabutone, ketolorac, ketorolac tromethamine, naproxen, oxaprozin, diclofenac, etodolac, indomethacin, sulindac, tolmetin, meclofenamate, meclofenamate sodium, mefenamic acid, piroxicam, meloxicam, celecoxib, rofecoxib, valdecoxib, parecoxib, etoricoxib, lumiracoxib, CS-502 (Sankyo), JTE-522 (Japan Tobacco Inc.), L-745,337 (Almirall), NS398 (Sigma), betamethasone (Celestone), prednisone (Deltasone), alclometasone, aldosterone, amcinonide, beclometasone, betamethasone, budesonide, ciclesonide, clobetasol, clobetasone, clocortolone, cloprednol, cortisone, cortivazol, deflazacort, deoxycorticosterone, desonide, desoximetasone, desoxycortone, dexamethasone, diflorasone, diflucortolone, difluprednate, fluclorolone, fludrocortisone, fludroxycortide, flumetasone, flunisolide, fluocinolone acetonide, fluocinonide, fluocortin, fluocortolone, fluorometholone, fluperolone, fluprednidene, fluticasone, formocortal, formoterol, halcinonide, halometasone, hydrocortisone, hydrocortisone aceponate, hydrocortisone buteprate, hydrocortisone butyrate, loteprednol, medrysone, meprednisone, methylprednisolone, methylprednisolone aceponate, mometasone furoate, paramethasone, prednicarbate, prednisone, rimexolone, tixocortol, triamcinolone, ulobetasol; Actos® (Pioglitazone), Avandia® (Rosiglitazone), Amaryl®
(Glimepiride), Sulfonylurea-types, Diabeta® (Glyburide), Diabinese® (Chlorpropamide),
Glucotrol® (Glipizide), Glynasec (glyburide), Micronase® (glyburide), Orinase® (Tolbutamide), Tolinase® (Tolazamide), Glucophage, Riomet® (Metformin), Glucovance® (glyburide + metformin), Avandamet® (Rosiglitazone + metformin), Avandaryl® (Rosiglitazone+glimepiride), Byetta® (Exenatide), Insulins, Januvia® (Sitagliptin), Metaglip® (glipizide and metformin), Prandin® (Repaglinide), Precose® (Acarbose), Starlix® (Nateglinide), Xenical® (Orlistat), cisplatin; carboplatin; oxaliplatin; mechlorethamine; cyclophosphamide; chlorambucil; vincristine; vinblastine; vinorelbine; vindesine; azathioprine; mercaptopurine; fludarabine; pentostatin;
cladribine; 5-fluorouracil (5FU); floxuridine (FUDR); cytosine arabinoside; methotrexate;
trimethoprim; pyrimethamine; pemetrexed; paclitaxel; docetaxel; etoposide; teniposide; irinotecan; topotecan; amsacrine; etoposide; etoposide phosphate; teniposide; dactinomycin; doxorubicin; daunorubicin; valrubicine; idarubicine; epirubicin; bleomycin; plicamycin; mitomycin; trastuzumab; cetuximab; rituximab; bevacizumab; finasteride; goserelin; aminoglutethimide; anastrozole;
letrozole; vorozole; exemestane; 4-androstene-3,6,17-trione ("6-OXO"; l,4,6-androstatrien-3,17- dione (ATD); formestane; testolactone; fadrozole; A-81834 (3-(3-(l,l-dimethylethylthio-5- (quinoline-2- ylmethoxy)- 1 -(4-chloromethylphenyl)indole-2-yl)-2,2-dimethylpropionaldehyde oxime-O-2-acetic acid; AM103 (Amira); AM803 (Amira); atreleuton; BAY-x-1005 ((R)-(+)-alpha- cyclopentyl-4-(2-quinolinylmethoxy)-Benzeneacetic acid); CJ-13610 (4-(3-(4-(2-Methyl-imidazol-l- yl)-phenylsulfanyl)- phenyl)-tetrahydro-pyran-4-carboxylic acid amide); DG-031 (DeCode); DG- 051 (DeCode); MK886 (l-[(4-chlorophenyl)methyl]3-[(l,l-dimethylethyl)thio]-a,a-dimethyl-5-(l - methylethyl)-lH-indole-2-propanoic acid, sodium salt); MK591 (3-(l -4[(4-chlorophenyl)methyl]-3- [(t-butylthio)-5-((2-quinoly)methoxy)-lH-indole-2]-, dimehtylpropanoic acid); RP64966 ([4-[5-(3- Phenyl-propyl)thiophen-2- yl]butoxy] acetic acid); SA6541 ((R)-S-[[4-
(dimethylamino.)phenyl]methyl] -N-(3-mercapto-2methyl- 1 -oxopropyl-L-cycteine); SC-56938 (ethyl- l-[2-[4-(phenylmethyl)phenoxy] ethyl] -4-piperidine- carboxylate); VIA-2291 (Via
Pharmaceuticals); WY-47,288 (2-[(l-naphthalenyloxy)methyl]quinoline); zileuton; ZD-2138 (6-((3- fluoro-5- (tetrahydro-4-methoxy-2H-pyran-4yl)phenoxy)methyl)-l-methyl-2(lH)-quinlolinone); busulphan; alemtuzumab; belatacept (LEA29Y); posaconazole; fingolimod (FTY720); an anti-CD40 ligand antibody (e.g., BG 9588); CTLA4Ig (BMS 188667); abetimus (UP 394); an anti-ILlO antibody; an anti-CD20 antibody (e.g. rituximab); an anti-C5 antibody (e.g., eculizumab); or combinations thereof.
[00234] In some embodiments, the second active agent is selected from: a niacin; a fibrate; a statin; an Apolipoprotein A-l modulator; an AC AT modulator; a CETP modulator; a glycoprotein Ilb/IIIa modulator; a P2Y12 modulator; an Lp-PLA2 modulator; or combinations thereof.
[00235] In some embodiments, the second active agent is a statin. In some embodiments, the second active agent is atorvastatin; cerivastatin; fluvastatin; lovastatin; mevastatin; pitavastatin; pravastatin; rosuvastatin; simvastatin; simvastatin and ezetimibe; lovastatin and niacin, extended-release;
atorvastatin and amlodipine besylate; simvastatin and niacin, extended-release; or combinations thereof.
[00236] In some embodiments, the second active agent is niacin. [00237] In some embodiments, the second active agent is a fibrate. In some embodiments, the second active agent is bezafibrate; ciprofibrate; clofibrate; gemfibrozil; fenofibrate; or combinations thereof.
[00238] In some embodiments, the second active agent is DF4 (Ac-D-W-F-K-A-F-Y-D-K-V-A-E- K-F-K-E-A-F-NH2); DF5; RVX-208 (Resverlogix); or combinations thereof.
[00239] In some embodiments, the second active agent is an ACAT inhibitor. In some embodiments, the second active agent is avasimibe; pactimibe sulfate (CS-505); CI-1011 (2,6-diisopropylphenyl [(2, 4,6-triisopropylphenyl)acetyl]sulfamate); CI-976 (2,2-dimethyl-N-(2,4,6- trimethoxyphenyl)dodecanamide); VULM1457 (l-(2,6-diisopropyl-phenyl)-3-[4-(4'- nitrophenylthio)phenyl] urea); CI-976 (2,2-dimethyl-N-(2,4,6- trimethoxyphenyl)dodecanamide); E- 5324 (n-butyl-N'-(2-(3-(5-ethyl-4-phenyl-lH-imidazol-l-yl)propoxy)-6-methylphenyl)urea); HL- 004 (N-(2,6-diisopropylphenyl) tetradecylthioacetamide); KY-455 (N-(4,6- dimethyl-1 - pentylindolin-7-yl)-2,2-dimethylpropanamide); FY-087 (N-[2-[N'-pentyl-(6,6-dimethyl-2,4- heptadiynyl)amino] ethyl] -(2-methyl-l-naphthyl-thio)acetamide); MCC-147 (Mitsubishi Pharma); F 12511 ((S)-2',3',5'-trimethyl-4'-hydroxy-alpha-dodecylthioacetanilide); SMP-500 (Sumitomo Pharmaceuticals); CL 277082 (2,4-difluoro-phenyl-N[[4-(2,2-dimethylpropyl)phenyl]methyl]-N- (hepthyl)urea); F-1394 ((ls,2s)-2-[3-(2,2-dimethylpropyl)-3-nonylureido]aminocyclohexane-l-yl 3- [N-(2,2,5,5-tetramethyl-l,3-dioxane-4-carbonyl)amino]propionate); CP- 113818 (N-(2,4- bis(methylthio)-6-methylpyridin-3-yl)-2-(hexylthio)decanoic acid amide); YM-750; or combinations thereof.
[00240] In some embodiments, the second active agent (partially or completely) inhibits the activity of Cholesteryl Ester Transfer Protein (CETP). In some embodiments, the second active agent is torcetrapib; anacetrapib; JTT-705 (Japan Tobacco/Roche); or combinations thereof.
[00241] In some embodiments, the second active agent inhibits (partially or fully) the activity of glycoprotein Ilb/IIIa. In some embodiments, the cardiovascular disorder agent is a Glycoprotein (GP) Ilb/IIIa receptor antagonist. In some embodiments, the second active agent is abciximab; eptifibatide; tirofiban; roxifiban; variabilin; XV 459 (N(3)-(2-(3-(4-formamidinophenyl)isoxazolin- 5-yl)acetyl)-N(2)-(l-butyloxycarbonyl)-2,3-diaminopropionate); SR 121566A (3-[N- {4-[4- (aminoiminomethyl)phenyl ]-l ,3-thiazol-2-yl}-N-(l -carboxymethylpiperid-4-yl) aminol propionic acid, trihydrochloride); FK419 ((S)-2-acetylamino-3-[(R)-[l-[3-(piperidin-4-yl) propionyl] piperidin-3-ylcarbonyl] amino] propionic acid trihydrate); or combinations thereof.
[00242] In some embodiments, the second active agent antagonizes P2Y12. In some embodiments, the second active agent is a P2Y12 receptor antagonist. In some embodiments, the second active agent is clopidogrel; prasugrel; cangrelor; AZD6140 (AstraZeneca); MRS 2395 (2,2-Dimethyl- propionic acid 3-(2-chloro-6-methylaminopurin-9-yl)- 2-(2,2-dimethyl-propionyloxymethyl)-propyl ester); BX 667 (Berlex Biosciences); BX 048 (Berlex Biosciences) or combinations thereof. [00243] In some embodiments, the second active agent inhibits (partially or fully) the activity of lipoprotein-associated phospholipase A2 (lp-PLA2). In some embodiments, the second active agent is an Lp-PLA2 antagonist. In some embodiments, the second active agent is darapladib (SB
480848); SB-435495 (GlaxoSmithKline); SB-222657 (Glaxo SmithKline); SB-253514
(Glaxo SmithKline); or combinations thereof.
[00244] In some embodiments, the second active agent inhibits a leukotriene (e.g., by antagonizing LTA4, LTB4, LTC4, LTD4, LTE4, LTF4, LTA4R; LTB4R; LTB4R1, LTB4R2, LTC4R, LTD4R, LTE4R, CYSLTRl, or CYSLTR2; or by inhibiting the synthesis of a leukotriene via 5- LO, FLAP, LTA4H, LTA4S, or LTC4S). In some embodiments, the second active agent is an antagonist of 5- LO. In some embodiments, the second active agent is an antagonist of FLAP. In some embodiments, the second active agent is A-81834 (3-(3-(l,l-dimethylethylthio-5-(quinoline-2- ylmethoxy)- 1 -(4- chloromethylphenyl)indole-2-yl)-2,2-dimethylpropionaldehyde oxime-O-2-acetic acid; AMI 03 (Amira); AM803 (Amira); atreleuton; BAY-x-1005 ((R)-(+)-alpha-cyclopentyl-4-(2- quinolinylmethoxy)-Benzeneacetic acid); CJ-13610 (4-(3-(4-(2-Methyl-imidazol-l-yl)- phenylsulfanyl)- phenyl)-tetrahydro-pyran-4-carboxylic acid amide); DG-031 (DeCode); DG-051 (DeCode); MK886 (l-[(4-chlorophenyl)methyl]3-[(l,l-dimethylethyl)thio]-a,a-dimethyl-5-(l - methylethyl)-lH-indole-2-propanoic acid, sodium salt); MK591 (3-(l -4[(4-chlorophenyl)methyl]-3- [(t-butylthio)-5-((2-quinoly)methoxy)-lH-indole-2]-, dimehtylpropanoic acid); RP64966 ([4-[5-(3- Phenyl-propyl)thiophen-2- yl]butoxy] acetic acid); SA6541 ((R)-S-[[4-
(dimethylamino.)phenyl]methyl] -N-(3-mercapto-2methyl- 1 -oxopropyl-L-cycteine); SC-56938 (ethyl- l-[2-[4-(phenylmethyl)phenoxy] ethyl] -4-piperidine- carboxylate); VIA-2291 (Via
Pharmaceuticals); WY-47,288 (2-[(l-naphthalenyloxy)methyl]quinoline); zileuton; ZD-2138 (6-((3- fluoro-5- (tetrahydro-4-methoxy-2H-pyran-4yl)phenoxy)methyl)-l-methyl-2(lH)-quinlolinone); or combinations thereof.
Gene Therapy
[00245] In some embodiments, are methods and pharmaceutical compositions for modulating a disorder of a cardiovascular system, comprising a synergistic combination of (a) a therapeutically- effective amount of a first active agent that inhibits the interaction between RANTES and Platelet Factor 4; and (b) gene therapy.
[00246] In some embodiments, the gene therapy comprises modulating the concentration of a lipid and/or lipoprotein (e.g., HDL) in the blood of an individual in need thereof. In some embodiments, modulating the concentration of a lipid and/or lipoprotein (e.g., HDL) in the blood comprises transfecting DNA into an individual in need thereof. In some embodiments, the DNA encodes part or all of an Apo Al gene, an LCAT gene, and/or an LDL gene. In some embodiments, the DNA is transfected into a liver cell. [00247] In some embodiments, the DNA is transfected into a liver cell via use of ultrasound. For disclosures of techniques related to transfecting ApoAl DNA via use of ultrasound see U.S. Patent No. 7,211,248, which is hereby incorporated by reference for those disclosures.
[00248] In some embodiments, an individual is administered a vector engineered to carry the human gene (the "gene vector"). For disclosures of techniques for creating an LDL gene vector see U.S. Patent No. 6,784,162, which is hereby incorporated by reference for those disclosures. In some embodiments, the gene vector is a retrovirus. In some embodiments, the gene vector is not a retrovirus (e.g. it is an adenovirus; or a polymeric delivery system such as METAFECTENE, SUPERFECT®, EFFECTENE®, or MIRUS TRANSIT). In certain instances, a retrovirus (e.g., a lentivirus) or an adenovirus will have a mutation such that the virus is rendered incompetent.
[00249] In some embodiments, the vector is administered in vivo (i.e., the vector is injected directly into the individual, for example into a liver cell), ex vivo (i.e., cells from the individual are grown in vitro and transduced with the gene vector, embedded in a carrier, and then implanted in the individual), or a combination thereof.
[00250] In certain instances, after administration of the gene vector, the gene vector infects the cells at the site of administration (e.g. the liver). In certain instances the gene sequence is incorporated into the subject's genome (e.g. when the gene vector is a retrovirus). In certain instances the therapy will need to be periodically re-administered (e.g. when the gene vector is not a retrovirus). In some embodiments, the therapy is re-administered annually. In some embodiments, the therapy is re- administered semi-annually. In some embodiments, the therapy is re-administered when the subject's HDL level decreases below about 60 mg/dL. In some embodiments, the therapy is re- administered when the subject's HDL level decreases below about 50 mg/dL. In some embodiments, the therapy is re-administered when the subject's HDL level decreases below about 45 mg/dL. In some embodiments, the therapy is re-administered when the subject's HDL level decreases below about 40 mg/dL. In some embodiments, the therapy is re-administered when the subject's HDL level decreases below about 35 mg/dL. In some embodiments, the therapy is re-administered when the subject's HDL level decreases below about 30 mg/dL.
RNAi Therapies
[00251] In some embodiments, are methods and pharmaceutical compositions for modulating a disorder of a cardiovascular system, comprising a synergistic combination of (a) a therapeutically- effective amount of a first active agent that targets PF4 and/or RANTES and (b) silencing the expression of a gene that increases the concentration of a lipid in blood (the "target gene"). In some embodiments, the target gene is Apolipoprotein B (Apo B), Heat Shock Protein 110 (Hsp 110), and Proprotein Convertase Subtilisin Kexin 9 (Pcsk9) (ALN-PCS, BMS-PCSK9RX). In some embodiments, the target gene is C-reactive protein (CRP) (ISIS-CRPRX). [00252] In some embodiments, the target gene is silenced by RNA interference (RNAi). In some embodiments, the RNAi therapy comprises use of an siRNA molecule. In some embodiments, a double stranded RNA (dsRNA) molecule with sequences complementary to an mRNA sequence of a gene to be silenced (e.g., Apo B, Hsp 110 and Pcsk9) is generated (e.g by PCR). In some embodiments, a 20-25 bp siRNA molecule with sequences complementary to an mRNA sequence of a gene to be silenced is generated. In some embodiments, the 20-25 bp siRNA molecule has 2-5 bp overhangs on the 3' end of each strand, and a 5' phosphate terminus and a 3' hydroxyl terminus. In some embodiments, the 20-25 bp siRNA molecule has blunt ends. For techniques for generating RNA sequences see Molecular Cloning: A Laboratory Manual, second edition (Sambrook et al., 1989) and Molecular Cloning: A Laboratory Manual, third edition (Sambrook and Russel, 2001), jointly referred to herein as "Sambrook"); Current Protocols in Molecular Biology (F. M. Ausubel et al., eds., 1987, including supplements through 2001); Current Protocols in Nucleic Acid Chemistry John Wiley & Sons, Inc., New York, 2000) which are hereby incorporated by reference for such disclosure.
[00253] In some embodiments, an siRNA molecule is "fully complementary" (i.e., 100%
complementary) to the target gene. In some embodiments, an antisense molecule is "mostly complementary" (e.g., 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, or 70%) complementary) to the target gene. In some embodiments, there is a 1 bp mismatch, a 2 bp mismatch, a 3 bp mismatch, a 4 bp mismatch, or a 5 bp mismatch.
[00254] In certain instances, after administration of the dsRNA or siRNA molecule, cells at the site of administration (e.g. the cells of the liver and/or small intestine) are transformed with the dsRNA or siRNA molecule. In certain instances following transformation, the dsRNA molecule is cleaved into multiple fragments of about 20-25 bp to yield siRNA molecules. In certain instances, the fragments have about 2bp overhangs on the 3 ' end of each strand.
[00255] In certain instances, an siRNA molecule is divided into two strands (the guide strand and the anti-guide strand) by an RNA-induced Silencing Complex (RISC). In certain instances, the guide strand is incorporated into the catalytic component of the RISC (i.e. argonaute). In certain instances, the guide strand binds to a complementary RB 1 mRNA sequence. In certain instances, the RISC cleaves an mRNA sequence of a gene to be silenced. In certain instances, the expression of the gene to be silenced is down-regulated.
[00256] In some embodiments, a sequence complementary to an mRNA sequence of a target gene is incorporated into a vector. In some embodiments, the sequence is placed between two promoters. In some embodiments, the promoters are orientated in opposite directions. In some embodiments, the vector is contacted with a cell. In certain instances, a cell is transformed with the vector. In certain instances following transformation, sense and anti-sense strands of the sequence are generated. In certain instances, the sense and anti-sense strands hybridize to form a dsRNA molecule which is cleaved into siRNA molecules. In certain instances, the strands hybridize to form an siRNA molecule. In some embodiments, the vector is a plasmid (e.g pSUPER; pSUPER.neo;
pSUPER.neo+gfp).
[00257] In some embodiments, an siRNA molecule is administered in vivo (i.e., the vector is injected directly into the individual, for example into a liver cell or a cell of the small intestine, or into the blood stream).
[00258] In some embodiments, a siRNA molecule is formulated with a delivery vehicle (e.g., a liposome, a biodegradable polymer, a cyclodextrin, a PLGA microsphere, a PLCA microsphere, a biodegradable nanocapsule, a bioadhesive microsphere, or a proteinaceous vector), carriers and diluents, and other pharmaceutically-acceptable excipients. For methods of formulating and administering a nucleic acid molecule to an individual in need thereof see Akhtar et al., 1992, Trends Cell Bio., 2, 139; Delivery Strategies for Antisense Oligonucleotide Therapeutics, ed.
Akhtar, 1995; Maurer et al., 1999, Mol. Membr. Biol., 16, 129-140; Holland and Huang, 1999, Handb. Exp. Pharmacol., 137, 165-192; Lee et al., 2000, ACS Symp. Ser., 752, 184-192; Beigelman et al., U.S. Pat. No. 6,395,713;Sullivan et al., PCT WO 94/02595; Gonzalez et al., 1999,
Bioconjugate Chem., 10, 1068-1074; Wang et al., International PCT publication Nos. WO 03/47518 and WO 03/46185; U.S. Pat. No. 6,447,796; US Patent Application Publication No. US
2002130430; O'Hare and Normand, International PCT Publication No. WO 00/53722; and U.S. Patent Application Publication No. 20030077829; U.S. Provisional patent application No.
60/678,531, all of which are hereby incorporated by reference for such disclosures.
[00259] In some embodiments, an siRNA molecule described herein is administered to the liver by any suitable manner (see e.g., Wen et al., 2004, World J Gastroenterol., 10, 244-9; Murao et al., 2002, Pharm Res., 19, 1808-14; Liu et al., 2003, Gene Ther., 10, 180-7; Hong et al., 2003, J Pharm Pharmacol., 54, 51-8; Herrmann et al., 2004, Arch Virol., 149, 1611-7; and MatsuNo. et al., 2003, Gene Ther., 10, 1559-66).
[00260] In some embodiments, an siRNA molecule described herein is administered
iontophoretically, for example to a particular organ or compartment (e.g., the liver or small intestine). Non-limiting examples of iontophoretic delivery are described in, for example, WO 03/043689 and WO 03/030989, which are hereby incorporated by reference for such disclosures.
[00261] In some embodiments, an siRNA molecule described herein is administered systemically (i.e., in vivo systemic absorption or accumulation of an siRNA molecule in the blood stream followed by distribution throughout the entire body). Administration routes contemplated for systemic administration include, but are not limited to, intravenous, subcutaneous, portal vein, intraperitoneal, and intramuscular. Each of these administration routes exposes the siRNA molecules of the invention to an accessible diseased tissue (e.g., liver). [00262] In certain instances the therapy will need to be periodically re-administered. In some embodiments, the therapy is re-administered annually. In some embodiments, the therapy is re- administered semi-annually. In some embodiments, the therapy is administered monthly. In some embodiments, the therapy is administered weekly. In some embodiments, the therapy is re- administered when the subject's HDL level decreases below about 60 mg/dL. In some embodiments, the therapy is re-administered when the subject's HDL level decreases below about 50 mg/dL. In some embodiments, the therapy is re-administered when the subject's HDL level decreases below about 45 mg/dL. In some embodiments, the therapy is re-administered when the subject's HDL level decreases below about 40 mg/dL. In some embodiments, the therapy is re-administered when the subject's HDL level decreases below about 35 mg/dL. In some embodiments, the therapy is re- administered when the subject's HDL level decreases below about 30 mg/dL.
[00263] For disclosures of techniques related to silencing the expression of Apo B and/or Hspl 10 see U.S. Pub. No. 2007/0293451 which is hereby incorporated by reference for such disclosures. For disclosures of techniques related to silencing the expression of Pcsk9 see U.S. Pub. No.
2007/0173473, which is hereby incorporated by reference for such disclosures.
Antisense Therapies
[00264] In some embodiments, are methods and pharmaceutical compositions for modulating a disorder of a cardiovascular system, comprising a synergistic combination of (a) a therapeutically- effective amount of a first active agent that targets PF4 and/or RANTES and (b) inhibiting the expression of and/or activity of an RNA sequence that increases the concentration of a lipid in blood (the "target sequence"). In some embodiments, inhibiting the expression of and/or activity of a target sequence comprises use of an antisense molecule complementary to the target sequence. In some embodiments, the target sequence is microRNA-122 (miRNA-122 or mRNA-122). In certain instances, inhibiting the expression of and/or activity of miRNA-122 results (partially or fully) in a decrease in the concentration of cholesterol and/or lipids in blood.
[00265] In some embodiments, an antisense molecule that is complementary to a target sequence is generated (e.g. by PCR). In some embodiments, the antisense molecule is about 15 to about 30 nucleotides. In some embodiments, the antisense molecule is about 17 to about 28 nucleotides. In some embodiments, the antisense molecule is about 19 to about 26 nucleotides. In some
embodiments, the antisense molecule is about 21 to about 24 nucleotides. For techniques for generating RNA sequences see Molecular Cloning: A Laboratory Manual, second edition
(Sambrook et al., 1989) and Molecular Cloning: A Laboratory Manual, third edition (Sambrook and Russel, 2001), jointly referred to herein as "Sambrook"); Current Protocols in Molecular Biology (F. M. Ausubel et al., eds., 1987, including supplements through 2001); Current Protocols in Nucleic Acid Chemistry John Wiley & Sons, Inc., New York, 2000) which are hereby incorporated by reference for such disclosure. [00266] In some embodiments, the antisense molecules are single- stranded, double- stranded, circular or hairpin. In some embodiments, the antisense molecules contain structural elements (e.g., internal or terminal bulges, or loops).
[00267] In some embodiments, an antisense molecule is "fully complementary" (i.e., 100% complementary) to the target sequence. In some embodiments, an antisense molecule is "mostly complementary" (e.g., 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, or 70%) complementary) to the target RNA sequence. In some embodiments, there is a 1 bp mismatch, a 2 bp mismatch, a 3 bp mismatch, a 4 bp mismatch, or a 5 bp mismatch.
[00268] In some embodiments, the antisense molecule hybridizes to the target sequence. As used herein, "hybridize" means the pairing of nucleotides of an antisense molecule with corresponding nucleotides of the target sequence. In certain instances, hybridization involves the formation of one or more hydrogen bonds (e.g., Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding) between the pairing nucleotides.
[00269] In certain instances, hybridizing results (partially or fully) in the degradation, cleavage, and/or sequestration of the RNA sequence.
[00270] In some embodiments, a siRNA molecule is formulated with a delivery vehicle (e.g., a liposome, a biodegradable polymer, a cyclodextrin, a PLGA microsphere, a PLCA microsphere, a biodegradable nanocapsule, a bioadhesive microsphere, or a proteinaceous vector), carriers and diluents, and other pharmaceutically-acceptable excipients. For methods of formulating and administering a nucleic acid molecule to an individual in need thereof see Akhtar et al., 1992, Trends Cell Bio., 2, 139; Delivery Strategies for Antisense Oligonucleotide Therapeutics, ed.
Akhtar, 1995; Maurer et al., 1999, Mol. Membr. Biol., 16, 129-140; Hofland and Huang, 1999, Handb. Exp. Pharmacol., 137, 165-192; Lee et al., 2000, ACS Symp. Ser., 752, 184-192; Beigelman et al., U.S. Pat. No. 6,395,713;Sullivan et al., PCT WO 94/02595; Gonzalez et al., 1999,
Bioconjugate Chem., 10, 1068-1074; Wang et al., International PCT publication Nos. WO 03/47518 and WO 03/46185; U.S. Pat. No. 6,447,796; US Patent Application Publication No. US
2002130430; O'Hare and Normand, International PCT Publication No. WO 00/53722; and U.S. Patent Application Publication No. 20030077829; U.S. Provisional patent application No.
60/678,531, all of which are hereby incorporated by reference for such disclosures.
[00271] In some embodiments, an siRNA molecule described herein is administered to the liver by any suitable manner (see e.g., Wen et al., 2004, World J Gastroenterol., 10, 244-9; Murao et al., 2002, Pharm Res., 19, 1808-14; Liu et al., 2003, Gene Ther., 10, 180-7; Hong et al., 2003, J Pharm Pharmacol., 54, 51-8; Herrmann et al., 2004, Arch Virol., 149, 1611-7; and MatsuNo. et al., 2003, Gene Ther., 10, 1559-66).
[00272] In some embodiments, an siRNA molecule described herein is administered
iontophoretically, for example to a particular organ or compartment (e.g., the liver or small intestine). Non-limiting examples of iontophoretic delivery are described in, for example, WO 03/043689 and WO 03/030989, which are hereby incorporated by reference for such disclosures.
[00273] In some embodiments, an siRNA molecule described herein is administered systemically (i.e., in vivo systemic absorption or accumulation of an siRNA molecule in the blood stream followed by distribution throughout the entire body). Administration routes contemplated for systemic administration include, but are not limited to, intravenous, subcutaneous, portal vein, intraperitoneal, and intramuscular. Each of these administration routes exposes the siRNA molecules of the invention to an accessible diseased tissue (e.g., liver).
[00274] In certain instances the therapy will need to be periodically re-administered. In some embodiments, the therapy is re-administered annually. In some embodiments, the therapy is re- administered semi-annually. In some embodiments, the therapy is administered monthly. In some embodiments, the therapy is administered weekly. In some embodiments, the therapy is re- administered when the subject's HDL level decreases below about 60 mg/dL. In some embodiments, the therapy is re-administered when the subject's HDL level decreases below about 50 mg/dL. In some embodiments, the therapy is re-administered when the subject's HDL level decreases below about 45 mg/dL. In some embodiments, the therapy is re-administered when the subject's HDL level decreases below about 40 mg/dL. In some embodiments, the therapy is re-administered when the subject's HDL level decreases below about 35 mg/dL. In some embodiments, the therapy is re- administered when the subject's HDL level decreases below about 30 mg/dL.
[00275] For disclosures of techniques related to silencing the expression of miRNA-122 see WO 07/027775A2, which is hereby incorporated by reference for such disclosures.
Device-Mediated Therapies
[00276] In some embodiments, an agent disclosed herein (e.g., peptides, antibodies, peptidbodies, and small molecules) is combined with a stent (e.g., a drug-eluting stent). As used herein, a stent is any stent (a scaffold) placed into an artery. In some embodiments, the stent is coated with an active agent that is released from the stent into the surrounding environment. In some embodiments, the stent is combined with the peptide CKEYFYTSSKSSNLAVVFVTRC (SEQ ID NO: 13). In some embodiments, the stent is combined with the peptide CKEYF YTS GKS SNPGIVFITRC (SEQ ID NO: 16). Any suitable stent is used. In some embodiments, an agent disclosed herein (e.g., a peptide of SEQ ID No. 13, a peptide of SEQ ID NO: 16) is combined with the stent by use of a coating. In some embodiments, the coating (e.g., a polymer) holds and elutes the agent (e.g., a peptide of SEQ ID No. 13, a peptide of SEQ ID NO 16) into the arterial wall by contact transfer. In some embodiments, the coating is biodegreadable. In some embodiments, the coating is durable (i.e., it does not biodegrade). In some embodiments, there are multiple layers of coating and drug, each with a different release profile. For example, the out-most coating has an immediate release profile and a second coating has a controlled or delayed release profile. [00277] In some embodiments, the device mediated strategy comprises removing a lipid from an HDL molecule in an individual in need thereof (delipidation), removing an LDL molecule from the blood or plasma of an individual in need thereof (delipidation), or a combination thereof. For disclosures of techniques for removing a lipid from an HDL molecule and removing an LDL molecule from the blood or plasma of an individual in need thereof see U.S. Pub. No.
2008/0230465, which is hereby incorporated by reference for those disclosures.
[00278] In certain instances, the delipidation therapy will need to be periodically re-administered. In some embodiments, the delipidation therapy is re-administered annually. In some embodiments, the delipidation therapy is re-administered semi-annually. In some embodiments, the delipidation therapy is re-administered monthly. In some embodiments, the delipidation therapy is re- administered semi-weekly. In some embodiments, the therapy is re-administered when the subject's HDL level decreases below about 60 mg/dL. In some embodiments, the therapy is re-administered when the subject's HDL level decreases below about 50 mg/dL. In some embodiments, the therapy is re-administered when the subject's HDL level decreases below about 45 mg/dL. In some embodiments, the therapy is re-administered when the subject's HDL level decreases below about 40 mg/dL. In some embodiments, the therapy is re-administered when the subject's HDL level decreases below about 35 mg/dL. In some embodiments, the therapy is re-administered when the subject's HDL level decreases below about 30 mg/dL.
Pharmaceutical Compositions
[00279] Disclosed herein, in certain embodiments, is a pharmaceutical composition comprising a therapeutically-effective amount of an active agent that inhibits interactions between RANTES and Platelet Factor 4.
[00280] Disclosed herein, in certain embodiments, is a pharmaceutical composition comprising a synergistic combination of (a) a therapeutically-effective amount of a first active agent that inhibits interactions between RANTES and Platelet Factor 4; and (b) a second active agent.
[00281] Disclosed herein, in certain embodiments, is a pharmaceutical composition comprising (a) a peptide that mimics the RANTES binding domain of PF4, and inhibits the formation of a PF4 and RANTES heterodimer, or (b) a peptide that comprises an amino acid that is 85% homologous (i.e., is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homologous, including any amount of homology between 85% and 100%) to an amino acid sequence selected from:
Figure imgf000086_0001
QCLCVKTTSQVRPRH (SEQ ID NO. 50) ALPRAHVKEYFYTSS (SEQ ID NO. 73)
PPvHITSLEVIKAGPHC (SEQ ID NO. 51) SSKCSNLAWFVTRR (SEQ ID NO. 74)
PTAQLIATLKNGPvKIC (SEQ ID NO. 52) RRNRQVCANPEKKWV (SEQ ID NO. 75)
LDLQAPLYKKIIKKLLES (SEQ ID NO. 53) EKKWVQEYINYLEMS (SEQ ID NO. 76)
MSVAAVFPvGLPvPSPE (SEQ ID NO. 54) S SKS SNLA WFVTRCCKEYFYT (SEQ ID
NO. 77)
SPELLLLGLLFLPAV (SEQ ID NO. 55) SKS SNLA WFVTRCCKEYFYTS (SEQ ID
NO. 78)
PAVVAVTSAGPEESD (SEQ ID NO. 56) KS SNLA WFVTRCCKEYFYT S S (SEQ ID
NO. 79);
ESDGDLSCVCVKTIS (SEQ ID NO. 57) S SNLA WFVTRCCKEYFYT S SK (SEQ ID
NO. 80);
TISSGIHLKHITSLE (SEQ ID NO. 58) SNLA WFVTRCCKEYFYTS SKS (SEQ ID
NO. 81);
VIKAGRHCAVPQLIA (SEQ ID NO. 59) NLA WFVTRCCKEYFYT S SKS S (SEQ ID
NO. 82);
LIATLKNGRKICLDPv (SEQ ID NO. 60) SFKGTTVYALSNVRSYSFVKCC (SEQ ID
NO. 83);
LDRQAPLYKKVIKKI (SEQ ID NO. 61) FKGTTVYALSNVRSYSFVKCCS (SEQ ID
NO. 84);
QAPLYKKVIKKILES (SEQ ID NO. 62) SNVRSYSFVKCCSFKGTTVYAL (SEQ ID
NO. 85);
MKVSAAALAVILIAT (SEQ ID NO. 63) NVRSYSFVKCCSFKGTTVYALS (SEQ ID
NO. 86);
ATALCAPASASPYSS (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID
NO. 87);
SDTTPCCFAYIARPL (SEQ ID NO. 65) YSFVKCCSFKGTTVYALSNVRS (SEQ ID
NO. 88);
PLPRAHIKEYFYTSG (SEQ ID NO. 66) SFVKCCSFKGTTVYALSNVRSY (SEQ ID
NO. 89);
EYFYTSGKCSNPAW (SEQ ID NO. 67) FVKCCSFKGTTVYALSNVRSYS (SEQ ID
NO. 90)
VFVTRKNRQVCANPE (SEQ ID NO. 66)
[00282] Pharmaceutical compositions herein are formulated using one or more physiologically acceptable carriers including excipients and auxiliaries which facilitate processing of the active agents into preparations which are used pharmaceutically. Proper formulation is dependent upon the route of administration chosen. A summary of pharmaceutical compositions is found, for example, in Remington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania 1975; Liberman, H.A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams & Wilkins, 1999).
[00283] In certain embodiments, a pharmaceutical composition disclosed herein comprises a pharmaceutically acceptable diluent(s), excipient(s), or carrier(s). In some embodiments, the pharmaceutical compositions includes other medicinal or pharmaceutical agents, carriers, adjuvants, such as preserving, stabilizing, wetting or emulsifying agents, solution promoters, salts for regulating the osmotic pressure, and/or buffers. In addition, the pharmaceutical compositions also contain other therapeutically valuable substances.
[00284] The pharmaceutical formulations described herein are optionally administered to a subject by multiple administration routes, including but not limited to, oral, parenteral (e.g., intravenous, subcutaneous, intramuscular), intranasal, buccal, topical, rectal, or transdermal administration routes. The pharmaceutical formulations described herein include, but are not limited to, aqueous liquid dispersions, self-emulsifying dispersions, solid solutions, liposomal dispersions, aerosols, solid dosage forms, powders, immediate release formulations, controlled release formulations, fast melt formulations, tablets, capsules, pills, delayed release formulations, extended release formulations, pulsatile release formulations, multiparticulate formulations, and mixed immediate and controlled release formulations.
[00285] The pharmaceutical compositions described herein are formulated into any suitable dosage form, including but not limited to, aqueous oral dispersions, liquids, gels, syrups, elixirs, slurries, suspensions and the like, for oral ingestion by a individual to be treated, solid oral dosage forms, aerosols, controlled release formulations, fast melt formulations, effervescent formulations, lyophilized formulations, tablets, powders, pills, dragees, capsules, modified release formulations, delayed release formulations, extended release formulations, pulsatile release formulations, multiparticulate formulations, and mixed immediate release and controlled release formulations. Multi-P articulate Dosage Forms
[00286] In some embodiments, the pharmaceutical compositions described herein are formulated as multiparticulate formulations. In some embodiments, the pharmaceutical compositions described herein comprise a first population of particles and a second population of particles. In some embodiments, the first population comprises an active agent. In some embodiments, the second population comprises an active agent. In some embodiments, the dose of active agent in the first population is equal to the dose of active agent in the second population. In some embodiments, the dose of active agent in the first population is not equal to (e.g., greater than or less than) the dose of active agent in the second population.
[00287] In some embodiments, the active agent of the first population is released before the active agent of the second population. In some embodiments, the second population of particles comprises a modified-release (e.g., delayed-release, controlled-release, or extended release) coating. In some embodiments, the second population of particles comprises a modified-release (e.g., delayed-release, controlled-release, or extended release) matrix.
[00288] Coating materials for use with the pharmaceutical compositions described herein include, but are not limited to, polymer coating materials (e.g., cellulose acetate phthalate, cellulose acetate trimaletate, hydroxy propyl methylcellulose phthalate, polyvinyl acetate phthalate); ammonio methacrylate copolymers (e.g., Eudragit® RS and RL); poly acrylic acid and poly acrylate and methacrylate copolymers (e.g., Eudragite S and L, polyvinyl acetaldiethylamino acetate, hydroxypropyl methylcellulose acetate succinate, shellac); hydrogels and gel-forming materials (e.g., carboxyvinyl polymers, sodium alginate, sodium carmellose, calcium carmellose, sodium carboxymethyl starch, poly vinyl alcohol, hydroxyethyl cellulose, methyl cellulose, gelatin, starch, hydoxypropyl cellulose, hydroxypropyl methylcellulose, polyvinylpyrrolidone, crosslinked starch, microcrystalline cellulose, chitin, aminoacryl-methacrylate copolymer, pullulan, collagen, casein, agar, gum arabic, sodium carboxymethyl cellulose, (swellable hydrophilic polymers)
poly(hydroxyalkyl methacrylate) (m. wt. ~5 k-5,000 k), polyvinylpyrrolidone (m. wt. "10 k-360 k), anionic and cationic hydrogels, polyvinyl alcohol having a low acetate residual, a swellable mixture of agar and carboxymethyl cellulose, copolymers of maleic anhydride and styrene, ethylene, propylene or isobutylene, pectin (m. wt. ~30 k-300 k), polysaccharides such as agar, acacia, karaya, tragacanth, algins and guar, polyacrylamides, Polyox® polyethylene oxides (m. wt. ~100 k-5,000 k), AquaKeep® acrylate polymers, diesters of polyglucan, crosslinked polyvinyl alcohol and poly N- vinyl-2-pyrrolidone, sodium starch; hydrophilic polymers (e.g., polysaccharides, methyl cellulose, sodium or calcium carboxymethyl cellulose, hydroxypropyl methyl cellulose, hydroxypropyl cellulose, hydroxyethyl cellulose, nitro cellulose, carboxymethyl cellulose, cellulose ethers, polyethylene oxides, methyl ethyl cellulose, ethylhydroxy ethylcellulose, cellulose acetate, cellulose butyrate, cellulose propionate, gelatin, collagen, starch, maltodextrin, pullulan, polyvinyl pyrrolidone, polyvinyl alcohol, polyvinyl acetate, glycerol fatty acid esters, polyacrylamide, polyacrylic acid, copolymers of methacrylic acid or methacrylic acid, other acrylic acid derivatives, sorbitan esters, natural gums, lecithins, pectin, alginates, ammonia alginate, sodium, calcium, potassium alginates, propylene glycol alginate, agar, arabic gum, karaya gum, locust bean gum, tragacanth gum, carrageens gum, guar gum, xanthan gum, scleroglucan gum); or combinations thereof. In some embodiments, the coating comprises a plasticiser, a lubricant, a solvent, or combinations thereof. Suitable plasticisers include, but are not limited to, acetylated monoglycerides; butyl phthalyl butyl glycolate; dibutyl tartrate; diethyl phthalate; dimethyl phthalate; ethyl phthalyl ethyl glycolate; glycerin; propylene glycol; triacetin; citrate; tripropioin; diacetin; dibutyl phthalate; acetyl monoglyceride; polyethylene glycols; castor oil; triethyl citrate; polyhydric alcohols, glycerol, acetate esters, gylcerol triacetate, acetyl triethyl citrate, dibenzyl phthalate, dihexyl phthalate, butyl octyl phthalate, diisononyl phthalate, butyl octyl phthalate, dioctyl azelate, epoxidised tallate, triisoctyl trimellitate, diethylhexyl phthalate, di-n-octyl phthalate, di-i-octyl phthalate, di-i-decyl phthalate, di-n-undecyl phthalate, di-n-tridecyl phthalate, tri-2- ethylhexyl trimellitate, di-2-ethylhexyl adipate, di-2-ethylhexyl sebacate, di-2-ethylhexyl azelate, dibutyl sebacate.
[00289] In some embodiments, the second population of particles comprises a modified release matrix material. Materials for use with the pharmaceutical compositions described herein include, but are not limited to microcrytalline cellulose, sodium carboxymethylcellulose,
hydoxyalkylcelluloses (e.g., hydroxypropylmethylcellulose and hydroxypropylcellulose), polyethylene oxide, alkylcelluloses (e.g., methylcellulose and ethylcellulose), polyethylene glycol, polyvinylpyrrolidone, cellulose acetate, cellulose acetate butyrate, cellulose acetate phthalate, cellulose acetate trimellitate, polyvinylacetate phthalate, polyalkylmethacrylates, polyvinyl acetate, or combinations thereof.
[00290] In some embodiments, the first population of particles comprises a cardiovascular disorder agent. In some embodiments, the second population of particles comprises a (1) a modulator of MIF; (2) a modulator of an interaction between RANTES and Platelet Factor 4; or (3) combinations thereof. In some embodiments, the first population of particles comprises a (1) a modulator of MIF; (2) a modulator of an interaction between RANTES and Platelet Factor 4; or (3) combinations thereof. In some embodiments, the second population of particles comprises a cardiovascular disorder agent.
Additional Dosage Forms
[00291] Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions are generally used, which optionally contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments are optionally added to the tablets or dragee coatings for identification or to characterize different combinations of active agent doses.
[00292] In some embodiments, the solid dosage forms disclosed herein are in the form of a tablet, (including a suspension tablet, a fast-melt tablet, a bite-disintegration tablet, a rapid-disintegration tablet, an effervescent tablet, or a caplet), a pill, a powder (including a sterile packaged powder, a dispensable powder, or an effervescent powder), a capsule (including both soft or hard capsules, e.g., capsules made from animal-derived gelatin or plant-derived HPMC, or "sprinkle capsules"), solid dispersion, solid solution, bioerodible dosage form, controlled release formulations, pulsatile release dosage forms, multiparticulate dosage forms, pellets, granules, or an aerosol. In other embodiments, the pharmaceutical formulation is in the form of a powder. In still other embodiments, the pharmaceutical formulation is in the form of a tablet, including but not limited to, a fast-melt tablet. Additionally, pharmaceutical formulations disclosed herein are optionally administered as a single capsule or in multiple capsule dosage form. In some embodiments, the pharmaceutical formulation is administered in two, or three, or four, capsules or tablets.
[00293] In another aspect, dosage forms include microencapsulated formulations. In some embodiments, one or more other compatible materials are present in the microencapsulation material. Exemplary materials include, but are not limited to, pH modifiers, erosion facilitators, anti- foaming agents, antioxidants, flavoring agents, and carrier materials such as binders, suspending agents, disintegration agents, filling agents, surfactants, solubilizers, stabilizers, lubricants, wetting agents, and diluents.
[00294] Exemplary microencapsulation materials useful for delaying the release of the formulations including a MIF receptor inhibitor, include, but are not limited to, hydroxypropyl cellulose ethers (HPC) such as Klucel® or Nisso HPC, low-substituted hydroxypropyl cellulose ethers (L-HPC), hydroxypropyl methyl cellulose ethers (HPMC) such as Seppifilm-LC, Pharmacoat®, Metolose SR, Methocel®-E, Opadry YS, PrimaFlo, Benecel MP824, and Benecel MP843, methylcellulose polymers such as Methocel®-A, hydroxypropylmethylcellulose acetate stearate Aqoat (HF-LS, HF- LG,HF-MS) and Metolose®, Ethylcelluloses (EC) and mixtures thereof such as E461, Ethocel®, Aqualon®-EC, Surelease®, Polyvinyl alcohol (PVA) such as Opadry AMB, hydroxyethylcelluloses such as Natrosol®, carboxymethylcelluloses and salts of carboxymethylcelluloses (CMC) such as Aqualon®-CMC, polyvinyl alcohol and polyethylene glycol co-polymers such as Kollicoat IR®, monoglycerides (Myverol), triglycerides (KLX), polyethylene glycols, modified food starch, acrylic polymers and mixtures of acrylic polymers with cellulose ethers such as Eudragit® EPO, Eudragit® L30D-55, Eudragit® FS 30D, Eudragit® L100-55, Eudragit® L100, Eudragit® S100, Eudragit® RD100, Eudragit® E100, Eudragit® L12.5, Eudragit® S12.5, Eudragit® NE30D, and Eudragit® NE 40D, cellulose acetate phthalate, sepifilms such as mixtures of HPMC and stearic acid, cyclodextrins, and mixtures of these materials.
[00295] Liquid formulation dosage forms for oral administration are optionally aqueous suspensions selected from the group including, but not limited to, pharmaceutically acceptable aqueous oral dispersions, emulsions, solutions, elixirs, gels, and syrups. See, e.g., Singh et al., Encyclopedia of Pharmaceutical Technology, 2nd Ed., pp. 754-757 (2002). In addition to a MIF receptor inhibitor, the liquid dosage forms optionally include additives, such as: (a) disintegrating agents; (b) dispersing agents; (c) wetting agents; (d) at least one preservative; (e) viscosity enhancing agents; (f) at least one sweetening agent; and (g) at least one flavoring agent. In some embodiments, the aqueous dispersions further include a crystal-forming inhibitor. [00296] In some embodiments, the pharmaceutical formulations described herein are self- emulsifying drug delivery systems (SEDDS). Emulsions are dispersions of one immiscible phase in another, usually in the form of droplets. Generally, emulsions are created by vigorous mechanical dispersion. SEDDS, as opposed to emulsions or microemulsions, spontaneously form emulsions when added to an excess of water without any external mechanical dispersion or agitation. An advantage of SEDDS is that only gentle mixing is required to distribute the droplets throughout the solution. Additionally, water or the aqueous phase is optionally added just prior to administration, which ensures stability of an unstable or hydrophobic active ingredient. Thus, the SEDDS provides an effective delivery system for oral and parenteral delivery of hydrophobic active ingredients. In some embodiments, SEDDS provides improvements in the bioavailability of hydrophobic active ingredients. Methods of producing self-emulsifying dosage forms include, but are not limited to, for example, U.S. Pat. Nos. 5,858,401, 6,667,048, and 6,960,563.
[00297] Suitable intranasal formulations include those described in, for example, U.S. Pat. Nos. 4,476,116, 5,116,817 and 6,391,452. Nasal dosage forms generally contain large amounts of water in addition to the active ingredient. Minor amounts of other ingredients such as pH adjusters, emulsifiers or dispersing agents, preservatives, surfactants, gelling agents, or buffering and other stabilizing and solubilizing agents are optionally present.
[00298] For administration by inhalation, the pharmaceutical compositions disclosed herein are optionally in a form of an aerosol, a mist or a powder. Pharmaceutical compositions described herein are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit is determined by providing a valve to deliver a metered amount. Capsules and cartridges of, such as, by way of example only, gelatin for use in an inhaler or insufflator are formulated containing a powder mix and a suitable powder base such as lactose or starch.
[00299] Buccal formulations include, but are not limited to, U.S. Pat. Nos. 4,229,447, 4,596,795, 4,755,386, and 5,739,136. In addition, the buccal dosage forms described herein optionally further include a bioerodible (hydrolysable) polymeric carrier that also serves to adhere the dosage form to the buccal mucosa. The buccal dosage form is fabricated so as to erode gradually over a predetermined time period. Buccal drug delivery avoids the disadvantages encountered with oral drug administration, e.g., slow absorption, degradation of the active agent by fluids present in the gastrointestinal tract and/or first-pass inactivation in the liver. The bioerodible (hydrolysable) polymeric carrier generally comprises hydrophilic (water-soluble and water-swellable) polymers that adhere to the wet surface of the buccal mucosa. Examples of polymeric carriers useful herein include acrylic acid polymers and copolymers, e.g., those known as "carbomers" (Carbopol®, which is obtained from B.F. Goodrich, is one such polymer). Other components also incorporated into the buccal dosage forms described herein include, but are not limited to, disintegrants, diluents, binders, lubricants, flavoring, colorants, preservatives, and the like. For buccal or sublingual administration, the compositions optionally take the form of tablets, lozenges, or gels formulated in a conventional manner.
[00300] Transdermal formulations of pharmaceutical compositions disclosed here are administered for example by those described in U.S. Pat. Nos. 3,598,122, 3,598,123, 3,710,795, 3,731,683, 3,742,951, 3,814,097, 3,921,636, 3,972,995, 3,993,072, 3,993,073, 3,996,934, 4,031,894, 4,060,084, 4,069,307, 4,077,407, 4,201,211, 4,230,105, 4,292,299, 4,292,303, 5,336,168, 5,665,378, 5,837,280, 5,869,090, 6,923,983, 6,929,801 and 6,946,144.
[00301] The transdermal formulations described herein include at least three components: (1) an active agent; (2) a penetration enhancer; and (3) an aqueous adjuvant. In addition, transdermal formulations include components such as, but not limited to, gelling agents, creams and ointment bases, and the like. In some embodiments, the transdermal formulation further includes a woven or non-woven backing material to enhance absorption and prevent the removal of the transdermal formulation from the skin. In other embodiments, the transdermal formulations described herein maintain a saturated or supersaturated state to promote diffusion into the skin.
[00302] In some embodiments, formulations suitable for transdermal administration employ transdermal delivery devices and transdermal delivery patches and are lipophilic emulsions or buffered, aqueous solutions, dissolved and/or dispersed in a polymer or an adhesive. Such patches are optionally constructed for continuous, pulsatile, or on demand delivery of pharmaceutical agents. Still further, transdermal delivery is optionally accomplished by means of iontophoretic patches and the like. Additionally, transdermal patches provide controlled delivery. The rate of absorption is optionally slowed by using rate -controlling membranes or by trapping an active agent within a polymer matrix or gel. Conversely, absorption enhancers are used to increase absorption. An absorption enhancer or carrier includes absorbable pharmaceutically acceptable solvents to assist passage through the skin. For example, transdermal devices are in the form of a bandage comprising a backing member, a reservoir containing an active agent optionally with carriers, optionally a rate controlling barrier to deliver an active agent to the skin of the host at a controlled and predetermined rate over a prolonged period of time, and means to secure the device to the skin.
[00303] Formulations suitable for intramuscular, subcutaneous, or intravenous injection include physiologically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Examples of suitable aqueous and non-aqueous carriers, diluents, solvents, or vehicles including water, ethanol, polyols (propyleneglycol, polyethylene -glycol, glycerol, cremophor and the like), suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate. Proper fluidity is maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Formulations suitable for subcutaneous injection also contain optional additives such as preserving, wetting, emulsifying, and dispensing agents.
[00304] For intravenous injections, an active agent is optionally formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological saline buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. For other parenteral injections, appropriate formulations include aqueous or nonaqueous solutions, preferably with physiologically compatible buffers or excipients.
[00305] Parenteral injections optionally involve bolus injection or continuous infusion. Formulations for injection are optionally presented in unit dosage form, e.g., in ampoules or in multi dose containers, with an added preservative. In some embodiments, the pharmaceutical composition described herein are in a form suitable for parenteral injection as sterile suspensions, solutions or emulsions in oily or aqueous vehicles, and contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Pharmaceutical formulations for parenteral administration include aqueous solutions of an active agent in water soluble form. Additionally, suspensions are optionally prepared as appropriate oily injection suspensions.
[00306] In some embodiments, an active agent disclosed herein is administered topically and formulated into a variety of topically administrable compositions, such as solutions, suspensions, lotions, gels, pastes, medicated sticks, balms, creams or ointments. Such pharmaceutical compositions optionally contain solubilizers, stabilizers, tonicity enhancing agents, buffers and preservatives.
[00307] An active agent disclosed herein is also optionally formulated in rectal compositions such as enemas, rectal gels, rectal foams, rectal aerosols, suppositories, jelly suppositories, or retention enemas, containing conventional suppository bases such as cocoa butter or other glycerides, as well as synthetic polymers such as polyvinylpyrrolidone, PEG, and the like. In suppository forms of the compositions, a low-melting wax such as, but not limited to, a mixture of fatty acid glycerides, optionally in combination with cocoa butter is first melted.
[00308] In some embodiments, the pharmaceutical composition described herein is in unit dosage forms suitable for single administration of precise dosages. In unit dosage form, the formulation is divided into unit doses containing appropriate quantities of an active agent disclosed herein. In some embodiments, the unit dosage is in the form of a package containing discrete quantities of the formulation. Non-limiting examples are packaged tablets or capsules, and powders in vials or ampoules. In some embodiments, aqueous suspension compositions are packaged in single-dose non-reclosable containers. Alternatively, multiple-dose reclosable containers are used, in which case it is typical to include a preservative in the composition. By way of example only, formulations for parenteral injection are presented in unit dosage form, which include, but are not limited to ampoules, or in multi dose containers, with an added preservative.
Dosages and Administration
[00309] In some embodiments, the pharmaceutical compositions disclosed herein are administered to an individual in need thereof. In some embodiments, the pharmaceutical compositions disclosed herein are administered to an individual diagnosed with (i.e., satisfies the diagnostic criteria for) a cardiovascular disease (e.g., atherosclerosis, angina, stenosis, restenosis, high blood pressure, an aneurysm, an embolism, a blood clot, and/or an infarction (e.g., a myocardial infarction or stroke). In some embodiments, the pharmaceutical compositions disclosed herein are administered to an individual suspected of having a cardiovascular disease. In some embodiments, the pharmaceutical compositions disclosed herein are administered to an individual predisposed to develop a cardiovascular disease.
[00310] In certain instances, an individual is at risk of atherosclerosis if their c-reactive protein (CPvP) levels are above about 3.0 mg/L. In certain instances, an individual is at risk of
atherosclerosis if their homocysteine levels exceed about 15.9 mmol/L. In certain instances, an individual is at risk of atherosclerosis if their LDL levels exceed about 160 mg/dL. In certain instances, an individual is at risk of atherosclerosis if their HDL levels are below about 40 mg/dL. In certain instances, an individual is at risk of atherosclerosis if their serum creatinine levels exceed about 1.5 mg/dL. In certain instances, an individual is pre-disposed to develop atherosclerosis if they possess the "G" allele of SNP rsl0757278 and/or the "C" allele of SNP rsl333049 both of which are located at the locus 9p21. For disclosures regarding the "G" allele of SNP rsl0757278 and/or the "C" allele of SNP rsl333049 see Science, June 8th, 2007; 316(5830): 1491-93 which is herein incorporated by reference for such disclosures. In certain instances, an individual is pre-disposed to develop atherosclerosis if they possess LTA4H haplotypes Hap A, HapB, HapC, HapL, HapK, and/or HapQ. For disclosures regarding LTA4H haplotypes see International Publication No.
WO/2006/105439 which is herein incorporated by reference for such disclosures.
[00311] The daily dosages appropriate for an active agent disclosed herein are from about 0.01 to 3 mg/kg per body weight. An indicated daily dosage in the larger mammal, including, but not limited to, humans, is in the range from about 0.5 mg to about 100 mg, conveniently administered in divided doses, including, but not limited to, up to four times a day or in extended release form. Suitable unit dosage forms for oral administration include from about 1 to 50 mg active ingredient. The foregoing ranges are merely suggestive, as the number of variables in regard to an individual treatment regime is large, and considerable excursions from these recommended values are not uncommon. Such dosages are optionally altered depending on a number of variables, not limited to the activity of the active agents used, the diseases or conditions to be treated, the mode of administration, the requirements of the individual subject, the severity of the disease or condition being treated, and the judgment of the practitioner.
[00312] In the case wherein the individual's condition does not improve, upon the doctor's discretion the administration of an active agent disclosed herein is optionally administered chronically, that is, for an extended period of time, including throughout the duration of the individual's life in order to ameliorate or otherwise control or limit the symptoms of the individual's disease or condition.
[00313] In the case wherein the individual's status does improve, upon the doctor's discretion the administration of an active agent disclosed herein is optionally given continuously; alternatively, the dose of drug being administered is temporarily reduced or temporarily suspended for a certain length of time (i.e., a "drug holiday"). The length of the drug holiday optionally varies between 2 days and 1 year, including by way of example only, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days, 35 days, 50 days, 70 days, 100 days, 120 days, 150 days, 180 days, 200 days, 250 days, 280 days, 300 days, 320 days, 350 days, or 365 days. The dose reduction during a drug holiday includes from 10%- 100%, including, by way of example only, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.
[00314] Toxicity and therapeutic efficacy of such therapeutic regimens are optionally determined in cell cultures or experimental animals, including, but not limited to, the determination of the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between the toxic and therapeutic effects is the therapeutic index, which is expressed as the ratio between LD50 and ED50. An active agent disclosed herein exhibiting high therapeutic indices is preferred. The data obtained from cell culture assays and animal studies are optionally used in formulating a range of dosage for use in human. The dosage of such an active agent disclosed herein lies preferably within a range of circulating concentrations that include the ED50 with minimal toxicity. The dosage optionally varies within this range depending upon the dosage form employed and the route of administration utilized.
EXAMPLES
Material and methods
Cell culture
[00315] Endothelial cells from the human umbilical cord (HUVEC, human umbilical vein endothelial cells, PromoCell, Heidelberg) are cultivated in Endothelial Cell Growth Medium (PromoCell, Heidelberg) and used after 2 to 4 passages.
[00316] Monocyte MoNo. Mac 6-cells (MM6, DSMZ) are cultivated in RPMI 1640 Medium (PAA Laboratories, Pasching, Austria) with addition of 10% fetal calf serum, 2 mM of L-glutamine (Biowhittaker), 1 mM of sodium pyruvate, 50 μ^ηιΐ of Gentamycin and 9 μ^ηιΐ of insulin (MM6 medium). The cells are seeded with a density of 2 x 105/ml in 2 ml of MM6 medium in 24 well plates and are cultivated at 37 degrees C in a humidified atmosphere with 5% C02 for 3 to 4 days, before they are used for experiments.
Peptides
[00317] Peptides of the sequence SEQ ID NO: 3 per formula (3), its mouse orthologue, as well a control peptide of sequence are chemically synthesized by means of t-Boc based solid phase peptide synthesis making use of 4-methyl benzhydrylamine resin, purified by means of reverse-phase HPLC, and optionally formed into a ring in 6 M of guanidine HCl/Tris pH 8. The molecular mass is determined by means of electrospray mass spectrometry (Dawson PE, Kent SB. (2000) Annu Rev Biochem. 69: 923-960, Hackeng TM, Griffin JH, Dawson PE. (1999) Proc Natl Acad Sci U.S.A., Vol 96, p. 10068-10073).
Example 1
[00318] Plasmon resonance studies are used to analyze the inhibitory effect of the peptide of sequence SEQ ID NO: 3 per formula (3) on the formation of heteroaggregates of RANTES and PF4. The plasmon resonance studies are carried out using HBS-EP buffer (10 mM HEPES, 150 mM NaCl, 0.005% Tween 20, pH 7.4). Two flow cells of a CI chip (Biacore AB, Uppsala, Sweden) are activated by injection of 50 μΐ of ethyl(dimethylaminopropyl)carbodiimide/N-hydroxy-succinimide (0.2 M/0.05 M, Pierce Co.) and then 20 μΐ of Streptavidine (0.2 mg/ml, Sigma-Aldrich) is perfused over the activated surface. After this, the surface is inactivated by four consecutive injections of 20 μΐ ethylene diamine (1 M, pH 8, Sigma-Aldrich).
[00319] At the N-terminus, biotinylated human PF4 (bPF4) is chemically synthesized by means of t- Boc based solid phase peptide synthesis and native chemical ligation of PF4 (Dawson PE, Kent SB. (2000) Annu Rev Biochem. 69: 923-960, Hackeng TM, Griffin JH, Dawson PE. (1999) Proc Natl Acad Sci USA, Vol. 96, p. 10068-10073). The bPF4 is immobilized on the dextran surface of a CI sensor chip by injecting 200 μg/ml of bPF4 in HBS-EP across one of the flow chambers and registering 240 resonance units (RU). The second flow chamber is not treated with bPF4 and serves as a reference.
[00320] The binding to bPF4 to RANTES (0.5 μΜ, recombinant human RANTES, Peprotech, Rocky Hill, NJ, USA) or RANTES (0.5 μΜ) that is preincubated with various concentrations, 0 μΜ, 10 μΜ, 50 μΜ and 100 μΜ, of the peptide of sequence SEQ ID NO: 3 per formula (3) in HBS-EP buffer over night at room temperature is determined by means of injection of 15 μΐ of the particular peptide/RANTES mixture and observation of the binding for 180 seconds. The coupling sequence and the measurements are carried out in a Biacore 2000 (Biacore AB) device at a flow rate of 5 μΐ/min. Sensorgrams of the RANTES binding are corrected for nonspecific background signals by means of the software BIAevaluation 3.0 (Biacore AB) and equilibrium resonance units (RU) is determined for each injection.
Example 2: Inhibition of the monocyte arrest on activated endothelium
[00321] The interaction of monocyte MoNo. Mac 6 cells on activated endothelial cells is investigated as follows: Petri dishes with confluent HUVEC cell layers, which are activated with IL- 1B, Peprotech, 10 ng/ml, 12 hours), are placed in a flow chamber. MoNo. Mac 6 cells (0.5 x 106 cells per ml) are resuspended in properly proportioned Hank solution (HBSS with 10 mM Hepes (Gibco BRL), pH 7.3, 0.5% bovine serum albumen (Serva) and kept on ice. Five minutes before the experiment, there is added to the monocyte MM6 cells Ca2+ and Mg2+ to a final concentration of 1 mM each and 60 nM of the chemokines RANTES (Peprotech, Rocky Hill, NJ, USA) and PF4 (ChromaTec, Greifswald) and 6 μΜ each of the peptides of SEQ ID NO: 2 per formula (2), sequence SEQ ID NO: 3 per formula (3), or a control peptide and the materials are heated to 37 degrees C. The thus pretreated cells are then perfused across the endothelial cells at 1.5 dyn/cm3 on a microscope of type IX 50 of the Olympus Co. The number of monocytes that are adherent by interaction with the endothelial cells is determined after 4 minutes in various fields by means of image analysis of pictures of a video camera (3 CCD, JVC) and recorder. The data are evaluated as mean (n = 5) ± standard deviation (p < 0.02) against a control.
Example 3 : In vivo investigations in a mouse model of atherosclerosis
[00322] Female ApoE-/- littermate mice 9 to 12 weeks old (The Jackson Lab, Bar Harbor, Maine, USA) will serve as the model for atherosclerosis. These are given a fat-rich diet (21% fat; Altromin C1061) for 12 weeks. During this time, two groups of mice receive thrice weekly intraperitoneal injections of 50 μg of peptide of sequence SEQ ID NO: 13, given below:
CKEYFYT S SKS SNLAVVFVTRC (13) (SEQ ID NO: 13)
(n = 12 mice) or of a control peptide of sequence SEQ ID NO: 9 per formula (9), as given below:
KEYFYTSGK (9) (SEQ ID NO: 9)
(n = 7 mice) in saline solution. An untreated group of mice (n = 12) serve as an additional control.
[00323] The mice are sacrificed for histological studies. During the period of the experiment, the mice are maintained healthy. Blood samples are taken at the start and after the end of the experimental feeding. The leukocyte count is determined by hemocytometry and the sera are collected and the cholesterol level is determined by means of Infinity Cholesterol kits (Thermo Electron, Melbourne, Australia).
[00324] The extent of the atherosclerosis is determined at the aortal roots and thoracoabdominal aortas by staining the lipid deposits with oil red O stain (Veillard NR, Kwak B, Pelli G, Mulhaupt F, James RW, Proudfoot AE, Mach F. Antagonism of RANTES receptors reduces atherosclerotic plaque formation in mice. Circ Res. 2004; 94: 253-61) and is quantified by means of computerized image analysis (Diskus software, Hilgers, Aachen). Regions of atherosclerotic lesions are determined in 5 micron transverse sections through heart and aortal root. The determination is done for each aortal root by means of lipid-stained regions of 6 sections, at a distance of 50 μιη from each other. The regions of atherosclerotic lesions redivided by the entire surface of the valve of each section. The thoracoabdominal aorta is opened along the ventral midline and the regions of lesions restained in an en face preparation by means of oil red O staining. The proportion of lipid deposition is calculated as the stained region divided by the entire thoracoabdominal surface.
Example 4: Preparation of Multi-Particulate Dosage Form
[00325] A multiparticulate dosage form is prepared. The dosage form comprises an immediate release population of particles containing lovastatin. The dosage form further comprises a controlled-release population of particles comprising the peptide of SEQ ID NO: 2.
[00326] 10 kg of lovastatin, 23 kg of lactose, 0.7 kg of croscarmellose sodium, 0.7 kg
polyvinylpyrrolidone K25 are blended in a high-speed blender. The dry mixture is granulated with 4.3 kg of granulating solution (dissolve 0.02 kg of BHA in 1.7 kg of ethanol while mixing in the high-speed blender and add 2.6 kg of demineralized water to the resulting solution). The granulation is dried in a bed-fluid dryer. The dried granulation is sieved in a 0.5 mm sieve to obtain granulation particles of the desired size.
[00327] 5 mg of COR100140 26 kg of lactose, 0.8 kg of croscarmellose sodium, 0.8 kg
polyvinylpyrrolidone K25 are blended in a high-speed blender. The dry mixture is granulated with 34.3 kg of granulating solution (dissolve 0.02 kg of BHA in 1.7 kg of ethanol while mixing in the high-speed blender and add 2.6 kg of demineralized water to the resulting solution). The granulation is dried in a bed-fluid dryer. The dried granulation is sieved in a 0.5 mm sieve to obtain granulation particles of the desired size. The granules are then sprayed with a controlled -release coating composition comprising.
[00328] The immediate release granules and the controlled-release granules are mixed together. The resulting mixture is encapsulated in gelatin capsules.
Example 5: Preparation of a multi-particulate dosage form
[00329] 10 kg Methotrexate is first screened through a suitable screen (e.g. 500 micron). 25 kg Lactose monohydrate, 8 kg hydroxypropylmethyl cellulose, the screened methotrexate and 5 kg calcium hydrogen phosphate (anhydrous) are then added to a suitable blender (e.g. a tumble mixer) and blended. The blend is screened through a suitable screen (e.g. 500 micron) and reblended. About 50% of the lubricant (2.5 kg, magnesium stearate) is screened, added to the blend and blended briefly. The blend is roller compacted through a suitable roller compactor. The ribbon blend is then granulated, by screening through a suitable screen (e.g. 500 micron) and reblended. The remaining lubricant (2 kg, magnesium stearate) is screened, added to the blend and blended briefly. The granules are screened (e.g. 200 micron) to obtain granulation particles of the desired size.
[00330] Peptide granules are prepared by blending 2.8 kg of the peptide of SEQ ID NO: 2 with microcrystalline cellulose (Avicel® PH101, FMC Corp., Philadelphia, Pa.) in relative amounts of 95:5 (w/w), wet massing the blend in a Hobart mixer with water equivalent to approximately 27% of the weight of the blend, extruding the wet mass through a perforated plate (Luwa EXKS-1 extruder, Fuji Paudal Co., Osaka Japan), spheronizing the extrudate (Luwa QJ-230 marumerizer, Fuji Paudal Co.) and drying the final granules which are about 1 mm diameter. The granules are optionally coated with a plasticized ethylcellulose dispersion (Sure lease®, Colorcon, West Point, Pa., typically applied at 15% solids concentration) in a bottom spray Wurster fluid bed coater (Aeromatic Strea-1, Niro Inc., Bubendorf, Switzerland) to obtain sustained release granules. The amount of coating applied is varied to obtain different dissolution rate behavior. For example, an additional coating of 2% Opadry® is optionally applied over the Surelease® Coat.
[00331] The methotrexate immediate release granules and the peptide of SEQ ID NO: 2 sustained release granules are mixed together and the resulting mixture is encapsulated in gelatin capsules.
Example 6: Toxicity Study following Statin/the peptide of SEQ ID NO: 2 combination in Mouse Model
Study Design
[00332] Female Harlan Sprague-Dawley mice weighing 20 to 24 g are used. The animals used were within an age range of 6 to 8 weeks at the start of dosing.
[00333] The mice are divided into two groups: the experimental group (n=16) and the control group (n=16). The experimental group receives daily intraperitoneal injections of a combination of simvastatin (80 mg/kg) and the peptide of SEQ ID NO: 2 (1.5 mg/kg) (n = 16 mice) for 14 days. The experimental group receives daily intraperitoneal injections of a saline solution (n = 16 mice) for 14 days.
[00334] The mice are sacrificed for histological studies. Four mice from the experimental group are sacrificed on each of days 5, 7, 12, and 14. Four mice from the control group are sacrificed on each of days 5, 7, 12, and 14.
Necropsy and Histology
[00335] Tissue sample are taken from the (a) heart, (b) kidneys, (c) liver, (d) stomach, and (e) muscle tissues. The sampled muscles tissues are taken from (a) the right fore limb (the biceps femoris, extensor digitorum longus, tibialis cranialis, and vastus medialis); (b) the left hind limb (the biceps brachii, extensor carpi radialis longus, and flexor carpi ulnaris); the abdominal peritoneal; the diaphragm; the masseter superficialis; the tongue; and the trapezius).
[00336] Tissues are fixed in buffered 10% formalin, processed to wax blocks, and then sectioned and stained with haematoxylin and eosin for examination by light microscopy. Necrosis is graded subjectively. Minimal necrosis is up to 10 necrotic fibers in the whole section; mild is up to about 20% necrotic fibers; moderate is up to about 50% necrotic fibers; and severe is more than 50% necrotic fibers.
Electron Microscopy
[00337] Samples for ultrastructural assessment are immersion fixed in 2.5% glutaraldehyde fixative. Glutaraldehyde-fixed samples are postfixed in 1% osmium tetroxide and processed to Araldite resin blocks. Thin, 70-90-nm resin sections are cut and stained using uranyl acetate and lead citrate. Ultrastructural morphology is examined with a TEM.
Muscle Histochemistry
[00338] Muscle samples are trimmed, orientated on a cork disk, and frozen in isopentane (Fisher Scientific) pre-cooled with liquid nitrogen. Serial cryosections of 7-μιη thickness are cut from each sample for fiber typing. Sections are stained for mATPase activity following pre-incubation at high and low pH. One section is placed in an incubating solution at pH 9.4 consisting of 0.5% ATP (Sigma) in 0.1 M glycine/NaCl buffer with 0.75 M CaCl2 for 45 minutes at 37°C. A further section is pre -incubated in 0.1 M sodium acetate buffer with 10 mM ETDA (pH 4.1-4.3) for 10 minutes at 4°C before placing in the incubation solution noted previously. Following incubation the slides are transferred to 2% CoCl2 for 5 minutes followed by 30 seconds in 10% ammonium sulphide solution. Sections are washed thoroughly in distilled water between each step. Sections are lightly counterstained with Carazzi's haematoxylin before being dehydrated, cleared, and mounted in Histomount.
Muscle Immunohistochemistry
[00339] Serial cryostat sections are stained for fast and slow myosin heavy chains using antibodies (e.g., NCL-MHCf for fast myosin heavy chains, and NCL-MHCs for slow myosin heavy chains). The sections are incubated in the primary antibody for 60 minutes, then incubated in the secondary antibody (i.e., rabbit anti-mouse HRP conjugate) for 30 minutes, before being visualized by incubation with 3,3' diaminobenzidine tetrahydrochloride for 5 minutes. All incubations are at room temperature, and sections are washed thoroughly in tris-buffered saline between each step. Sections are counterstained with Carazzi's haematoxylin before being dehydrated, cleared, and mounted in Histomount. Dewaxed sections are subjected to 2 minutes full pressure in a microwave pressure cooker containing 0.01 M citrate buffer at pH 6.0, and then 5 minutes digestion at room temperature by proteinase K. Endogenous peroxidase activity is blocked by incubation in a peroxidase inhibitor for 20 minutes, followed by 15 minutes in 20% normal rabbit serum. Mouse monoclonal antibody is applied for 30 minutes, followed by 30 minutes in peroxidase-conjugated rabbit anti-mouse antibody. Vector Laboratory's SG peroxidase substrate kit (SK4700) is then applied for 10 minutes. Following an additional 15 minutes of incubation in 20% normal rabbit serum, a mouse mAB to fast myosin is applied. This is visualized using Vector Red alkaline phosphatase substrate kit (Vector Labs SK5100) for 10 minutes. All incubations were at room temperature, and sections are washed thoroughly in tris-buffered saline between each step. Sections are dehydrated, cleared, and mounted in Histomount.
Example 7: Statin/the peptide of SEP ID NO: 2 combination in mouse model of atherosclerosis
[00340] Female ApoE-/- littermate mice 9 to 12 weeks old (The Jackson Lab, Bar Harbor, Maine, USA) will serve as the model for atherosclerosis. These are given a fat-rich diet (21% fat; Altromin C1061) for 12 weeks. During this time, two groups of mice receive thrice weekly intraperitoneal injections of a combination of simvastatin (5 mL/kg) and the peptide of SEQ ID NO: 2 (1.5 mg/kg) (n = 12 mice) or a saline solution (n = 7 mice).
[00341] The mice are sacrificed for histological studies. During the period of the experiment, the mice are maintained healthy. Blood samples are taken at the start and after the end of the experimental feeding. The leukocyte count is determined by hemocytometry and the sera are collected and the cholesterol level is determined by means of Infinity Cholesterol kits (Thermo Electron, Melbourne, Australia).
[00342] The extent of the atherosclerosis is determined at the aortal roots and thoracoabdominal aortas by staining the lipid deposits with oil red O stain (Veillard NR, Kwak B, Pelli G, Mulhaupt F, James RW, Proudfoot AE, Mach F. Antagonism of RANTES receptors reduces atherosclerotic plaque formation in mice. Circ Res. 2004; 94: 253-61) and is quantified by means of computerized image analysis (Diskus software, Hilgers, Aachen). Regions of atherosclerotic lesions are determined in 5 micron transverse sections through heart and aortal root. The determination is done for each aortal root by means of lipid-stained regions of 6 sections, at a distance of 50 μιη from each other. The regions of atherosclerotic lesions redivided by the entire surface of the valve of each section. The thoracoabdominal aorta is opened along the ventral midline and the regions of lesions restained in an en face preparation by means of oil red O staining. The proportion of lipid deposition is calculated as the stained region divided by the entire thoracoabdominal surface.
Example 8: Human Clinical Trial of P4/RANTES Antagonist in Combination with Torcetrapib as a Treatment for Hypercholesterolemia
Study Objective(s): The primary objective of this study is to assess the efficacy of a combination of torcetrapib and the peptide of SEQ ID NO: 2 (C-KEYFYTSGKCSNPAWFVTR-C) (T/P2; 60mg/1.5 mg/kg) in subjects with homozygous familial hypercholesterolemia (HoFH) versus torcetrapib (60 mg) alone.
METHODS
[00343] Study Design: This study is a prospective, double -blind, multicenter, parallel-treatment trial comparing T/P2 versus T alone in male and female subjects >18 years of age with HoFH. After initial screening, eligible subjects enter a 4-week screening period, consisting of 2 visits (Weeks -4 and -1), during which all lipid-lowering drugs are discontinued (except for bile acid sequestrants and cholesterol absorption inhibitors) and therapeutic lifestyle change counseling (TLC) according to National Cholesterol Education Program (NCEP) Adult Treatment Panel (ATP-III) clinical guidelines or equivalent is initiated. Subjects already on apheresis continue their treatment regimen maintaining consistent conditions and intervals during the study. At Visit 3 (Week 0), subjects begin treatment with the T/P2 fixed combination once daily (QD) for 6 weeks or T alone. Final visit (Visit 6) occurs at Week 18. Study visits are timed with subjects' apheresis treatments to occur immediately before the visit procedures, where applicable. When the intervals between aphereses are misaligned with a study drug treatment period, the subjects are kept in the same drug treatment period until the next scheduled apheresis, and until the intervals are brought back to the original length of time. Efficacy measures are done at least 2 weeks after the previous apheresis and just before the apheresis procedure scheduled for the day of study visit.
[00344] Number of Subjects: 50 subjects divided into two groups - the experimental group (n= 25) and the control group (n= 25).
[00345] Diagnosis and Main Criteria for Inclusion: Men and women 18 years of age or older with definite evidence of the familial hypercholesterolemia (FH) homozygote per World Health
Organization guidelines, and with serum fasting triglyceride (TG) <400 mg/dL (4.52 mmol/L) for subjects aged >20 years and 200 mg/dL (2.26 mmol/L) for subjects aged 18-20 years, are screened for study participation.
[00346] Study Treatment: Subjects are randomized into two groups. During the three 6-week treatment period, subjects in the experimental group take 1 tablet of T/P2 QD, with food, immediately after the morning meal. Subjects in the control group take 1 tablet of T QD, with food, immediately after the morning meal.
[00347] Efficacy Evaluations: The primary endpoints are the mean percent changes in HDL-C and LDL-C from baseline to the end of each treatment period (ie, Weeks 6, 12 and 18). A lipid profile which included HDL-C and LDL-C is obtained at each study visit.
[00348] Safety Evaluations: Safety is assessed using routine clinical laboratory evaluations (hematology and urinalysis panels at Weeks -4, 0 and 18, and chemistry also at Weeks 6 and 12). Vital signs are monitored at every visit, and physical examinations and electrocardiograms (ECGs) are performed at Weeks 0 and 18. Urine pregnancy testing is carried out at every visit except Week -1. Subjects are monitored for adverse events (AEs) from Week 0 to Week 18. Week 18 safety assessments are completed at early termination if this took place.
[00349] Statistical Methods: The primary efficacy endpoints are the percent changes in HDL-C and LDL-C from baseline to the end of each treatment period (ie, Weeks 6, 12, and 18). The primary efficacy analysis population is the full analysis set (FAS) which includes all subjects who received at least 1 dose of study drug and had both a baseline and at least 1 valid post-baseline measurement at each analysis period.
[00350] The primary efficacy endpoints are analyzed through the computation of sample means of percent (or nominal) changes, their 95% confidence intervals (CIs), 1 -sample t-test statistics, and corresponding p-values. Incremental treatment differences between different dose levels are also estimated and 95% CIs obtained. Hypothesis testing is 2-sided with an overall family-wise type I error rate of 5% (ie, p = 0.05 significance level). Hochberg's procedure is used to control the family- wise error rate for multiple comparisons.
Example 9: Human Clinical Trial of MIF Antagonist in Combination with Atorvastatin as a Treatment for Atherosclerosis
[00351] Study Objective(s): To measure the effect of 18 months of treatment with lipid lowering treatment (atorvastatin 80-mg daily) versus 8 months of treatment with atorvastatin in combination with a peptide of SEQ ID NO: 2 (1.5 mg/kg) on coronary artery plaque using intravascular ultrasound (IVUS) imaging of the coronary arteries.
Study Design:
[00352] This study is a prospective, double-blind, multicenter, parallel-treatment trial comparing the effects of atorvastatin 80-mg versus atorvastatin in combination with (80-mg daily) a peptide of SEQ ID NO: 2 (1.5 mg/kg) as measured by IVUS.
[00353] The study consists of three phases: (1) subject identification and cardiac catheterization, (2) screening phase to determine eligibility, which includes a 2-week Placebo Run-in Period, and (3) an 18-month, randomized, double-blind treatment phase.
[00354] The study includes a total of up to 12 visits (nine required plus three optional) at which safety and/or efficacy assessments are performed: Qualifying IVUS Visit (Cath 1), Screening Visit 1 (SV1), Optional Screening Visits (SV2 and SV3), Randomization Visit (RV), and Clinic Visits for Month 3 (M3), M6, M9, M12, M15, M17 (optional), and M18.
[00355] The primary efficacy parameter is percent change in total plaque (atheroma) volume (TPV) by IVUS.
[00356] Secondary efficacy parameters include nominal change in TPV and change in percent plaque (atheroma) volume (PPV). Number of Patients:
[00357] Approximately 400 subjects (200 subjects per treatment group) are to be enrolled
Diagnosis and Main Criteria for Inclusion:
[00358] Male and female subjects between 30-75 years of age with CAD who have had a coronary catheterization. Precise angiographic inclusion criteria will determine subject eligibility, specifically the presence of at least one obstruction in a major cardiac vessel with at least a 20% luminal diameter narrowing by visual estimation. In addition, subjects must have had a "target vessel" for rV JS interrogation with no more than 50% luminal narrowing throughout a segment that was a minimum of 30 mm in length (the "target segment"). The target vessel must not have undergone previous intervention, nor have been a candidate for intervention at the time of Baseline catheterization. Lipid entry criterion require subjects to have a low-density lipoprotein cholesterol (LDL-C) between 125 and 210 mg/dL following a 4- to 10-week washout period if the subject is taking antihyperlipidemic medication.
Study Treatment:
[00359] Subjects are divided into the groups. The first group (n=200) receives atorvastatin. The second group (n=200) receives atorvastatin in combination with a peptide of SEQ ID NO: 2 (1.5 mg/kg).
[00360] Placebo Run-in Period: Subjects in the two groups are instructed to take two placebo tablets at bedtime each day and return to the Clinic in two weeks for the Randomization Visit. The time between visits during the Placebo Run-in Period is not to exceed 17 days. Subjects are also required to be at least 90%> compliant before randomization to the double-blind period.
[00361] Double-Blind Period: Subjects in group 1 are instructed to take 80-mg atorvastatin (2 x 40- mg tablet) and one placebo tablet daily at bedtime each day for 18 months. Subjects in group 2 are instructed to take 80-mg atorvastatin (2 x 40-mg tablet) in combination with a peptide of SEQ ID NO: 2 (1.5 mg/kg; 1 tablet) daily at bedtime each day for 18 months.
Efficacy Evaluations:
[00362] Primary efficacy variable: The percent change in total plaque volume for all slices of anatomically comparable segments of the target coronary artery from Baseline to Month 18 measured by IVUS.
[00363] Safety Evaluations: Safety of the treatment is assessed by an evaluation of type, frequency, intensity, and duration of all reported adverse events (AEs), monitoring of laboratory parameters, and changes in vital signs. Data for electrocardiogram (ECG) results and physical examination findings is collected.
Example 10: In vivo investigations in a rat model of arthritis disease to test combination of
Etanercept and the peptide of SEQ ID NO: 2 [00364] 31 Male Lewis rats are immunized with complete Freund's adjuvant on day 0 to induce an aggressive arthritis characterized by joint destruction and paw swelling.
[00365] From day 8 to 20, two groups of rats receive thrice weekly intraperitoneal injections of 50 μg of peptide of SEQ ID NO: 3 (n = 12 rats). During this time, the rats also receive weekly subcutaneous injections of 50 μg Etanercept. An untreated group of rats (n = 12) serve as a control.
[00366] Every week, paw swelling is determined by water displacement plethysmometry. The extent of arthritis is determined at the end of the study on day 21. Radiographs are obtained of the right hind paw to assess bone changes using a semi-quantitative scoring system: demineralization (0-2+), calcaneal erosion (0-1+), and heterotropic bone formation (0-1+), with a maximum possible score=6. Blood samples are tested for neutropenia.
Example 11 : In vivo investigations in a rat model of Crohn's disease to test combination of methotrexate and the peptide of SEP ID NO: 2
[00367] A modified animal model disclosed in Kirkil, C. et al., J Gastrointest Surg. 2008, 12, 1429- 35 is used. Twenty-eight Sprague-Dawley rats are divided into four groups. Groups I and II are used as sham-operated and control groups, respectively. Bowel inflammation is induced by intrajejunal injection of iodoacetamide in groups III and IV. Rats in group IV are treated with oral preparation of methotrexate (10 mg) and intravenous injection of 50 μg of peptide of sequence SEQ ID NO: 3 (n = 12 rats).
[00368] Three days after induction of the inflammation, partial resection of test loop and anastomosis is performed. Re-laparotomy is performed, anastomosis bursting pressures and peritonitis scores are measured, and tissue samples are obtained for the measurements of tissue hydroxylproline level and mucosal damage index 4 days later.
[00369] On the fourth day, measurements of tissue hydroxylproline level and mucosal damage index are obtained. The severity of iodoacetamide induced intestinal inflammation, wound healing in the inflamed intestinal tissue, and decrease in severity of peritonitis is also recorded.
Example 12: Human Clinical Trial in SLE to test combination of cyclophosphamide and the peptide of SEP ID NO: 2
[00370] Study Pbjective(s): The primary objective of this study is to assess efficacy of the fixed combination cyclophosphamide and the peptide of SEP ID NP: 2 (C/P2; 60/20 mg, 60/40 mg, 60/80 mg) in subjects with systemic lupus erythematosus (SLE) who are currently receiving
cyclophosphamide. This study will also determine if P2 is effective in decreasing disease activity in these patients.
METHPDS [00371] The first part of the study is a dose-escalation study in which participants will receive one of two doses of P2 (20 mg, or 40 mg,); this part of the study will last 60 days. At screening, patients will have an IV catheter inserted into their arms for administration of cyclophosphamide and P2. Patients will also have medical and medication history assessments, a comprehensive physical exam, and blood and urine tests. There are 5 study visits for the first part of the trial; these will occur at screening, at study entry, and Days 1, 14, and 28. Selected visits will include physical exam, vital signs measurement, blood and urine tests, and disease activity assessment. At Days 7 and 60, patients will be contacted by phone to report their medication history and any adverse effects they have experienced.
[00372] The second part of the study will evaluate a single 80 mg dose of P2; this part of the study will last 90 days. In the study, participants will be randomly assigned to one of two groups. At the start of the study, Group 1 participants will receive P2 and cyclophosphamide and Group 2 participants will receive cyclophosphamide only. There will be 9 study visits; these will occur at study screening, study entry, and Days 1, 4, 7, 14, 28, and 60. At selected visits, patients will undergo physical exam, vital signs measurement, blood tests and urine tests, and disease activity assessment.
[00373] Number of Subjects: It is planned to recruit between 30 and 50 subjects for each part of the study.
[00374] Diagnosis and Main Criteria for Inclusion: Diagnosis of SLE by American College of Rheumatology (ACR) criteria
[00375] Concurrent treatment with intravenous cyclophosphamide for at least one of the following manifestations of lupus: World Health Organization (WHO) class III, IV, or V lupus nephritis;
British Isles Lupus Assessment Group (BILAG) score of A for vasculitis; BILAG score of A for cytopenia; BILAG score of A for nervous system; Stable medication regimen for at least 4 weeks prior to study entry; Weight between 40 kg (88.2 lbs) and 125 kg (275.6 lb).
[00376] Study Treatment: During the study periods, subjects will have an IV catheter inserted into their arms for intravenous bi-weekly administration of cyclophosphamide and P2.
[00377] Efficacy Evaluations: The primary endpoint is SLE disease activity as measured by blood tests, urine tests, and disease activity assessment.
[00378] Safety Evaluations: Safety is assessed using routine clinical laboratory evaluations (lupus serology and renal function).
Example 13 : Human Clinical Trial in Rheumatoid Arthritis to test combination of infliximab and the peptide of SEP ID NO: 2
[00379] Study Objective(s): The primary objective of this study is to assess efficacy of the fixed combination infliximab/the peptide of SEQ ID NO: 2 (I/P2; 5 mg/kg/20 mg, 10 mg/kg/20 mg, 15 mg/kg/20 mg) in subjects with rheumatoid arthritis who are currently receiving infliximab for treatment of rheumatoid arthritis. This study will also determine if P2 is effective in decreasing disease activity in these patients.
METHODS
[00380] Participants will receive nine infusions of infliximab and P2 every three weeks during this 28-week study. The drug is given intravenously (IV, into a vein) over 2 hours. The first three infusions will be at a dose of 5 mg/kg of body weight. Patients will also receive 20 mg P2 in a saline solution (IV, into a vein) over 1 hour. Patients who improve on this regimen will receive another 6 infusions at the same dose. Patients who do not significantly improve on 5 mg/kg at the end of 6 weeks (the third infusion) may continue with phase 2 of the study, in which they will be randomly assigned to receive either: 1) 6 additional doses of infliximab at 5 mg/kg per dose, or 2) a gradually increased dose of inflilximab to a maximum of 15 mg/kg. In addition, all patients will continue to take P2 at the same dose as when they entered the study.
[00381] Patients will have imaging studies (x-rays, MRI and Dexa scan) at the beginning and end of the study and will collect a 24-hour urine sample before each infliximab and P2 infusion.
[00382] Number of Subjects: It is planned to recruit between 30 and 50 subjects for each part of the study.
[00383] Inclusion criteria: Patients must be at least 18 years old at the screening visit. Patients must have a diagnosis of adult-onset RA of at least six months duration but not longer than fifteen years as defined by the 1987 American College of Rheumatology classification criteria.
[00384] Patients must have active RA disease as defined by: 9 tender joints at Screening and
Baseline, 9 swollen joints at Screening and Baseline and fulfilling 1 of the following 2 criteria during the screening period, 30 mm/hour ESR (Westergren), or CRP >15 mg/L.
[00385] Patients must have received treatment with infliximab for at least 6 months prior to the
Baseline visit. The dose of infliximab and route of administration must have been stable for at least
2 months prior to the baseline visit. The minimum stable dose of infliximab allowed is 5 mg/kg weekly.
[00386] Exclusion criteria: Patients must not have a diagnosis of any other inflammatory arthritis (e.g., psoriatic arthritis or ankylosing spondylitis), Patients must not have a secondary, noninflammatory type of arthritis (e.g. OA or fibromyalgia), Female patients who are breast feeding, pregnant, or plan to become pregnant during the trial or for three months following last dose of study drug, Patients with a history of tuberculosis or positive chest X-ray for tuberculosis, Patients at a high risk of infection (e.g. leg ulcers, indwelling urinary catheter and persistent or recurrent chest infections and patients who are permanently bed ridden or wheelchair bound), Patients with known human immunodeficiency virus (HTV) infection, Patients with an active malignancy of any type or a history of malignancy (except basal cell carcinoma of the skin that has been excised prior to study start), Patients with a current or recent history, as determined by the Investigator, of severe, progressive, and/or uncontrolled renal, hepatic, hematological, gastrointestinal, endocrine, pulmonary, cardiac, neurological, or cerebral disease which would interfere with the patient's participation in the trial, Patients with a history of, or suspected, demyelinating disease of the central nervous system (e.g. multiple sclerosis or optic neuritis).
[00387] Primary Outcome measures: Compare efficacy of two dose regimens of infliximab in combination with P2 to infliximab alone in patients with RA measured by the ACR20 at week 28.
[00388] Secondary outcome measures: Assess Safety and Tolerability of two dose regimens of infliximab in combination with P2 and infliximab alone in patients with RA; prevention of joint damage in patients with RA; Health Outcomes Measures.
[00389] Study treatment: During the study periods, subjects will have an IV catheter inserted into their arms for intravenous administration of infliximab and P2.
[00390] Efficacy evaluations: The primary endpoint is rheumatoid arthritis disease activity as measured by blood tests, urine tests, x-rays and disease activity assessment.
[00391] Safety Evaluations: Safety is assessed using routine clinical laboratory evaluations (blood tests, urine tests).
EXAMPLE 14
Use of a Peptide Disclosed herein to Diagnose Abdominal Aortic Aneurysms (AAA)
[00392] A human patient is selected for inclusion in the trial if they have been diagnosed with atherosclerosis and meet 2 of the following criteria: 60 years of age or older; male; cigarette smoking; high blood pressure; high serum cholesterol; and diabetes mellitus.
[00393] Peptide 2 (P2; C -KEYFYTSGKCSNPAWFVTR-C) is radiolabeled using 18F. The peptide is then formulated for parenteral administration. The peptide composition is administered intravenously over 30 minutes. Two hours after administration of the peptide, the individual is imaged via use of a gamma-camera.
EXAMPLE 15
Animal Model for Treatment of Abdominal Aortic Aneurysms (AAA)
[00394] Animal models are prepared as follows. An adult, male rat is subjected to infusion of elastase for 2 hours. Histological analysis is performed 12-24 hours after infusion to confirm presence of fragmented and disorganized elastin. Ultrasound is performed daily to identify and monitor areas of aortic enlargement.
[00395] 2 weeks after administration of elastase, the rat is administered Peptide 2 (P2; C- KEYFYTSGKCSNPAWFVTR-C). The initial administration of P2 is infused into subject at a rate of 0.5 mg/hr. In the absence of infusion toxicity, increase infusion rate by 0.5 mg/hr increments every 30 minutes, to a maximum of 2.0 mg/hr. Each week thereafter, P2 is infused at a rate of 1.0 mg/hr. In the absence of infusion toxicity, increase rate by 1.0 mg/hr increments at 30-minute intervals, to a maximum of 4.0 mg/hr.
[00396] Efficacy Evaluations: The primary endpoints are the mean percent changes in Abdominal Aortic Aneurysm (AAA) size (i.e., aortic diameter) from baseline to weeks 3, 6, and 12.
EXAMPLE 16
Human Clinical Trial for Treatment of Abdominal Aortic Aneurysms (AAA)
[00397] Study Objective(s): The primary objective of this study is to assess efficacy of Peptide 2
(P2; C-KEYFYTSGKCSNPAWFVTR-C) in individuals with early AAA.
METHODS
[00398] Study Design: This is a multi-center, open-label, single-group study of P2 in male and female individuals >18 years of age with early AAA. Presence of early Abdominal Aortic Aneurysm (AAA) is confirmed with serial cross-sectional imaging. At Week 0, baseline efficacy/safety values are determined and individuals begin treatment with the initial dose of P2. Subjects are administered P2 once a week for 12 weeks.
[00399] Number of Participants: Between 30 and 50 individuals.
[00400] Study Treatment: The initial administration of P2 is infused into subject at a rate of 50 mg/hr. In the absence of infusion toxicity, increase infusion rate by 50 mg/hr increments every 30 minutes, to a maximum of 400 mg/hr. Each week thereafter, P2 is infused at a rate of 100 mg/hr. In the absence of infusion toxicity, increase rate by 100 mg/hr increments at 30-minute intervals, to a maximum of 400 mg/hr.
[00401] Efficacy Evaluations: The primary endpoints are the mean percent changes in Abdominal Aortic Aneurysm (AAA) size (i.e., aortic diameter) from baseline to weeks 3, 6, and 12.
EXAMPLE 17
Animal Model for Treatment of Abdominal Aortic Aneurysms (AAA)
[00402] The aim of this study was to evaluate whether a peptide antagonist of the CXCL4-CCL5 interaction effects the development of AAA in the porcine pancreatic elastase (PPE) infusion mouse model of human AAA.
[00403] Male C57BL/6 mice at 10 weeks of age were injected i.v. with Peptide 2(P2;
CKE YF YT S GKC SNP A WF VTRC, 10 mg/kg, n=5, or 20 mg/kg, n=7) or the vehicle for 3 days. AAA was then created in these mice by infusion of the infrarenal aorta with PPE. Mice were continuously treated i.v. with peptide antagonist or vehicle for 13 days. Ultrasonography was used to measure infrarenal aortic diameter before PPE infusion and several time points thereafter, and the AAA was defined as a 50% increase in the aortic diameter. On day 14 after PPE infusion, mice were sacrificed, and the aortae were collected for evaluating mural inflammation by histopathology.
[00404] P2 showed a dose-dependent inhibition of the increase in aorta diameter caused by PPE infusion.
[00405] AAA developed in all mice treated with the vehicle within 7 days. In contrast, AAA developed in 3 mice and 1 mouse treated with P2 at the dose of 10 and 20 mg /kg/day, respectively, within 14 days.
[00406] The elastic Masson staining and the anti-smooth muscle cell alpha actin antibody immunostaining revealed that medial elastin fibers and smooth muscle cell layers were well preserved in P2 -treated mice as compared to the vehicle -treated mice.
[00407] P2 treatment significantly reduced the numbers of macrophages, CD4+ T cells and neutrophils and newly formed blood vessels in the media and adventitia by immunostaining against leukocyte subsets and endothelial cells.
[00408] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

CLAIMS WHAT IS CLAIMED IS:
1. A peptide that (a) mimics the RANTES binding domain of PF4 and (b) inhibits the formation of a PF4 and RANTES heterodimer.
2. The peptide of claim 1, comprising (a) 15-25 amino acids and (b) an amino acid sequence that is 85% homologous to an amino acid sequence selected from:
Figure imgf000112_0001
- I l l - NO. 87);
SDTTPCCFAYIARPL (SEQ ID NO. 65) YSFVKCCSFKGTTVYALSNVRS (SEQ ID
NO. 88);
PLPRAHIKEYFYTSG (SEQ ID NO. 66) SFVKCCSFKGTTVYALSNVRSY (SEQ ID
NO. 89);
EYFYTSGKCSNPAW (SEQ ID NO. 67) FVKCCSFKGTTVYALSNVRSYS (SEQ ID
NO. 90)
VFVTRKNPvQVCANPE (SEQ ID NO. 66)
3. The peptide of claim 2, comprising an amino acid sequence is at least 90% homologous to an amino acid sequence selected from:
MSSAAGFCASRP (SEQ ID NO. 46) EKKWVREYINSLEMS (SEQ ID NO. 69)
RPGLLFLGLLLLPL (SEQ ID NO. 47) MKISAAALTIILTAA (SEQ ID NO. 70)
PLWAFASAEA (SEQ ID NO. 48) AALCTPAPASPYGSD (SEQ ID NO. 71)
SAEAEEDGDLQC (SEQ ID NO. 49) SDTTPCCFAYLSLAL (SEQ ID NO. 72)
QCLCVKTTSQVRPRH (SEQ ID NO. 50) ALPRAHVKEYFYTSS (SEQ ID NO. 73)
PRHITSLEVIKAGPHC (SEQ ID NO. 51) SSKCSNLAWFVTRR (SEQ ID NO. 74)
PTAQLIATLKNGRKIC (SEQ ID NO. 52) RRNRQVCANPEKKWV (SEQ ID NO. 75)
LDLQAPLYKKIIKKLLES (SEQ ID NO. 53) EKKWVQEYINYLEMS (SEQ ID NO. 76)
MSVAAVFRGLRPSPE (SEQ ID NO. 54) S SKS SNLA WFVTRCCKEYFYT (SEQ ID
NO. 77)
SPELLLLGLLFLPAV (SEQ ID NO. 55) SKS SNLA WFVTRCCKEYFYTS (SEQ ID
NO. 78)
PAVVAVTSAGPEESD (SEQ ID NO. 56) KS SNLA WFVTRCCKEYFYT S S (SEQ ID
NO. 79);
ESDGDLSCVCVKTIS (SEQ ID NO. 57) S SNLA WFVTRCCKEYFYT S SK (SEQ ID
NO. 80);
TISSGIHLKHITSLE (SEQ ID NO. 58) SNLA WFVTRCCKEYFYTS SKS (SEQ ID
NO. 81);
VIKAGRHCAVPQLIA (SEQ ID NO. 59) NLA WFVTRCCKEYFYT S SKS S (SEQ ID
NO. 82);
LIATLKNGRKICLDR (SEQ ID NO. 60) SFKGTTVYALSNVRSYSFVKCC (SEQ ID
NO. 83);
LDRQAPLYKKVIKKI (SEQ ID NO. 61) FKGTTVYALSNVRSYSFVKCCS (SEQ ID
NO. 84);
QAPLYKKVIKKILES (SEQ ID NO. 62) SNVRSYSFVKCCSFKGTTVYAL (SEQ ID NO. 85);
MKVSAAALAVILIAT (SEQ ID NO. 63) NVRSYSFVKCCSFKGTTVYALS (SEQ ID
NO. 86);
ATALCAPASASPYSS (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID
NO. 87);
SDTTPCCFAYIARPL (SEQ ID NO. 65) YSFVKCCSFKGTTVYALSNVRS (SEQ ID
NO. 88);
PLPRAHIKEYFYTSG (SEQ ID NO. 66) SFVKCCSFKGTTVYALSNVRSY (SEQ ID
NO. 89);
EYFYTSGKCSNPAW (SEQ ID NO. 67) FVKCCSFKGTTVYALSNVRSYS (SEQ ID
NO. 90)
VFVTRKNPvQVCANPE (SEQ ID NO. 66)
4. The peptide of claim 2, comprising an amino acid sequence is at least 95% homologous to an amino acid sequence selected from:
MSSAAGFCASRP (SEQ ID NO. 46) EKKWVREYINSLEMS (SEQ ID NO. 69)
RPGLLFLGLLLLPL (SEQ ID NO. 47) MKISAAALTIILTAA (SEQ ID NO. 70)
PLWAFASAEA (SEQ ID NO. 48) AALCTPAPASPYGSD (SEQ ID NO. 71)
SAEAEEDGDLQC (SEQ ID NO. 49) SDTTPCCFAYLSLAL (SEQ ID NO. 72)
QCLCVKTTSQVRPRH (SEQ ID NO. 50) ALPRAHVKEYFYTSS (SEQ ID NO. 73)
PRHITSLEVIKAGPHC (SEQ ID NO. 51) SSKCSNLAWFVTRR (SEQ ID NO. 74)
PTAQLIATLKNGRKIC (SEQ ID NO. 52) RRNRQVCANPEKKWV (SEQ ID NO. 75)
LDLQAPLYKKIIKKLLES (SEQ ID NO. 53) EKKWVQEYINYLEMS (SEQ ID NO. 76)
MSVAAVFRGLRPSPE (SEQ ID NO. 54) S SKS SNLA WFVTRCCKEYFYT (SEQ ID
NO. 77)
SPELLLLGLLFLPAV (SEQ ID NO. 55) SKS SNLA WFVTRCCKEYFYTS (SEQ ID
NO. 78)
PAVVAVTSAGPEESD (SEQ ID NO. 56) KS SNLA WFVTRCCKEYFYT S S (SEQ ID
NO. 79);
ESDGDLSCVCVKTIS (SEQ ID NO. 57) S SNLA WFVTRCCKEYFYT S SK (SEQ ID
NO. 80);
TISSGIHLKHITSLE (SEQ ID NO. 58) SNLA WFVTRCCKEYFYTS SKS (SEQ ID
NO. 81);
VIKAGRHCAVPQLIA (SEQ ID NO. 59) NLA WFVTRCCKEYFYT S SKS S (SEQ ID
NO. 82);
LIATLKNGRKICLDR (SEQ ID NO. 60) SFKGTTVYALSNVRSYSFVKCC (SEQ ID NO. 83);
LDRQAPLYKKVIKKI (SEQ ID NO. 61) FKGTTVYALSNVRSYSFVKCCS (SEQ ID
NO. 84);
QAPLYKKVIKKILES (SEQ ID NO. 62) SNVRSYSFVKCCSFKGTTVYAL (SEQ ID
NO. 85);
MKVSAAALAVILIAT (SEQ ID NO. 63) NVRSYSFVKCCSFKGTTVYALS (SEQ ID
NO. 86);
ATALCAPASASPYSS (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID
NO. 87);
SDTTPCCFAYIARPL (SEQ ID NO. 65) YSFVKCCSFKGTTVYALSNVRS (SEQ ID
NO. 88);
PLPRAHIKEYFYTSG (SEQ ID NO. 66) SFVKCCSFKGTTVYALSNVRSY (SEQ ID
NO. 89);
EYFYTSGKCSNPAW (SEQ ID NO. 67) FVKCCSFKGTTVYALSNVRSYS (SEQ ID
NO. 90)
VFVTRKNRQVCANPE (SEQ ID NO. 66)
5. A peptide of claim 1, wherein comprising an amino acid sequence selected from:
MSSAAGFCASRP (SEQ ID NO. 46) EKKWVREYINSLEMS (SEQ ID NO. 69)
RPGLLFLGLLLLPL (SEQ ID NO. 47) MKISAAALTIILTAA (SEQ ID NO. 70)
PLWAFASAEA (SEQ ID NO. 48) AALCTPAPASPYGSD (SEQ ID NO. 71)
SAEAEEDGDLQC (SEQ ID NO. 49) SDTTPCCFAYLSLAL (SEQ ID NO. 72)
QCLCVKTTSQVRPRH (SEQ ID NO. 50) ALPRAHVKEYFYTSS (SEQ ID NO. 73)
PRHITSLEVIKAGPHC (SEQ ID NO. 51) SSKCSNLAWFVTRR (SEQ ID NO. 74)
PTAQLIATLKNGRKIC (SEQ ID NO. 52) RRNRQVCANPEKKWV (SEQ ID NO. 75)
LDLQAPLYKKIIKKLLES (SEQ ID NO. 53) EKKWVQEYINYLEMS (SEQ ID NO. 76)
MSVAAVFRGLRPSPE (SEQ ID NO. 54) S SKS SNLA WFVTRCCKEYFYT (SEQ ID
NO. 77)
SPELLLLGLLFLPAV (SEQ ID NO. 55) SKS SNLA WFVTRCCKEYFYTS (SEQ ID
NO. 78)
PAVVAVTSAGPEESD (SEQ ID NO. 56) KS SNLA WFVTRCCKEYFYT S S (SEQ ID
NO. 79);
ESDGDLSCVCVKTIS (SEQ ID NO. 57) S SNLA WFVTRCCKEYFYT S SK (SEQ ID
NO. 80);
TISSGIHLKHITSLE (SEQ ID NO. 58) SNLA WFVTRCCKEYFYTS SKS (SEQ ID
NO. 81); VIKAGRHCAVPQLIA (SEQ ID NO. 59) NLAVVFVTRCCKEYF YT S SKS S (SEQ ID
NO. 82);
LIATLKNGRKICLDPv (SEQ ID NO. 60) SFKGTTVYALSNVRSYSFVKCC (SEQ ID
NO. 83);
LDRQAPLYKKVIKKI (SEQ ID NO. 61) FKGTTVYALSNVRSYSFVKCCS (SEQ ID
NO. 84);
QAPLYKKVIKKILES (SEQ ID NO. 62) SNVRSYSFVKCCSFKGTTVYAL (SEQ ID
NO. 85);
MKVSAAALAVILIAT (SEQ ID NO. 63) NVRSYSFVKCCSFKGTTVYALS (SEQ ID
NO. 86);
ATALCAPASASPYSS (SEQ ID NO. 64) SYSFVKCCSFKGTTVYALSNVR (SEQ ID
NO. 87);
SDTTPCCFAYIARPL (SEQ ID NO. 65) YSFVKCCSFKGTTVYALSNVRS (SEQ ID
NO. 88);
PLPRAHIKEYFYTSG (SEQ ID NO. 66) SFVKCCSFKGTTVYALSNVRSY (SEQ ID
NO. 89);
EYFYTSGKCSNPAW (SEQ ID NO. 67) FVKCCSFKGTTVYALSNVRSYS (SEQ ID
NO. 90)
VFVTRKNRQVCANPE (SEQ ID NO. 66)
6. A peptide according to any of claims 1-5, comprising (a) N- and/or C-terminal chemical modifications to improve ADME-PK or (b) non-natural amino acids.
7. A peptide according to any of claims 1-5, wherein the peptide is a cyclical variant.
8. A peptide according to any of claims 1-7, for use in the treatment of a disease characterized by inflammation mediated by the binding of PF4 and RANTES.
9. A peptide for use as claimed in claim 8, wherein the disease is atherosclerosis, cystic fibrosis, AAA, RSV infection, emphysema, moderate asthma, severe asthma, or any combination thereof.
10. A peptide for use as claimed in claim 8, wherein the use further comprises the coadministration of an active agent selected from: niacin, a fibrate, a statin, an Apo-Al mimetic peptide, an ApoA-I transcriptional up-regulator, an ACAT inhibitor, a CETP modulator,
Glycoprotein (GP) Ilb/IIIa receptor antagonists, P2Y12 receptor antagonists, Lp-PLA2-inhibitors, an anti-TNF agent, an IL-1 receptor antagonist, an IL-2 receptor antagonist, a cytotoxic agent, an immunomodulatory agent, an antibiotic, a T-cell co-stimulatory blocker, a disorder-modifying antirheumatic agent, a B cell depleting agent, an immunosuppressive agent, an anti-lymphocyte antibody, an alkylating agent, an anti-metabolite, a plant alkaloid, a terpenoid, a topoisomerase inhibitor, an antitumor antibiotic, a monoclonal antibody, a hormone, or combinations thereof.
11. A pharmaceutical composition which comprises a peptide as set forth in any of claims 1 -7.
12. A method of diagnosing an abdominal aortic aneurysm in an individual in need thereof, comprising (a) administering to the individual an agent that (i) specifically binds to all or a portion of PF4, RANTES, or a combination thereof, and (ii) is labeled with an imaging agent; (b) exposing the individual to an imaging device for a period of time sufficient to generate an image of the agent; and (c) diagnosing the individual with an abdominal aortic aneurysm if the image of the agent shows a concentration of the agent in the abdominal aorta that exceeds the concentration in a control.
13. The method of claim 12, wherein the agent localizes to the site of an abdominal aortic aneurysm.
14. The method of claim 12, wherein the agent is an antibody, a peptibody, a small molecule, or a peptide.
15. The method of claim 12, wherein the agent is a peptide that (a) mimics all or a portion of PF4, or (b) all or a portion of RANTES.
16. The method of claim 12, wherein the agent is a peptide that (a) mimics all or a portion of the RANTES binding domain of PF4, or (b) all or a portion of PF4 binding domain of RANTES.
17. The method of claim 12, wherein the agent is a peptide that comprises an amino acid sequence that is at least 85% homologous to the sequence: CKEYFYTSSKSSNLAVVFVTRC (SEQ ID NO: 13).
18. The method of claim 12, wherein the agent is a peptide that comprises an amino acid sequence that is at least 85% homologous to the sequence: CKEYFYTSGKSSNPGIVFITRC (SEQ ID NO: 16).
19. The method of claim 12, wherein the agent is an anti-PF4 antibody, an anti-RANTES antibody, or a combination thereof.
20. The method of claim 12, wherein the agent is labeled for radio-imaging, PET imaging, MRI imaging, or fluorescent imaging.
21. The method of claim 12, wherein the agent is imaged by use of a gamma camera, MRI, PET scanner, x-ray computed tomography (CT), functional magnetic resonance imaging (fMRI), or single photon emission computed tomography (SPECT).
22. The method of claim 12, wherein the imaging agent is a radiolabel.
23. The method of claim 12, wherein the imaging agent is a fluorescent label.
24. The method of claim 12, wherein the imaging agent is magnetic, paramagnetic or superparamagnetic.
25. The method of claim 12, wherein the imaging agent is 17F, 18F, U7I, 118I, 119I, 120I, 121I, 122I, 124I, 126L 1281 131I, 10C, UC, 130, 140, 150, 74Br, 75Br, 76Br, 77Br, 78Br, 82Br, mIn, 225Ac, 13N, 82Rb, 32C1, CI, CI, Cu, monobromobimane (mBBr), dibromobimane,
monobromotrimethylammoniobimane, indocyanine green acylthiazolidinethione (ICG-ATT), 7- amino -4 -methyl coumarin-3-acetic acid (AMCA), guanidinoethanethiol (GET), ferucarbotran, UC- dimethylamine, perfluoro-15-crown-5-ether (PFCE), perfluorooctylbromide (PFOB), Pvhodamine 6G, Pvhodamine B, Rhodamine 123, N-[2-(4-18F-fluorobenzamido)ethyl]maleimide, colloidal gold particles, colloidal silver particles, colloidal iron particles, colloidal gadolinium particles, iron dextran particles, gold dextran particles, silver dextran particles, or a combination thereof.
26. A method of monitoring an abdominal aortic aneurysm in an individual in need thereof, comprising (a) administering to the individual an agent that (i) specifically binds to all or a portion of PF4, RANTES, or a combination thereof, and (ii) is labeled with an imaging agent; (b) exposing the individual to an imaging device for a period of time sufficient to generate an image of the agent; wherein (i) a decrease in the concentration of the agent in the abdominal aorta as compared to an earlier image of the agent indicates that the Abdominal Aortic Aneurysm (AAA) is regressing, (ii) an increase in the concentration of the agent in the abdominal aorta as compared to an earlier image of the agent indicates that the Abdominal Aortic Aneurysm (AAA) is progressing, or (iii) no change in the concentration of the agent in the abdominal aorta as compared to an earlier image of the agent indicates that the Abdominal Aortic Aneurysm (AAA) is neither regressing or progressing.
27. The method of claim 26, wherein the agent localizes to the site of an abdominal aortic aneurysm.
28. The method of claim 26, wherein the agent is an antibody, a peptibody, a small molecule, or a peptide.
29. The method of claim 26, wherein the agent is a peptide that (a) mimics all or a portion of PF4, or (b) all or a portion of RANTES.
30. The method of claim 26, wherein the agent is a peptide that (a) mimics all or a portion of the RANTES binding domain of PF4, or (b) all or a portion of PF4 binding domain of RANTES.
31. The method of claim 26, wherein the agent is a peptide that comprises an amino acid sequence that is at least 85% homologous to the sequence: CKEYFYTSSKSSNLAVVFVTRC (SEQ ID NO: 13).
32. The method of claim 26, wherein the agent is a peptide that comprises an amino acid sequence that is at least 85% homologous to the sequence: CKEYFYTSGKSSNPGTVFITRC (SEQ ID NO: 16).
33. The method of claim 26, wherein the agent is an anti-PF4 antibody, an anti-RANTES antibody, or a combination thereof.
34. The method of claim 26, wherein the agent is labeled for radio-imaging, PET imaging, MRI imaging, or fluorescent imaging.
35. The method of claim 26, wherein the agent is imaged by use of a gamma camera, MRI, PET scanner, x-ray computed tomography (CT), functional magnetic resonance imaging (fMRI), or single photon emission computed tomography (SPECT).
36. The method of claim 26, wherein the imaging agent is a radiolabel.
37. The method of claim 26, wherein the imaging agent is a fluorescent label.
38. The method of claim 26, wherein the imaging agent is magnetic, paramagnetic or superparamagnetic.
39. The method of claim 26, wherein the imaging agent is 17F, 18F, U7I, 118I, 119I, 120I, 121I, 122I, 124I, 126L 1281 131I, 10C, UC, 130, 140, 150, 74Br, 75Br, 76Br, 77Br, 78Br, 82Br, l uIn, 225Ac, 13N, 82Rb, 32C1, 33C1, 34C1, 64Cu, monobromobimane (mBBr), dibromobimane,
monobromotrimethylammoniobimane, indocyanine green acylthiazolidinethione (ICG-ATT), 7- amino -4 -methyl coumarin-3-acetic acid (AMCA), guanidinoethanethiol (GET), ferucarbotran, UC- dimethylamine, perfluoro-15-crown-5-ether (PFCE), perfluorooctylbromide (PFOB), Rhodamine 6G, Rhodamine B, Rhodamine 123, N-[2-(4-18F-fluorobenzamido)ethyl]maleimide, colloidal gold particles, colloidal silver particles, colloidal iron particles, colloidal gadolinium particles, iron dextran particles, gold dextran particles, silver dextran particles, or a combination thereof.
40. A stent comprising a peptide comprising an amino acid sequence that is at least 85% homologous to the amino acid sequence CKEYFYTSSKSSNLAWFVTRC (SEQ ID NO: 13), or CKEYFYTSGKSSNPGIVFITRC (SEQ ID NO: 16).
41. The stent of claim 40, wherein the peptide is CKEYFYTSSKSSNLAWFVTRC (SEQ ID NO: 13), or CKEYFYTSGKSSNPGIVFITRC (SEQ ID NO: 16).
42. A method of treating RSV infection, comprising administering an agent that specifically binds to all or a portion of PF4, RANTES, or a combination thereof.
43. The method of claim 42, wherein the agent is an antibody, a peptibody, a small molecule, or a peptide.
44. The method of claim 42, wherein the agent (a) mimics all or a portion of PF4, or (b) all or a portion of RANTES.
45. The method of claim 42, wherein the agent (a) mimics all or a portion of the RANTES binding domain of PF4, or (b) all or a portion of PF4 binding domain of RANTES.
46. The method of claim 42, wherein the agent is a peptide comprising an amino acid sequence that is at least 85% homologous to the amino acid sequence: CKEYFYTSSKSSNLAWFVTRC (SEQ ID NO: 13).
47. The method of claim 42, wherein the agent is a peptide comprising an amino acid sequence that is at least 85% homologous to the amino acid sequence: CKEYFYTSGKSSNPGIVFITRC (SEQ ID NO: 16).
48. The method of claim 42, wherein he agent is CKEYFYTSSKSSNLAWFVTRC (SEQ ID NO: 13), or CKEYFYTSGKSSNPGIVFITRC (SEQ ID NO: 16).
49. A method of treating emphysema, comprising administering an agent that specifically binds to all or a portion of PF4, RANTES, or a combination thereof.
50. The method of claim 49, wherein the agent is an antibody, a peptibody, a small molecule, or a peptide.
51. The method of claim 49, wherein the agent (a) mimics all or a portion of PF4, or (b) all or a portion of RANTES.
52. The method of claim 49, wherein the agent (a) mimics all or a portion of the RANTES binding domain of PF4, or (b) all or a portion of PF4 binding domain of RANTES.
53. The method of claim 49, wherein the agent is a peptide comprising an amino acid sequence that is at least 85% homologous to the amino acid sequence: CKEYFYTSSKSSNLAWFVTRC (SEQ ID NO: 13).
54. The method of claim 49, wherein the agent is a peptide comprising an amino acid sequence that is at least 85% homologous to the amino acid sequence: CKEYFYTSGKSSNPGIVFITRC (SEQ ID NO: 16).
55. The method of claim 49, wherein he agent is CKEYFYTSSKSSNLAWFVTRC (SEQ ID NO: 13), or CKEYFYTSGKSSNPGIVFITRC (SEQ ID NO: 16).
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