WO2008100567A2 - Thrombin peptide derivatives for treating fractures in osteopenic patients - Google Patents

Thrombin peptide derivatives for treating fractures in osteopenic patients Download PDF

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Publication number
WO2008100567A2
WO2008100567A2 PCT/US2008/001953 US2008001953W WO2008100567A2 WO 2008100567 A2 WO2008100567 A2 WO 2008100567A2 US 2008001953 W US2008001953 W US 2008001953W WO 2008100567 A2 WO2008100567 A2 WO 2008100567A2
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asp
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ala
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PCT/US2008/001953
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WO2008100567A3 (en
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James T. Ryaby
Gary M. Kiebzak
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Orthologic Corp.
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A61K38/482—Serine endopeptidases (3.4.21)
    • A61K38/4833—Thrombin (3.4.21.5)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/6429—Thrombin (3.4.21.5)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21005—Thrombin (3.4.21.5)

Definitions

  • Osteopenia is characterized by decreased density of bone. Similar to osteoporosis, people with osteopenia have a higher risk and frequency of suffering bone fractures. Clearly, an agent that accelerates fracture healing would reduce costs due to disability and improve the quality of life for those who sustain a fracture. With a projected increase in incidence worldwide, early therapeutic intervention should mitigate costs associated with complications of healing.
  • TP508 a polypeptide which stimulates or activates the non-proteolytically activated thrombin receptor (hereinafter "NPAR"), can promote the healing of bone fractures in osteopenic human patients (Example 1). Osteopenic females with distal radius fractures were treated with TP508 and the data showed statistically significant effects in the primary endpoint (time to immobilization removal) and certain secondary endpoints, including time to clinical evaluation of fracture healing, time to overall radiographic healing, time to radial cortical bridging, and range of motion assessed by 30 degrees flexion and extension.
  • NPAR non-proteolytically activated thrombin receptor
  • osteopenic women have statistically slower healing than the normal women, based on time to immobilization removal and time to clinical evaluation of fracture healing (Example 2). Based on this observation, methods of promoting healing of bone fractures with NPAR agonists are disclosed herein.
  • the present invention is directed to methods of promoting healing of a bone fracture in an osteopenic human patient, comprising administering to the patient a therapeutically effective amount of an NPAR agonist.
  • the NPAR agonist is a thrombin peptide derivative disclosed herein. More specifically, one thrombin peptide derivative comprises the amino acid sequence of Arg-Gly-Asp-Ala-Cys-Xi-Gly-Asp-Ser-Gly-Gly-Pro-X 2 -Val (SEQ ID NO:1), or a C-terminal truncated fragment thereof comprising at least six amino acids.
  • the thrombin peptide derivative comprises the amino acid sequence of SEQ ID NO:2: Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Cys-Xi-Gly- Asp-Ser-Gly-Gly-Pro-X 2 -Val, an N-terminal truncated fragment of the thrombin peptide derivative having at least fourteen amino acids, or a C-terminal truncated fragment of the thrombin peptide derivative comprising at least eighteen amino acids.
  • Xj is GIu or GIn and X 2 is Phe, Met, Leu, His or VaI.
  • the thrombin peptide derivative is the polypeptide SEQ ID NO:3: H-Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys- Arg-Gly-Asp-Ala-Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe- VaI-NH 2 (TP508).
  • the NPAR agonist is a modified thrombin peptide derivative disclosed herein.
  • the modified thrombin peptide derivative comprises the amino acid sequence of SEQ ID NO:4: Arg-Gly-Asp-Ala-Xaa-Xi-Gly-Asp- Ser-Gly-Gly-Pro-X 2 -Val, or a C-terminal truncated fragment thereof having at least six amino acids.
  • the modified thrombin peptide derivative comprises the amino acid sequence of SEQ ID NO: 5: Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly- Lys-Arg-Gly-Asp-Ala-Xaa-Xi-Gly-Asp-Ser-Gly-Gly-Pro-X 2 -Val, or a fragment thereof comprising amino acids 10-18 of SEQ ID NO: 5.
  • the pharmaceutical compositions comprising thrombin peptide derivatives or modified thrombin peptide derivatives of the present invention can also include a dimerization inhibitor.
  • a dimerization inhibitor is a compound that inhibits or reduces dimerization of a thrombin peptide derivative or a modified thrombin peptide derivative. Dimerization inhibitors include chelating agents and/or thio-containing compounds. In another embodiment, the NPAR agonist is a thrombin peptide derivative dimer of two thrombin peptide derivatives disclosed herein.
  • a thrombin peptide derivative of a dimer comprises the amino acid sequence Arg-Gly-Asp-Ala-Cys-Xi-Gly- Asp-Ser-Gly-Gly-Pro-X 2 -Val (SEQ ID NO:1) or a C-terminal truncated fragment thereof having at least six amino acids.
  • the thrombin peptide derivative of the dimer comprises a polypeptide having the amino acid sequence of SEQ ID NO:2: Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Cys-Xi-Gly-Asp-Ser- Gly-Gly-Pro-X 2 -Val, or a fragment thereof comprising amino acids 10-18 of SEQ ID NO:2.
  • the thrombin peptide derivative of the dimer is the polypeptide H-Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly- Asp- Ala-Cys-Glu-Gly- Asp-Ser-Gly-Gly-Pro-Phe- VaI-NH 2 (SEQ ID NO:3).
  • the thrombin peptide derivative dimer is represented by the structural formula (IV).
  • the NPAR agonist is an antibody or antigen-binding fragment thereof that binds to and activates the non-proteolytically activated thrombin receptor or binds to a complementary peptide, wherein the complementary peptide is encoded by the complement of a nucleotide sequence encoding a portion of thrombin.
  • the thrombin referred to above can be a mammalian thrombin, and in particular, a human thrombin.
  • the portion of thrombin can be a thrombin receptor binding domain or a portion thereof.
  • the thrombin receptor binding domain or portion thereof comprises the amino acid sequence Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg- Gly-Asp-Ala-Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val (SEQ ID NO:6).
  • Another portion of a thrombin receptor binding domain comprises the amino acid sequence Glu-Gly-Lys- Arg-Gly-Asp-Ala-Cys-Glu-Gly (SEQ ID NO:7).
  • the complementary peptide to which the antibody or the antigen-binding fragment thereof binds can be encoded by the 5 '-3' sequence of the antisense RNA strand or encoded by the 3 '-5' sequence of the antisense RNA strand.
  • the complementary peptide comprises the amino acid sequence Lys-Gly-Ser-Pro-Thr-Val-Thr-Phe-Thr-Gly-Ile-Pro-Cys-Phe-Pro-Phe-Ile-Arg-Leu- Val-Thr-Ser (SEQ ID NO:8) or Thr-Phe-Thr-Gly-Ile-Pro-Ser-Phe-Pro-Phe (SEQ ID NO:9) or Arg-Pro-Met-Phe-Gly-Leu-Leu-Pro-Phe-Ala-Pro-Leu-Arg-Thr-Leu-Pro-Leu-Ser-Pro-Pro- Gly-Lys-Gln (SEQ ID NO: 10) or Lys-Pro-Phe- Ala-Pro-Leu- Arg-Thr-Leu-Pro (SEQ ID NO:11).
  • the NPAR agonist to be used in the methods of the invention can be a polyclonal antibody, or a monoclonal antibody or antigen-binding fragment thereof. In particular embodiments, these are human antibodies. Monoclonal antibodies to be used as NPAR agonists in methods of therapy can be humanized antibodies, chimeric antibodies or antigen- binding fragments of any of the foregoing, which can include Fab fragments, Fab' fragments, F(ab') 2 fragments and Fv fragments.
  • FIG. 1 is a graph showing time to immobilization removal for osteopenic patients treated with TP508 and patients treated with placebo.
  • FIG. 2 is a graph showing time to overall radiographic healing for osteopenic patients treated with TP508 and patients treated with placebo.
  • FIG. 3 is a graph showing time for 30 degree or greater in flexion for osteopenic patients treated with TP508 and patients treated with placebo.
  • FIG. 4 is graph showing time for 30 degree extension in osteopenic patients treated with TP508 and patients treated with placebo.
  • FIG. 5 is a graph showing time to immobilization removal for osteopenic patients and normal patients treated with placebo.
  • FIG. 6 is a graph showing time to clinical evaluation of fracture healing for osteopenic patients and normal patient treated with placebo.
  • the invention includes methods of promoting healing of a bone fracture in an osteopenic human, comprising administering to the patient a therapeutically effective amount of an agonist of the non-proteolytically activated thrombin receptor (NPAR).
  • NPAR non-proteolytically activated thrombin receptor
  • NPAR neuropeptide derived peptides
  • NPAR neuropeptide derived peptides
  • NPAR appears to mediate a number of cellular signals that are initiated by thrombin independent of its proteolytic activity.
  • An example of one such signal is the upregulation of annexin V and other molecules identified by subtractive hybridization (see Sower, et. al, Experimental Cell Research 247:422 (1999)).
  • NPAR is therefore characterized by its high affinity interaction with thrombin at cell surfaces and its activation by proteolytically inactive derivatives of thrombin and thrombin derived peptide agonists as described below.
  • NPAR activation can be assayed based on the ability of molecules to stimulate cell proliferation when added to fibroblasts in the presence of submitogenic concentrations of thrombin or molecules that activate protein kinase C, as disclosed in U.S. Patent Nos. 5,352,664 and 5,500,412. The entire teachings of these patents are incorporated herein by reference.
  • NPAR agonists can be identified by this activation or by their ability to compete with 125 I-thrombin binding to cells.
  • a thrombin receptor binding domain is defined as a polypeptide or portion of a polypeptide which directly binds to the thrombin receptor and/or competitively inhibits binding between high-affinity thrombin receptors and alpha-thrombin.
  • NPAR agonists of the present invention include thrombin derivative peptides, modified thrombin derivative peptides, thrombin derivative peptide dimers and NPAR agonist antibodies to complementary peptides of thrombin as disclosed herein.
  • thrombin peptide derivatives also: "thrombin derivative peptides"
  • thrombin derivative peptides are analogs of thrombin that have an amino acid sequence derived at least in part from that of thrombin and are active at the non-proteolytically activated thrombin receptor.
  • Thrombin peptide derivatives include, for example, peptides that are produced by recombinant DNA methods, peptides produced by enzymatic digestion of thrombin, and peptides produced synthetically, which can comprise amino acid substitutions compared to thrombin and/or modified amino acids, especially at the termini.
  • NPAR agonists of the present invention include thrombin derivative peptides described in U.S. Patent Nos. 5,352,664 and 5,500,412.
  • the NPAR agonist of the present invention is a thrombin peptide derivative or a physiologically functional equivalent, i.e., a polypeptide with no more than about fifty amino acids, preferably no more than about thirty amino acids and having sufficient homology to the fragment of human thrombin corresponding to thrombin amino acids 508-530 (Ala-Gly-Tyr- Lys-Pro- Asp-Glu-Gly-Lys- Arg-Gly- Asp- Ala-Cys-Glu-Gly- Asp-Ser-Gly-Gly-Pro-Phe- VaI ; SEQ ID NO:6) that the polypeptide activates NPAR.
  • thrombin peptide derivatives or modified thrombin peptide derivatives described herein preferably have from about 12 to about 23 amino acid residues, more preferably from about 19 to about 23 amino acid residues.
  • the NPAR agonist of the present invention is a thrombin peptide derivative comprising a moiety represented by Structural Formula (I):
  • R is a serine esterase conserved domain.
  • Serine esterases e.g., trypsin, thrombin, chymotrypsin and the like, have a region that is highly conserved.
  • Serine esterase conserved domain refers to a polypeptide having the amino acid sequence of one of these conserved regions or is sufficiently homologous to one of these conserved regions such that the thrombin peptide derivative retains NPAR activating ability.
  • a physiologically functional equivalent of a thrombin derivative encompasses molecules which differ from thrombin derivatives in particulars which do not affect the function of the thrombin receptor binding domain or the serine esterase conserved amino acid sequence.
  • Such particulars may include, but are not limited to, conservative amino acid substitutions and modifications, for example, amidation of the carboxyl terminus, acetylation of the amino terminus, conjugation of the polypeptide to a physiologically inert carrier molecule, or sequence alterations in accordance with the serine esterase conserved sequences.
  • a domain having a serine esterase conserved sequence can comprise a polypeptide sequence containing at least 4-12 of the N-terminal amino acids of the dodecapeptide previously shown to be highly conserved among serine proteases (Asp-Xi -CyS-X 2 -GIy-ASp- Ser-Gly-Gly-Pro-X 3 -Val; SEQ ID NO: 13); wherein X 1 is either Ala or Ser; X 2 is either GIu or GIn; and X 3 is Phe, Met, Leu, His, or VaI).
  • the serine esterase conserved sequence comprises the amino acid sequence of SEQ ID NO: 14 (Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val) or a C-terminal truncated fragment of a polypeptide having the amino acid sequence of SEQ ID NO: 14. It is understood, however, that zero, one, two or three amino acids in the serine esterase conserved sequence can differ from the corresponding amino acid in SEQ ID NO: 14.
  • the amino acids in the serine esterase conserved sequence which differ from the corresponding amino acid in SEQ ID NO: 14 are conservative substitutions, and are more preferably highly conservative substitutions.
  • a "C-terminal truncated fragment” refers to a fragment remaining after removing an amino acid or block of amino acids from the C- terminus, said fragment having at least six and more preferably at least nine amino acids.
  • the serine esterase conserved sequence comprises the amino acid sequence of SEQ ID NO: 15 (Cys-Xi-Gly-Asp-Ser-Gly-Gly-Pro-XrVal; Xi is GIu or GIn and X 2 is Phe, Met, Leu, His or VaI) or a C-terminal truncated fragment thereof having at least six amino acids, preferably at least nine amino acids.
  • the thrombin peptide derivative comprises a serine esterase conserved sequence and a polypeptide having a more specific thrombin amino acid sequence Arg-Gly-Asp-Ala (SEQ ID NO: 16).
  • thrombin peptide derivative of this type comprises Arg-Gly-Asp-Ala-Cys-Xi-Gly-Asp-Ser-Gly-Gly-Pro-X 2 -Val (SEQ ID NO:1).
  • Xi and X 2 are as defined above.
  • the thrombin peptide derivative can comprise the amino acid sequence of SEQ ID NO:6 (Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly- Asp-Ala-Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val) or an N-terminal truncated fragment thereof, provided that zero, one, two or three amino acids at positions 1-9 in the thrombin peptide derivative differ from the amino acid at the corresponding position of SEQ ID NO:6.
  • amino acid residues in the thrombin peptide derivative which differ from the corresponding amino acid residues in SEQ ID NO: 6 are conservative substitutions, and are more preferably highly conservative substitutions.
  • An 'W-terminal truncated fragment refers to a fragment remaining after removing an amino acid or block of amino acids from the TV-terminus, preferably a block of no more than six amino acids, more preferably a block of no more than three amino acids.
  • the thrombin peptide derivatives described herein can be amidated at the C-terminus and/or acylated at the N-terminus.
  • the thrombin peptide derivatives comprise a C-terminal amide and optionally comprise an acylated N- terminus, wherein said C-terminal amide is represented by -C(O)NR a R b , wherein R a and Rb are independently hydrogen, a C 1 - C 10 substituted or unsubstituted aliphatic group, or R a and R b , taken together with the nitrogen to which they are bonded, form a C 1 -C 10 non-aromatic heterocyclic group, and said N-terminal acyl group is represented by R c C(O)-, wherein R c is hydrogen, a C 1 -C 10 substituted or unsubstituted aliphatic group, or a C 1 -C io substituted or unsubstituted aromatic
  • the N-terminus of the thrombin peptide derivative is free (i.e., unsubstituted) and the C-terminus is free (i.e., unsubstituted) or amidated, preferably as a carboxamide (i.e., -C(O)NH 2 ).
  • the thrombin peptide derivative comprises the following amino acid sequence: Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Cys-Glu-Gly-Asp-Ser-Gly-Gly- Pro-Phe-Val (SEQ ID NO:6).
  • the thrombin peptide derivative comprises the amino sequence of Arg-Gly-Asp-Ala-Cys-Glu-Gly-Asp-Ser-Gly- Gly-Pro-Phe-Val (SEQ ID NO: 17).
  • the thrombin peptide derivative comprises the amino acid sequence of SEQ ID NO: 18: Asp-Asn-Met-Phe-Cys-Ala-Gly-Tyr- Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val- Met-Lys-Ser-Pro-Phe.
  • the thrombin peptide derivatives comprising the amino acids of SEQ ID NO: 6, 17, or 18 can optionally be amidated at the C-terminus and/or acylated at the N- terminus.
  • the N-terminus is free (i.e., unsubstituted) and the C-terminus is free (i.e., unsubstituted) or amidated, preferably a carboxamide (i.e., -C(O)NH 2 ). It is understood, however, that zero, one, two or three amino acids at positions 1-9 and 14-23 in the thrombin peptide derivative can differ from the corresponding amino acid in SEQ ID NO:6.
  • zero, one, two or three amino acids at positions 1-14 and 19-33 in the thrombin peptide derivative can differ from the corresponding amino acid in SEQ ID NO: 18.
  • the amino acids in the thrombin peptide derivative which differ from the corresponding amino acid in SEQ ID NO: 6 or SEQ ID NO: 18 are conservative substitutions, and are more preferably highly conservative substitutions.
  • an N- terminal truncated fragment of the thrombin peptide derivative having at least fourteen amino acids or a C-terminal truncated fragment of the thrombin peptide derivative having at least eighteen amino acids is a thrombin peptide derivative to be used as an NP AR agonist.
  • C-terminal truncated fragment refers to a fragment remaining after removing an amino acid or block of amino acids from the C-terminus.
  • N-terminal truncated fragment refers to a fragment remaining after removing an amino acid or block of amino acids from the N-terminus. It is to be understood that the terms "C-terminal truncated fragment” and 'W-terminal truncated fragment” encompass acylation at the N-terminus and/or amidation at the C-terminus, as described above.
  • a preferred thrombin peptide derivative for use in the disclosed method comprises the amino acid sequence SEQ ID ⁇ O:2: Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly- Asp-Ala-Cys-Xi-Gly-Asp-Ser-Gly-Gly-Pro-X 2 -Val.
  • Another preferred thrombin peptide derivative for use in the disclosed method comprises the amino acid sequence of SEQ ID NO: 19: Asp- Asn-Met-Phe-Cys-Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly- Asp- AIa- CyS-X 1 -GIy- Asp-Ser-Gly-Gly-Pro-X 2 -Val-Met-Lys-Ser-Pro-Phe.
  • Xi is GIu or GIn;
  • X 2 is Phe, Met, Leu, His or VaI.
  • the thrombin peptide derivatives of SEQ ID NO:2 and SEQ ID NO: 19 can optionally comprise a C-terminal amide and/or acylated N-terminus, as defined above.
  • the N-terminus is free (i.e., unsubstituted) and the C-terminus is free (i.e., unsubstituted) or amidated, preferably as a carboxamide (i.e., -C(O)NH 2 ).
  • N- terminal truncated fragments of these preferred thrombin peptide derivatives can also be used in the disclosed method.
  • TP508 is an example of a thrombin peptide derivative and is 23 amino acid residues long, wherein the N-terminal amino acid residue Ala is unsubstituted and the COOH of the C-terminal amino acid VaI is modified to an amide represented by
  • thrombin peptide derivative comprises the amino acid sequence of SEQ ID NO: 6, wherein both N- and C-termini are unsubstituted ("deamide TP508").
  • Other examples of thrombin peptide derivatives which can be used in the disclosed method include N-terminal truncated fragments of TP508 (or deamide TP508), the N-terminal truncated fragments having at least fourteen amino acids, or C-terminal truncated fragments of TP5O8 (or deamide TP508), the C-terminal truncated fragments having at least eighteen amino acids.
  • a "conservative substitution" in a polypeptide is the replacement of an amino acid with another amino acid that has the same net electronic charge and approximately the same size and shape.
  • Amino acids with aliphatic or substituted aliphatic amino acid side chains have approximately the same size when the total number of carbon and heteroatoms in their side chains differs by no more than about four. They have approximately the same shape when the number of branches in their side chains differs by no more than one.
  • Amino acids with phenyl or substituted phenyl groups in their side chains are considered to have about the same size and shape. Listed below are five groups of amino acids. Replacing an amino acid in a polypeptide with another amino acid from the same group results in a conservative substitution:
  • Group I glycine, alanine, valine, leucine, isoleucine, serine, threonine, cysteine, and non-naturally occurring amino acids with C 1 -C4 aliphatic or C 1 -C4 hydroxyl substituted aliphatic side chains (straight chained or monobranched).
  • Group II glutamic acid, aspartic acid and non-naturally occurring amino acids with carboxylic acid substituted C1-C4 aliphatic side chains (unbranched or one branch point).
  • Group III lysine, ornithine, arginine and non-naturally occurring amino acids with amine or guanidino substituted C1-C4 aliphatic side chains (unbranched or one branch point).
  • Group IV glutamine, asparagine and non-naturally occurring amino acids with amide substituted C1-C4 aliphatic side chains (unbranched or one branch point).
  • Group V phenylalanine, phenylglycine, tyrosine and tryptophan.
  • a "highly conservative substitution" in a polypeptide is the replacement of an amino acid with another amino acid that has the same functional group in the side chain and nearly the same size and shape.
  • Amino acids with aliphatic or substituted aliphatic amino acid side chains have nearly the same size when the total number of carbon and heteroatoms in their side chains differs by no more than two. They have nearly the same shape when they have the same number of branches in the their side chains.
  • Examples of highly conservative substitutions include valine for leucine, threonine for serine, aspartic acid for glutamic acid and phenylglycine for phenylalanine.
  • substitutions which are not highly conservative include alanine for valine, alanine for serine and aspartic acid for serine.
  • the NPAR agonists are modified relative to the thrombin peptide derivatives described above, wherein cysteine residues of aforementioned thrombin peptide derivatives are replaced with amino acids having similar size and charge properties to minimize dimerization of the peptides.
  • suitable amino acids include alanine, glycine, serine, or an 5-protected cysteine.
  • cysteine is replaced with alanine.
  • the modified thrombin peptide derivatives have about the same biological activity as the unmodified thrombin peptide derivatives.
  • modified thrombin peptide derivatives disclosed herein can optionally comprise C-terminal amides and/or iV-terminal acyl groups, as described above.
  • the N-terminus of a thrombin peptide derivative is free (i.e., unsubstituted) and the C-terminus is free (i.e., unsubstituted) or amidated, preferably as a carboxamide (i.e., -C(O)NH 2 ).
  • the modified thrombin peptide derivative comprises a polypeptide having the amino acid sequence of SEQ ID NO:4: Arg-Gly-Asp-Ala-Xaa-Xj- Gly-Asp-Ser-Gly-Gly-Pro-X 2 -Val, or a C-terminal truncated fragment thereof having at least six amino acids.
  • the thrombin peptide derivative comprises the amino acid sequence of SEQ ID NO:20: Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp- Ala-Xaa-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val or a fragment thereof comprising amino acids 10-18 of SEQ ID NO:20.
  • the thrombin peptide derivative comprises the amino acid sequence SEQ ID NO:5: Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys- Arg-Gly-Asp-Ala-Xaa-Xi-Gly-Asp-Ser-Gly-Gly-Pro-X 2 -Val, or a fragment thereof comprising amino acids 10-18 of SEQ ID NO:5.
  • Xaa is alanine, glycine, serine or an S- protected cysteine.
  • Xi is GIu or GIn and X 2 is Phe, Met, Leu, His or VaI.
  • Xi is GIu
  • X 2 is Phe
  • Xaa is alanine.
  • a thrombin peptide derivative of this type is a polypeptide having the amino acid sequence Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly- Lys-Arg-Gly- Asp-Ala- Ala-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe- VaI (SEQ ID NO:21).
  • a further example of a thrombin peptide derivative of this type is the polypeptide H-AIa-GIy- Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Ala-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe- VaI-NH 2 (SEQ ID NO:22).
  • Zaa is alanine, glycine, serine or an ⁇ -protected cysteine.
  • the difference is conservative.
  • the thrombin peptide derivative comprises a polypeptide having the amino acid sequence SEQ ID NO:23: Asp-Asn-Met-Phe-Xbb-Ala- Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Xaa-Glu-Gly-Asp-Ser-Gly-Gly-Pro- Phe-Val-Met-Lys-Ser-Pro-Phe, or a fragment thereof comprising amino acids 6-28.
  • the thrombin peptide derivative comprises a polypeptide having the amino acid sequence SEQ ID NO: 24: Asp-Asn-Met-Phe-Xbb-Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys- Arg-Gly-Asp-Ala-Xaa-Xi-Gly-Asp-Ser-Gly-Gly-Pro-X 2 -Val-Met-Lys-Ser-Pro-Phe, or a fragment thereof comprising amino acids 6-28.
  • Xaa and Xbb are independently alanine, glycine, serine or an S-protected cysteine.
  • Xi is GIu or GIn and X 2 is Phe, Met, Leu, His or VaI.
  • Xi is GIu
  • X 2 is Phe
  • Xaa and Xbb are alanine.
  • a thrombin peptide derivative of this type is a polypeptide comprising the amino acid sequence Asp-Asn-Met-Phe-Ala-Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Ala-Glu- Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val-Met-Lys-Ser-Pro-Phe (SEQ ID NO:25).
  • a further example of a thrombin peptide derivative of this type is the polypeptide H-Asp-Asn-Met- Phe-Ala-Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Ala-Glu-Gly-Asp-Ser- Gly-Gly-Pro-Phe-Val-Met-Lys-Ser-Pro-Phe-NH 2 (SEQ ID NO:26).
  • Zero, one, two or three amino acids in the thrombin peptide derivative can differ from the amino acid at the corresponding position of SEQ ID NO:23, 24, 25 or 26.
  • Xaa and Xbb are independently alanine, glycine, serine or an S-protected cysteine. Preferably, the difference is conservative.
  • An "S-protected cysteine” is a cysteine residue in which the reactivity of the thiol moiety, -SH, is blocked with a protecting group. Suitable protecting groups are are known in the art and are disclosed, for example, in T. W. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 3 rd Edition, John Wiley & Sons, (1999), pp. 454-493, the teachings of which are incorporated herein by reference in their entirety.
  • Suitable protecting groups should be non-toxic, stable in pharmaceutical formulations and have minimum additional functionality to maintain the activity of the thrombin peptide derivative.
  • a free thiol can be protected as a thioether, a thioester, or can be oxidized to an unsymmetrical disulfide.
  • the thiol is protected as a thioether.
  • Suitable thioethers include, but are not limited to, S-alkyl thioethers (e.g., C 1 -C 5 alkyl), and S-benzyl thioethers (e.g, cysteine-S- S-t-Bu).
  • the protective group is an alkyl thioether.
  • the S- protected cysteine is an S-methyl cysteine.
  • the protecting group can be: 1) a cysteine or a cysteine-containing peptide (the "protecting peptide") attached to the cysteine thiol group of the thrombin peptide derivative by a disulfide bond; or 2) an amino acid or peptide ("protecting peptide") attached by a thioamide bond between the cysteine thiol group of the thrombin peptide derivative and a carboxylic acid in the protecting peptide (e.g., at the C-terminus or side chain of aspartic acid or glutamic acid).
  • the protecting peptide can be physiologically inert (e.g., a polyglycine or polyalanine of no more than about fifty amino acids optionally interrupted by a cysteine), or can have a desirable biological activity.
  • the thrombin peptide derivatives or the modified thrombin peptide derivatives of the present invention can be mixed with a dimerization inhibitor for the preparation of a pharmaceutical composition comprising the thrombin peptide derivatives or the modified thrombin peptide derivatives of the present invention.
  • Dimerization inhibitors can include chelating agents and/or thiol-containing compounds. An antioxidant can also be used in combination with the chelating agent and/or the thiol-containing compound.
  • a "chelating agent,” as used herein, is a compound having multiple sites (two, three, four or more) which can simultaneously bind to a metal ion or metal ions such as, for example, lead, cobalt, iron or copper ions.
  • the binding sites typically comprise oxygen, nitrogen, sulfur or phosphorus.
  • salts of EDTA ethylenediaminetetraacetic acid
  • a chelating agent also includes a polymer which has multiple binding sites to a metal or metal ions.
  • a chelating agent of the invention is non-toxic and does not cause unacceptable side effects at the dosages being administered.
  • a copper-chelating agent is preferable.
  • a "copper-chelating agent” refers to a chelating agent which can bind to a copper ion or copper ions.
  • copper-chelating agent examples include ethylenediaminetetraacetic acid (EDTA), penicillamine, trientine, N, TV- diethyldithiocarbamate (DDC), 2,3,2'-tetraamine (2,3,2'-tet), neocuproine, N.N.N'.N- tetrakis(2-pyridylmethyl)ethylenediamine (TPE ⁇ ), 1,10-phenanthroline (PHE), tetraethylenepentamine (TEPA), triethylenetetraamine and tris(2-carboxyethyl) phosphine (TCEP).
  • EDTA ethylenediaminetetraacetic acid
  • DDC TV- diethyldithiocarbamate
  • 2,3,2'-tetraamine (2,3,2'-tet) neocuproine N.N.N'.N- tetrakis(2-pyridylmethyl)ethylenediamine (TPE ⁇
  • chelating agents are diethylenetriaminepentacetic acid (DTPA) and bathophenanthroline disulfonic acid (BPADA).
  • EDTA is a preferred chelating agent.
  • Typical amounts of a chelating agent present in the pharmaceutical compositions of the instant invention is in a range of between about 0.00001 % and about 0.1 % by weight, preferably between about 0.0001 % and about 0.05 % by weight.
  • a "pharmaceutically acceptable thiol-containing compound,” as used herein, is a compound which comprises at least one thiol (-SH) group and which does not cause unacceptable side effects at the dosages which are being administered.
  • Examples of the pharmaceutically acceptable thiol-containing compound include thioglycerol, mercaptoethanol, thioglycol, thiodiglycol, cysteine, thioglucose, dithiothreitol (DTT) and dithio-bis-maleimidoethane (DTME).
  • thioglycerol mercaptoethanol
  • thioglycol thiodiglycol
  • cysteine thioglucose
  • DTT dithiothreitol
  • DTME dithio-bis-maleimidoethane
  • antioxidant is a compound which is used to prevent or reduce an oxidation reaction caused by an oxidizing agent such as oxygen.
  • examples of the antioxidant include tocopherol, cystine, methionine, glutathione, tocotrienol, dimethyl glycine, betaine, butylated hydroxyanisole, butylated hydroxytoluene, vitamin E, ascorbic acid, ascorbyl palmitate, thioglycolic acid and antioxidant peptides such as, for example, turmerin.
  • examples of the antioxidant include tocopherol, cystine, methionine, glutathione, tocotrienol, dimethyl glycine, betaine, butylated hydroxyanisole, butylated hydroxytoluene, vitamin E, ascorbic acid, ascorbyl palmitate, thioglycolic acid and antioxidant peptides such as, for example,
  • certain chelating agents or thiol-containing compounds may also function as an antioxidant, for example, tris(2-carboxyethyl) phosphine, cysteine or dithiothreitol. Other types of commonly used antioxidants, however, do not contain a thiol group. It is also understood that certain thiol-containing compounds may also function as a chelating agent, for example, dithiothreitol. Other types of commonly used chelating agents, however, do not contain a thiol group. It is also understood that the pharmaceutical compositions of the instant invention can comprise more than one chelating agent, thiol- containing compound or antioxidant. That is, for example, a chelating agent can be used either alone or in combination with one or more other suitable chelating agents.
  • the NPAR agonists of the methods are thrombin peptide derivative dimers.
  • the dimers essentially do not revert to monomers and still have about the same biological activity as the thrombin peptide derivatives monomer described above.
  • a "thrombin peptide derivative dimer” is a molecule comprising two thrombin peptide derivatives linked by a covalent bond, preferably a disulfide bond between cysteine residues.
  • Thrombin peptide derivative dimers are typically essentially free of the corresponding monomer, e.g., greater than 95% free by weight and preferably greater than 99% free by weight.
  • the polypeptides are the same and covalently linked through a disulfide bond.
  • the thrombin peptide derivative dimers of the present invention comprises the thrombin peptide derivatives described above. Specifically, thrombin peptide derivatives have less than about fifty amino acids, preferably less than about thirty-three amino acids. Thrombin peptide derivatives also have sufficient homology to the fragment of human thrombin corresponding to thrombin amino acid residues 508-530: Ala-Gly-Tyr-Lys-Pro- Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val (SEQ ID NO: 6) so that the polypeptide activates NPAR.
  • the thrombin peptide derivative dimers described herein are formed from polypeptides typically having at least six amino acids and preferably from about 12 to about 33 amino acid residues, and more preferably from about 12 to about 23 amino acid residues.
  • each thrombin peptide derivative comprising a dimer comprises a polypeptide having the amino acid sequence SEQ ID NO: 1 : Arg-Gly-Asp-Ala- Cys-Xi-Gly-Asp-Ser-Gly-Gly-Pro-X 2 -Val, or a C-terminal truncated fragment thereof comprising at least six amino acids.
  • each thrombin peptide derivative comprises the amino acid sequence of SEQ ID NO:6: Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly- Lys-Arg-Gly-Asp-Ala-Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val, or a fragment thereof comprising amino acids 10-18 of SEQ ID NO: 5.
  • the thrombin peptide derivative comprises the amino acid sequence SEQ ID NO:2: Ala-Gly-Tyr-Lys-Pro- Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Cys-Xi-Gly-Asp-Ser-Gly-Gly-Pro-X 2 -Val, or a fragment thereof comprising amino acids 10-18 of SEQ ID NO:2.
  • X 1 is GIu or GIn and X 2 is Phe, Met, Leu, His or VaI.
  • X 1 is GIu
  • X 2 is Phe.
  • thrombin peptide derivative of this type is a polypeptide comprising the amino acid sequence Ala-Gly- Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe- VaI (SEQ ID NO:6).
  • a further example of a thrombin peptide derivative of this type is a polypeptide having the amino acid sequence H-Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys- Arg-GIy-ASp-AIa-CyS-GIu-GIy-ASp-Ser-GIy-GIy-PrO-Phe-VaI-NH 2 (SEQ ID NO:3).
  • Zero, one, two or three amino acids in the thrombin peptide derivative differ from the amino acid at the corresponding position of SEQ ID NO:6, 1, 2, or 3.
  • the difference is conservative.
  • thrombin peptide derivative dimer of the present invention is represented by Formula (IV):
  • each thrombin peptide derivative comprising a dimer comprises a polypeptide comprising the amino acid sequence SEQ ID NO:27: AIa- Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro- Phe-Val-Met-Lys-Ser-Pro-Phe-Asn-Asn-Arg-Trp-Tyr, or a C-terminal truncated fragment thereof having at least twenty-three amino acids.
  • each thrombin peptide derivative comprises the amino acid sequence SEQ ID NO:28: Ala-Gly-Tyr-Lys-Pro-Asp- Glu-Gly-Lys-Arg-Gly-Asp-Ala-Cys-X,-Gly-Asp-Ser-Gly-Gly-Pro-X 2 -Val-Met-Lys-Ser- Pro-Phe-Asn-Asn- Arg-Trp-Tyr, or a C-terminal truncated fragment thereof comprising at least twenty-three amino acids.
  • Xi is GIu or GIn and X 2 is Phe, Met, Leu, His or VaI.
  • Xi is GIu
  • X 2 is Phe.
  • a thrombin peptide derivative of this type is a polypeptide comprising the amino acid sequence Ala-Gly-Tyr-Lys-Pro-Asp-Glu- Gly-Lys-Arg-Gly-Asp-Ala-Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val-Met-Lys-Ser-Pro- Phe-Asn-Asn- Arg-Trp-Tyr (SEQ ID NO:27).
  • a further example of a thrombin peptide derivative of this type is a polypeptide comprising the amino acid sequence H-Ala-Gly-Tyr- Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val- Met-Lys-Ser-Pro-Phe-Asn-Asn-Arg-Trp-Tyr-NH 2 (SEQ ID NO:29).
  • Zero, one, two or three amino acids in the thrombin peptide derivative differ from the amino acid at the corresponding position of SEQ ID NO:27, 28 or 29.
  • the difference is conservative.
  • NPAR agonists includes antibodies and antigen-binding fragments that can both bind to and activate the non-proteolytically activated thrombin receptor (NPAR).
  • Agonist antibodies that bind to thrombin receptors have been described in the art. For example, Frost et al. teach that a monoclonal antibody, TR-9, can mimic the effects of thrombin's high affinity interaction with the high affinity thrombin receptor (Frost, G.H., et al., J. Cell Biol. 105 (6 PT. 1):2551-58(1987)).
  • Antibodies or antigen-binding fragments thereof that are NPAR agonists can be found by their binding to a complementary peptide that is encoded by the complement of a nucleotide sequence encoding a portion of thrombin. See Molecular Recognition Theory below.
  • the NPAR agonist antibody or antigen-binding fragment binds to a complementary peptide that is encoded by the complement of a nucleotide sequence encoding a thrombin.
  • An NPAR agonist antibody or antigen-binding fragment can be found by its binding to a complementary peptide that is encoded by the complement of a nucleotide sequence encoding a portion of thrombin.
  • the thrombin or portion thereof (which is encoded by the sense or +RNA strand and is the complement of the RNA strand encoding the complementary peptide to which the antibody or antigen-binding fragment binds) is a mammalian thrombin or a portion of a mammalian thrombin. In another embodiment, the thrombin or portion thereof is a human thrombin or a portion of a human thrombin.
  • Antibodies or antigen-binding fragments thereof that bind to a complementary peptide, wherein the complementary peptide is encoded by the complement of a nucleotide sequence encoding thrombin or a portion thereof, can be NPAR agonists.
  • the portion of thrombin (which is encoded by the sense or +RNA strand and is the complement of the RNA strand encoding the complementary peptide to which the antibody or antigen-binding fragment binds) is a thrombin receptor binding domain or a portion thereof.
  • a thrombin receptor binding domain or a portion thereof is a segment of thrombin that is capable of selectively binding to the high-affinity non- proteolytically activated thrombin receptor (NPAR).
  • NPAR non- proteolytically activated thrombin receptor
  • Such thrombin receptor binding domains contain a portion of a domain (represented by amino acid residues 517-520 of human thrombin.
  • the amino acid sequence of human prothrombin includes a sequence homologous to the tripeptide cell binding domain of fibronectin, Arg-Gly- Asp.
  • the thrombin receptor binding domain or portion thereof comprises the amino acid sequence AGYKPDEGKRGDACEGDSGGPFV (i.e., amino acids 508-530 of human thrombin (SEQ ID NO:6)).
  • the thrombin receptor binding domain or portion thereof is a portion of the thrombin receptor binding domain and comprises the amino acid sequence EGKRGDACEG (SEQ ID NO:7).
  • complementary peptides of domains of thrombin that are encoded by both the 5'-3' sequence of the antisense RNA strand and the 3'-5' sequence of the antisense RNA strand can be used to produce the NPAR agonist antibodies and antigen- binding domains of the invention.
  • the complementary peptide (to which the antibodies and antigen-binding fragments bind) is encoded by the 5 '-3' sequence of the antisense RNA strand. In another embodiment, the complementary peptide is encoded by the 3 '-5' sequence of the antisense RNA strand.
  • a complementary peptide (to which the NPAR agonist antibodies and antigen-binding fragments of the invention bind) comprises the amino acid sequence KGSPTVTFTGIPCFPFIRLVTS (AC-23; SEQ ID NO:30).
  • the complementary peptide comprises the amino acid sequence KGSPTVTFTGIPSFPFIRLVTS (23C53; SEQ ID NO:31).
  • the complementary peptide comprises the amino acid sequence TFTGIPSFPF (C1053; SEQ ID NO:32).
  • the complementary peptide comprises the amino acid sequence RPMFGLLPFAPLRTLPLSPPGKQ [AC-23rev (SEQ ID NO:33), which is the complementary 5 '-3' peptide corresponding to AC-23].
  • the complementary peptide comprises the amino acid sequence LPF APLRTLP [C1053rev (SEQ ID NO: 12), which is the complementary 5 '-3' peptide corresponding to C 1053].
  • an NPAR agonist antibody or an antigen-binding fragment thereof binds to a cysteine-altering complementary peptide comprising the amino acid sequence KGSPTVTFTGIPSFPFIRLVTS (23C53; SEQ ID NO:31).
  • 23C53 which differs from AC- 23 by a single amino acid, is the complementary peptide of TP508, except that it possesses a single amino acid alteration from Cys to Ser.
  • the stimulatory (agonistic) thrombin polypeptide derivatives possess a domain (represented by amino acid residues 519- 530 of human thrombin) with a high degree of homology to a number of serine esterases.
  • the inhibitory (antagonistic) thrombin polypeptide derivatives do not include the serine esterase domain.
  • Thrombin peptide derivatives from amino acid residues 508-530 of human thrombin have been described for promoting thrombin receptor mediated cell stimulation.
  • stimulatory (agonistic) thrombin polypeptide derivatives containing both fibronectin- and serine protease-homologous domains bind to thrombin receptors with high-affinity and substitute for DIP-alpha-thrombin as an initiator of receptor occupancy-related mitogenic signals.
  • DIP-alpha-thrombin is a proteolytically inactive derivative of thrombin that retains receptor binding activity.
  • inhibitory (antagonistic) thrombin polypeptide derivatives containing only the fibronectin-homologous domain (p517-520) (but not the serine protease-homologous domain) bind to the thrombin receptor without inducing mitogenesis.
  • An intermediate thrombin peptide derivative (p519- 530) retains the ability to mediate mitogenesis but to a much lesser degree than p508-530.
  • Hydropathy is an index of the affinity of an amino acid for a polar environment; hydrophilic residues yielding a more negative score, while hydrophobic residues exhibit more positive scores.
  • Kyte and Doolittle (1982) conceived a hydropathy scale that is widely used (Kyte, J., and Doolittle, R.F., J. MoI. Biol. 5:105-32 (1982)).
  • the observed relationship between the middle base of a triplet codon and residue hydropathy entails that peptides encoded by complementary DNA will exhibit complementary, or inverted, hydropathic profiles.
  • Gho and Chae describe peptide antagonists of human angiogenin that are complementary peptides encoded by the antisense RNA sequence corresponding to the receptor binding site of angiogenin (Gho, Y. S. and Chae, CB. J. Biol. Chem. 272(39):24294-99 (1997)).
  • Ghiso et al. describe a peptide complementary to a region of cystatin C that exhibits inhibitory activity (Ghiso, J., et al., Proc. Natl. Acad. Sci.
  • Bost and Blalock describe the production of anti-idiotypic antibodies by immunization with a pair of complementary peptides (Bost. K.L., and Blalock, J.E., J. Molec. Recognit. 1 :179-83 (1989)).
  • MRT Molecular Recognition Theory
  • Blalock suggested that it is the linear pattern of amino acid hydropathy scores in a sequence (rather than the combination of specific residue identities), that defines the secondary structure environment. Furthermore, he suggested that sequences with inverted hydropathic profiles are complementary in shape by virtue of inverse forces that determine their steric relationships.
  • NPAR agonists as referred to herein encompass antibodies and antigen-binding fragments thereof that bind to the complementary peptides described herein and activate the non-proteolytically activated thrombin receptor.
  • the antibodies as referred to herein can be polyclonal or monoclonal, and the term "antibody” is intended to encompass both polyclonal and monoclonal antibodies.
  • the terms polyclonal and monoclonal refer to the degree of homogeneity of an antibody preparation, and are not intended to be limited to particular methods of production.
  • the antibody or antigen-binding fragment is a monoclonal antibody or antigen-binding fragment thereof.
  • monoclonal antibody or “monoclonal antibody composition” as used herein, refers to a population of antibody molecules that contain only one species of an antigen binding site capable of immunoreacting with a particular epitope of a polypeptide of the invention.
  • a monoclonal antibody composition thus typically displays a single binding affinity for a particular polypeptide of the invention with which it immunoreacts.
  • antibody as used herein also encompasses functional fragments of antibodies, including fragments of chimeric, humanized, primatized, veneered or single chain antibodies.
  • Functional fragments include antigen-binding fragments of antibodies that bind to the complementary peptides, wherein complementary peptides are encoded by the complement of a nucleotide sequence encoding thrombin or a portion thereof.
  • antibody fragments capable of binding to a complementary peptide include, but are not limited to Fv, Fab, Fab' and F(ab') 2 fragments.
  • Such fragments can be produced by enzymatic cleavage or by recombinant techniques. For example, papain or pepsin cleavage can generate Fab or F(ab') 2 fragments, respectively. Other proteases with the requisite substrate specificity can also be used to generate Fab or F(ab') 2 fragments.
  • Antibodies can also be produced in a variety of truncated forms using antibody genes in which one or more stop codons have been introduced upstream of the natural stop site.
  • a chimeric gene encoding a F(ab') 2 heavy chain portion can be designed to include DNA sequences encoding the CHi domain and hinge region of the heavy chain.
  • Single chain antibodies, and chimeric, humanized or primatized (CDR-grafted), or veneered antibodies, as well as chimeric, CDR-grafted or veneered single chain antibodies, comprising portions derived from different species, are also encompassed by the term antibody.
  • the various portions of these antibodies can be joined together chemically by conventional techniques, or can be prepared as a contiguous protein using genetic engineering techniques.
  • nucleic acids encoding a chimeric or humanized chain can be expressed to produce a contiguous protein. See, e.g., Cabilly et al., U.S. Patent No. 4,816,567; Cabilly et al., European Patent No.
  • Boss et al. U.S. Patent No. 4,816,397; Boss et al., European Patent No. 0,120,694 B 1 ; Neuberger, M.S. et al., WO 86/01533; Neuberger, M.S. et al., European Patent No. 0,194,276 Bl; Winter, U.S. Patent No. 5,225,539; Winter, European Patent No. 0,239,400 Bl; Queen et al., European Patent No. 0 451 216 Bl; and Padlan, E.A. et al., EP 0 519 596 Al . See also, Newman, R.
  • Humanized antibodies can be produced using synthetic or recombinant DNA technology using standard methods or other suitable techniques.
  • Nucleic acid (e.g., cDNA) sequences coding for humanized variable regions can also be constructed using PCR mutagenesis methods to alter DNA sequences encoding a human or humanized chain, such as a DNA template from a previously humanized variable region (see e.g., Kamman, M., et al., Nucl. Acids Res., 17: 5404 (1989)); Sato, K., et al., Cancer Research, 53: 851-856 (1993); Daugherty, B.L. et al., Nucleic Acids Res., 19(9): 2471-2476 (1991); and Lewis, A.P.
  • variants can also be readily produced.
  • cloned variable regions can be mutated, and sequences encoding variants with the desired specificity can be selected (e.g., from a phage library; see e.g., Krebber et al., U.S. 5,514,548; Hoogenboom et al., WO 93/06213).
  • the antibody can be a humanized antibody comprising one or more immunoglobulin chains [e.g., an antibody comprising a complementarity-determining region (CDR) of nonhuman origin (e.g., one or more CDRs derived from an antibody of nonhuman origin)] and a framework region derived from a light and/or heavy chain of human origin (e.g., CDR- grafted antibodies with or without framework changes)].
  • CDR complementarity-determining region
  • the antibody or antigen-binding fragment thereof comprises the light chain CDRs (CDRl, CDR2 and CDR3) and heavy chain CDRs (CDRl , CDR2 and CDR3) of a particular immunoglobulin.
  • the antibody or antigen-binding fragment further comprises a human framework region.
  • Antibodies that are specific for a complementary peptide, wherein the complementary peptide is encoded by the complement of a nucleotide sequence encoding thrombin or a portion thereof, can be raised against an appropriate immunogen, such as a synthetic or recombinant complementary peptide or a portion thereof.
  • Antibodies can also be raised by immunizing a suitable host (e.g., mouse) with transfected cells that express a complementary peptide. Such cells can also be used in a screen for an antibody that binds thereto (See e.g., Chuntharapai et al., J. Immunol., 152: 1783-1789 (1994); Chuntharapai et al., U.S. Patent No. 5,440,021).
  • Preparation of immunizing antigen, and polyclonal and monoclonal antibody production can be performed using any suitable technique (e.g., as exemplified herein).
  • Any suitable technique e.g., as exemplified herein.
  • a variety of methods have been described (see e.g., Kohler et al., Nature, 256: 495-497 (1975) and Eur. J. Immunol. 6: 511-519 (1976); Milstein et al., Nature 266: 550-552 (1977); Koprowski et al., U.S. Patent No. 4,172,124; Harlow, E. and D. Lane, 1988, Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory: Cold Spring Harbor, NY); Current Protocols In Molecular Biology, Vol.
  • a hybridoma is produced by fusing a suitable immortal cell line (e.g., a myeloma cell line, such as SP2/0, P3X63Ag8.653 or a heteromyeloma) with antibody-producing cells.
  • a suitable immortal cell line e.g., a myeloma cell line, such as SP2/0, P3X63Ag8.653 or a heteromyeloma
  • Antibody-producing cells can be obtained from the peripheral blood or, preferably the spleen or lymph nodes, of humans or other suitable animals immunized with a complementary peptide.
  • the fused cells (hybridomas) can be isolated using selective culture conditions, and cloned by limiting dilution. Cells that produce antibodies with the desired specificity can be selected by a suitable assay (e.g., ELISA).
  • Suitable methods of producing or isolating antibodies of the requisite specificity can be used, including, for example, methods that select recombinant antibody from a library (e.g., a phage display library).
  • a library e.g., a phage display library.
  • Transgenic animals capable of producing a repertoire of human antibodies e.g., Xenomouse ® (Abgenix, Fremont, CA)
  • suitable methods see e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90: 2551-2555 (1993); Jakobovits et al., Nature, 362: 255-258 (1993)).
  • the invention also encompasses bispecific antibodies, or functional fragments thereof (e.g., F(ab') 2 ), which bind to a complementary peptide as described herein and at least one other antigen (e.g., a tumor antigen, a viral antigen).
  • Bispecific antibodies can be secreted by triomas and hybrid hybridomas.
  • triomas are formed by fusion of a hybridoma and a lymphocyte (e.g., antibody-secreting B cell) and hybrid hybridomas are formed by fusion of two hybridomas.
  • Each of the fused cells i.e., hybridomas, lymphocytes
  • triomas and hybrid hybridomas can produce an antibody containing antigen-binding sites that recognize different antigens.
  • triomas and hybrid hybridomas can be assayed for bispecific antibody using a suitable assay (e.g., ELISA), and bispecific antibodies can be purified using conventional methods, (see, e.g., U.S. Patent No. 5,959,084 (Ring et al.), U.S. Patent No. 5,141,736 (Iwasa et al.), U.S. Patent Nos. 4,444,878, 5,292,668, 5,523,210 (all to Paulus et al.) and U.S. Patent No. 5,496,549 (Yamazaki et al.)).
  • a suitable assay e.g., ELISA
  • the present invention is directed to methods of promoting, stimulating or accelerating healing of a bone fracture in an osteopenic human comprising administering to the human a therapeutically effective amount of an NPAR agonist.
  • Promoting, stimulating or accelerating healing of a bone fracture means causing, after the administration of the NPAR agonist or composition comprising an NPAR agonist, compared to fractures not treated with the NPAR agonist, a decreased time to any one or more of the following: (1) trabecular bridging (e.g., at least 1%, 2%, 3%, 4%, 5%, 6%, 7% or 2-50%, or 2-40%, or 2-20% or 2-15%, or 2-10%, or 5-10% shorter in time for fractures treated with the NPAR agonist compared to that for untreated fractures); (2) overall radiographic healing (e.g., at least 1%, 2%, 5%, 7%, 10%, 11%, 12%, 13%, or 2-50%, or 5- 40%, or 5-30%, or 10-25%, or 10-20% shorter in time for fractures treated with the healing agent and untreated fractures); (3) cortical bridging (e.g., at least 1%, 2%,
  • a “bone fracture” refers to a fracture in a bone where healing would occur without treatment, where healing would occur after immobilization or where healing would occur after bone fragments have been aligned (or reduction).
  • “bone fracture” includes hairline fractures, simple non-displaced fractures, and non-displaced complex fractures. It also includes displaced simple, displaced complex and non-union fractures, in which the bone fragments have been aligned.
  • “Bone fracture” does not include gaps between bones where bone healing would not spontaneously occur (e.g. gaps typically greater than 0.5, 0.6, 0.7, 0.8, 0.9, 1.0 or 1.5 cm). Such gaps occur, for example, after removal of a bone tumor or cyst or before alignment of a non-union fracture. (See, for example, U. S. Patent No. 6.914,050, incorporated by reference by its entirety).
  • the disclosed methods can be used as a primary treatment method, where the method is used shortly after the occurrence of the bone fracture.
  • the disclosed methods can also be used as a secondary treatment method, where the bone fractures have been previously treated, but have not properly healed.
  • the disclosed methods are not limited to any particular kind of bone(s) or bone location(s).
  • fractures which can be treated by the disclosed methods include fractures of the humerus, radius, ulna, femur, tibia, fibula, spine, pelvis, carpus, metacarpus, phalanx (hand), talus, calcaneus, navicular, cuneiform, cuboid, metatarsus, phalanx (foot), clavicle, scapula, or patella.
  • Bone fractures at different bone segments can also be treated by the present methods.
  • the bone segments include proximal, diaphyseal, or distal for long bone.
  • a fourth segment is known as malleolar segment.
  • the two segments are pelvic ring and acetabulum.
  • osteoopenic human or “human with osteopenia” refers to a human having decreased density of bone. Osteopenia is classified based on bone mineral density (BMD). The BMD is reported in units called "T-scores.”
  • an osteopenic human is a human with a T-score between and including -1.1 and -2.4 in the hip and/or spine. Osteopenic humans can be males or females.
  • a “therapeutically effective amount” is the quantity of the NPAR agonist that results in a decreased time to any one or more of the following: (1) trabecular bridging; (2) overall radiographic healing; (3) cortical bridging; (4) clinical evaluation of fracture healing; and (5) removal of immobilization devices.
  • the amount of the NPAR agonist administered will depend on the degree, severity, and type of the bone fracture, and the release characteristics of the pharmaceutical formulation. It will also depend on the human's health, size, weight, age, sex and tolerance to drugs.
  • the NPAR agonists can be administered to the bone fracture site for 1, 2, 3, 4, 5, 6, or 7 or more times during the period in which the fracture is healing or daily during the period in which the fracture is healing.
  • the NPAR agonists are preferably administered at evenly spaced intervals; each dose can be the same or different, but is preferably the same.
  • a dose delivered to the bone fracture site can be, for example, 0.1-500 ⁇ g, preferably 1- 50 ⁇ g of NPAR agonist, and is commonly 3, 5, 10, 30 or 50 ⁇ g.
  • the first administration of NPAR agonist is shortly (less than 11 days) after the occurrence of the bone fracture.
  • the NPAR agonist is administered after an closed or open reduction surgery (within 11 days).
  • the NPAR agonist is administered during an open reduction surgery.
  • the NPAR agonist is administered conincident with or before immobilization (within 3, 2, or 1 day).
  • the disclosed NPAR agonists can be administered by any suitable route, including, for example, by local introduction to the bone fracture by, for example, percutaneous injection.
  • the NPAR agonist can be advantageously administered to the fracture in a sustained release formulation, or can be delivered by a pump or an implantable device, or implantable carrier, such as the polymers discussed below.
  • "Administered to the bone fracture” means delivered between the fractured ends or on to the surfaces of the bones at the fracture, as seen, for example, by fluoroscopy. Alternatively, the point of delivery of the
  • NPAR agonist can be in sufficient proximity to the fractured ends or surfaces of the bones so that the agonist can diffuse and contact the fractured ends or surfaces, for example, within 1 cm of one or both fractured ends or surfaces of the bone.
  • the disclosed method can be used in combination with immobilization devices such as a cast.
  • the disclosed method can be used in combination with measures that align the bone fracture, for example, reduction surgery.
  • the disclosed method can be used in combination with measures that fasten the bone fracture together, for example, through the use of a wire, a pin, a rod or a plate.
  • the disclosed method can used to treat displaced simple, displaced complex or non-union fractures in combination with measures that re-align the bone fractures, typically through immobilization, reduction surgery or a combination thereof.
  • the fracture are held together through the use of a wire, a pin, a rod or a plate.
  • the NPAR agonists can be administered to the human in conjunction with an acceptable pharmaceutical carrier as part of a pharmaceutical composition.
  • the formulation of the pharmaceutical composition will vary according to the mode of administration selected.
  • Suitable pharmaceutical carriers may contain inert ingredients which do not interact with the compound.
  • the carriers should be biocompatible, i.e., non-toxic, noninflammatory, non-immunogenic and devoid of other undesired reactions at the administration site.
  • Examples of pharmaceutically acceptable carriers include, for example, saline, commercially available inert gels, or liquids supplemented with albumin, methyl cellulose or a collagen matrix.
  • compositions may include gels.
  • Gels are comprising a base selected from an oleaginous base, water, or an emulsion-suspension base.
  • a gelling agent which forms a matrix in the base, increasing its viscosity to a semisolid consistency.
  • examples of gelling agents are hydroxypropyl cellulose, acrylic acid polymers, and the like.
  • the active ingredients are added to the formulation at the desired concentration at a point preceding addition of the gelling agent or can be mixed after the gelation process.
  • the NPAR agonists are administered in a sustained release formulation.
  • Polymers are often used to form sustained release formulations. Examples of these polymers include poly ⁇ -hydroxy esters such as polylactic acid/polyglycolic acid homopolymers and copolymers, polyphosphazenes (PPHOS), polyanhydrides and poly(propylene fumarates).
  • Polylactic acid/polyglycolic acid (PLGA) homo and copolymers are well known in the art as sustained release vehicles.
  • the rate of release can be adjusted by the skilled artisan by variation of polylactic acid to polyglycolic acid ratio and the molecular weight of the polymer (see Anderson, et al, Adv. Drug Deliv. Rev. 28:5 (1997), the entire teachings of which are incorporated herein by reference).
  • the incorporation of poly(ethylene glycol) into the polymer as a blend to form microparticle carriers allows further alteration of the release profile of the active ingredient (see Cleek et al, J. Control Release 48:259 (1997), the entire teachings of which are incorporated herein by reference).
  • Ceramics such as calcium phosphate and hydroxyapatite can also be incorporated into the formulation to improve mechanical qualities.
  • PPHOS polymers contain alternating nitrogen and phosphorous with no carbon in the polymer backbone, as shown below in Structural Formula (II):
  • the properties of the polymer can be adjusted by suitable variation of side groups R and R' that are bonded to the polymer backbone.
  • the degradation of and drug release by PPHOS can be controlled by varying the amount of hydrolytically unstable side groups.
  • an increase in degradation rate is observed (see Laurencin et al., J Biomed Mater. Res. 27:963 (1993), the entire teachings of which are incorporated herein by reference), thereby increasing the rate of drug release.
  • Polyanhydrides shown in Structural Formula (III), have well defined degradation and release characteristics that can be controlled by including varying amounts of hydrophobic or hydrophilic monomers such as sebacic acid and l,3-bis(p- carboxyphenoxy)propane (see Leong et al, J. Biomed. Mater. Res. /9:941 (1985), the entire teachings of which are incorporated herein by reference).
  • hydrophobic or hydrophilic monomers such as sebacic acid and l,3-bis(p- carboxyphenoxy)propane (see Leong et al, J. Biomed. Mater. Res. /9:941 (1985), the entire teachings of which are incorporated herein by reference).
  • anhydrides are often copolymerized with imides to form polyanhydride-co-imides.
  • polyanhydride-co-imides that are suitable for orthopaedic applications are poly(trimellitylimido-glycine-co- 1 ,6-bis(carboxyphenoxy)hexane and pyromellityimidoalanine: 1 ,6-bis(p-carboxyphenoxy)hexane copolymers.
  • Poly(propylene fumarates) are highly desirable biocompatible implantable carriers because they are an injectable, in situ polymerizable, biodegradable material.
  • injectable means that the material can be injected by syringe through a standard needle used for injecting pastes and gels.
  • PPF combined with a vinyl monomer (N-vinyl pyrrolidinone) and an initiator (benzoyl peroxide), forms an injectable solution that can be polymerized in situ. It is particularly suited for filling skeletal defects of a wide variety of sizes and shapes (see Suggs et al., Macromolecules 50.4318 (1997), Peter et al., J. Biomater. ScL Poly,. Ed.
  • compositions can be administered to the site in the form of microparticles or microspheres.
  • the microparticles are placed in contact or in close proximity to the bone fracture site either by surgically exposing the site and applying the microparticles on or in close proximity to the site by painting, pipetting, spraying, injecting or the like.
  • Microparticles can also be delivered to the site by laparoscopy or by percutaneous injection.
  • Compositions which are injectable include the solutions of poly(propylene fumarate) copolymers described above and pastes of calcium phosphate ceramics (see Schmitz et al. , J. Oral Maxillofacial Surgery 57:1122 (1999), the entire teachings of which are incorporated herein by reference). Injectable compositions can be injected directly to the bone fracture site.
  • Bone fractures are often accompanied by symptoms and infirmities such as pain and infection.
  • managing pain and inflammation may require co-administration with analgesic or an anti-inflammatory agents.
  • Managing infection may require co-administration with antimicrobial, antibiotic or disinfectant agents.
  • Thrombin peptide derivatives and modified thrombin peptide derivatives can be synthesized by solid phase peptide synthesis (e.g., BOC or FMOC) method, by solution phase synthesis, or by other suitable techniques including combinations of the foregoing methods.
  • BOC and FMOC methods which are established and widely used, are described in Merrifield, J. Am.
  • Thrombin peptide derivative dimers can be prepared by oxidation of the monomer.
  • Thrombin peptide derivative dimers can be prepared by reacting the thrombin peptide derivative with an excess of oxidizing agent.
  • a well-known suitable oxidizing agent is iodine.
  • non-aromatic heterocyclic group is a non-aromatic carbocyclic ring system that has 3 to 10 atoms and includes at least one heteroatom, such as nitrogen, oxygen, or sulfur.
  • non-aromatic heterocyclic groups include piperazinyl, piperidinyl, pyrrolidinyl, morpholinyl, thiomorpholinyl.
  • aryl group includes both carbocyclic and heterocyclic aromatic ring systems. Examples of aryl groups include phenyl, indolyl, furanyl and imidazolyl.
  • an "aliphatic group” is a straight chain, branched or cyclic non-aromatic hydrocarbon.
  • An aliphatic group can be completely saturated or contain one or more units of unsaturation (e.g., double and/or triple bonds), but is preferably saturated, i.e., an alkyl group.
  • a straight chained or branched aliphatic group has from 1 to about 10 carbon atoms, preferably from 1 to about 4, and a cyclic aliphatic group has from 3 to about 10 carbon atoms, preferably from 3 to about 8.
  • Aliphatic groups include, for example, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, tert-butyl, pentyl, cyclopentyl, hexyl, cyclohexyl, octyl and cyclooctyl.
  • Suitable substituents for an aliphatic group, an aryl group or a non-aromatic heterocyclic group are those which do not significantly lower therapeutic activity of the NPAR agonist, for example, those found on naturally occurring amino acids.
  • Each R e is independently an alkyl group or an aryl group.
  • a substituted aliphatic group can have more than one substituent.
  • EXAMPLE 1 A PHASE 3 STUDY SHOWS THAT TP508 HAS BENEFICIAL EFFECTS
  • a double blind, randomized, placebo-controlled Phase 3 clinical trial was carried out to evaluate the efficacy and safety of a single percutaneous injection of TP508 at 10 ⁇ g formulated in 20 mg/ml mannitol and 10 ⁇ M EDTA on the rate of healing in distal radius fractures.
  • Adult females with unstable and/or displaced intra- or extra-articular fractures of the distal radius requiring closed reduction and stabilization with both cast and K-wires or with external fixation with or without K-wires were enrolled into two parallel study arms.
  • One group of patients received a single percutaneous injection of TP508 at 10 ⁇ g/ml and the other group received a single percutaneous injection of placebo control.
  • BMD bone mineral density
  • DXA dual-energy x-ray absorptiometry
  • ROI regions of interest
  • Investigators were not restricted with respect to the type of DXA system used. Quality assurance records were obtained from each center to document stability of the DXA system used. DXA systems were not cross-calibrated across all study centers.
  • DXA scan data were obtained on 394 patients in this study. Among them, a total of 302 female patients had DXA scans. The coordination of scans, assessment of scan quality and accuracy, interface with DXA technicians, and DXA data analysis were conducted. T-scores, reflecting BMD relative to normative values, were tabulated for each patient.
  • T-scores from the radius were not used to classify patients due to substantial inter-manufacturer discrepancies and lack of a standardized database for radius (Kiebzak et al., (2004) Journal of Clinical Densitometry 7(2): 143-152).
  • T-score Classification of bone status was based on the lowest T-score from total hip or spine and defined as: normal, T-score > - 1.0; osteopenia, T-score between and including -1.1 and -2.4; osteoporosis, T-score ⁇ -2.5 (Assessment of Fracture Risk and its Application to Screening for Postmenopausal Osteoporosis (1994) WHO Technical Report Series 843; Hamdy RC, Petak SA, Lenchik L (2002) Journal of Clinical Densitometry 5(Suppl 1): Sl 1-17).
  • the study's primary, protocol-specified outcome was time to removal of immobilization. Baseline comparability of the two study groups, osteopenic patients treated with TP508 and osteopenic patients treated with placebo, was tested using t-tests and chi-squares.
  • the primary analysis of the time-to-removal outcome was performed using a Cox proportional hazards model, with terms for treatment, type of fixation (cast or external fixator), dominance (injured hand was or was not dominant), intra- vs. extra-articular fracture, smoking status (current vs. not current), body mass index (BMI), age, and gender.
  • Secondary analyses of time-to-healing outcomes time to radiographic healing and time to clinical evaluation of healing) were performed using similar Cox models.
  • a second model for each outcome added a term for the interaction of treatment group with intra- vs. extra- articular fracture. The assumption of proportional hazards was validated graphically. Analyses were performed with SAS statistical software, version 8.1.
  • osteoporotic women differed in the same way compared to osteopenic women, with the latter group in an intermediate position (regarding age and size) between women with normal BMD and osteoporotic women.
  • Table 2 displays demographic variables in osteopenic women by treatment, TP508 or placebo.
  • TP5O8-treated and placebo-treated osteopenic females Variables that might affect injury dynamics such as weight, BMI, and age, appear to have been well balanced between the randomization groups.
  • the only significant difference between treatment groups is seen in the percentage of patients who were treated with external fixation (42% in the placebo group vs. 24% in the TP508 group). Additionally, an imbalance in the percentage of extra-articular fractures (42% in the placebo group vs. 54% in TP508 treated) was observed.
  • Time to immobilization removal is defined as the time elapsed between the date of final fracture reduction (date of surgery) and the first study visit at which the investigator, based on clinical and radiographic assessments of healing, removes all rigid immobilization hardware used to stabilize the fracture.
  • Time to Clinical Evaluation of Bon healing is based on the patient's assessment of pain at the fracture site using a 10cm pain Visual Analog Scale (VAS), the investigator's palpation of the fracture to evaluate the degree of motion, inflammation, and extent of edema, and a confirmation of clinical findings by a brief review of the radiographs.
  • VAS pain Visual Analog Scale
  • Cox model estimate of the placebo-to-TP508 hazard ratio adjusted for reduction method (Cast/Ex-fix vs. splint), injured hand dominant, fracture classification (extra-articular), body mass index, smoking status (current smoker vs. non-smoker/ex-smoker), and age.
  • Radiographic progression of healing at each evaluable time point was based on:
  • TP508-treated osteopenic women had a 17.5 percent shorter median healing time than placebo recipients (log-rank p- value 0.021, Cox model p-value 0.022, hazard ratio 1.48).
  • TP508- treated osteopenic women showed a very statistically significant decrease compared to the placebo group (log-rank p-value 0.0024, Cox model p-value 0.0028, hazard ratio 1.64), as shown in FIG. 2.
  • a 13% decrease in median time for overall radiographic healing was observed for TP508-treated osteopenic women compared to the placebo group.
  • Range of motion of the affected and contralateral limbs was evaluated by measurements of wrist extension, flexion, ulnar and radial deviation, pronation and supination commencing at the time of rigid immobilization removal and continuing at all subsequent postoperative evaluations.
  • Grip strength in the affected and contralateral limb was assessed using a calibrated Jamar hand dynamometer, which is a hand instrument with high instrument stability. Grip strength was evaluated by recording the results of 3 grip strength trials for each of the hands with the dynamometer set at the appropriate grip setting for the patient. Upon the selection of the initial appropriate dynamometer setting, the same setting was used for the duration of the study for an individual patient. In addition, for each patient, the dynamometer setting was the same for both the affected and the contralateral limbs for the duration of the study. The measurement was performed for all patients commencing at the time of rigid immobilization removal and continuing at all subsequent postoperative evaluations. Grip strength was adjusted to balance for non-dominant versus dominant hand fractures.
  • PRWE Patient Rated Wrist Evaluation
  • radiographic endpoints such as time to radial cortical bridging, time to overall radiographic healing, time to trabeculae bridging, however, did not show this same statistically significant effect.
  • time to radial cortical bridging time to overall radiographic healing
  • time to trabeculae bridging did not show this same statistically significant effect.
  • osteopenic women require longer time to reach radiographic endpoints than normal women (FIGs 7-9).

Abstract

The present invention is directed to methods of promoting healing of a bone fracture in an osteopenic human patient, comprising administering to the patient a therapeutically effective amount of an non-proteolytically activated thrombin receptor agonist.

Description

THROMBIN PEPTIDE DERIVATIVES FOR TREATING FRACTURES IN
OSTEOPENIC PATIENTS
RELATED APPLICATION
This application claims the benefit of U.S. Provisional Application No. 60/901,441, filed on February 15, 2007. The entire teachings of the above application are incorporated herein by reference.
BACKGROUND OF THE INVENTION
Fracture incidence is expected to increase with the aging population in industrialized countries. The most problematic fractures, those of the wrist, spine, and hip, are directly linked to the rising incidence of osteopenia and osteoporosis as people age. The World Health Organization (WHO) estimates that the worldwide lifetime risk for osteoporotic fractures is approximately 40% for women and 10% for men. In 2000 there were an estimated 9 million osteoporotic fractures worldwide, of which 1.6 million were fractures of the hip, 1.7 million wrist, and 1.4 million vertebral fractures. By 2050 the number of people in the age group most at risk for osteopenic and osteoporotic fracture (> 65 years old) will increase threefold. The worldwide incidence of hip fracture is expected to increase from 1.9 million in 1990 to 6.3 million in 2050. Hip fractures alone were estimated to have direct hospital costs in 1996 of $4. OB and $5.7B in the European Union and US, respectively. By 2050, it is estimated that the worldwide cost of hip fractures alone is projected to be over $130 billion.
Osteopenia is characterized by decreased density of bone. Similar to osteoporosis, people with osteopenia have a higher risk and frequency of suffering bone fractures. Clearly, an agent that accelerates fracture healing would reduce costs due to disability and improve the quality of life for those who sustain a fracture. With a projected increase in incidence worldwide, early therapeutic intervention should mitigate costs associated with complications of healing.
Currently, no pharmaceuticals have been approved by FDA to be used to stimulate bone healing in human, let alone for people with osteopenia. Therefore, there is a need for the development of new drugs that stimulate bone healing in people with osteopenia. SUMMARY OF THE INVENTION
Applicants have discovered that TP508, a polypeptide which stimulates or activates the non-proteolytically activated thrombin receptor (hereinafter "NPAR"), can promote the healing of bone fractures in osteopenic human patients (Example 1). Osteopenic females with distal radius fractures were treated with TP508 and the data showed statistically significant effects in the primary endpoint (time to immobilization removal) and certain secondary endpoints, including time to clinical evaluation of fracture healing, time to overall radiographic healing, time to radial cortical bridging, and range of motion assessed by 30 degrees flexion and extension. In the same study, it was found that, in the placebo group, osteopenic women have statistically slower healing than the normal women, based on time to immobilization removal and time to clinical evaluation of fracture healing (Example 2). Based on this observation, methods of promoting healing of bone fractures with NPAR agonists are disclosed herein.
The present invention is directed to methods of promoting healing of a bone fracture in an osteopenic human patient, comprising administering to the patient a therapeutically effective amount of an NPAR agonist.
In one embodiment, the NPAR agonist is a thrombin peptide derivative disclosed herein. More specifically, one thrombin peptide derivative comprises the amino acid sequence of Arg-Gly-Asp-Ala-Cys-Xi-Gly-Asp-Ser-Gly-Gly-Pro-X2-Val (SEQ ID NO:1), or a C-terminal truncated fragment thereof comprising at least six amino acids. In another specific embodiment, the thrombin peptide derivative comprises the amino acid sequence of SEQ ID NO:2: Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Cys-Xi-Gly- Asp-Ser-Gly-Gly-Pro-X2-Val, an N-terminal truncated fragment of the thrombin peptide derivative having at least fourteen amino acids, or a C-terminal truncated fragment of the thrombin peptide derivative comprising at least eighteen amino acids. Xj is GIu or GIn and X2 is Phe, Met, Leu, His or VaI. In another specific embodiment, the thrombin peptide derivative is the polypeptide SEQ ID NO:3: H-Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys- Arg-Gly-Asp-Ala-Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe- VaI-NH2 (TP508).
In another embodiment, the NPAR agonist is a modified thrombin peptide derivative disclosed herein. In a specific embodiment, the modified thrombin peptide derivative comprises the amino acid sequence of SEQ ID NO:4: Arg-Gly-Asp-Ala-Xaa-Xi-Gly-Asp- Ser-Gly-Gly-Pro-X2-Val, or a C-terminal truncated fragment thereof having at least six amino acids. In another specific embodiment, the modified thrombin peptide derivative comprises the amino acid sequence of SEQ ID NO: 5: Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly- Lys-Arg-Gly-Asp-Ala-Xaa-Xi-Gly-Asp-Ser-Gly-Gly-Pro-X2-Val, or a fragment thereof comprising amino acids 10-18 of SEQ ID NO: 5. The pharmaceutical compositions comprising thrombin peptide derivatives or modified thrombin peptide derivatives of the present invention can also include a dimerization inhibitor. A dimerization inhibitor is a compound that inhibits or reduces dimerization of a thrombin peptide derivative or a modified thrombin peptide derivative. Dimerization inhibitors include chelating agents and/or thio-containing compounds. In another embodiment, the NPAR agonist is a thrombin peptide derivative dimer of two thrombin peptide derivatives disclosed herein. More specifically, a thrombin peptide derivative of a dimer comprises the amino acid sequence Arg-Gly-Asp-Ala-Cys-Xi-Gly- Asp-Ser-Gly-Gly-Pro-X2-Val (SEQ ID NO:1) or a C-terminal truncated fragment thereof having at least six amino acids. In another specific embodiment, the thrombin peptide derivative of the dimer comprises a polypeptide having the amino acid sequence of SEQ ID NO:2: Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Cys-Xi-Gly-Asp-Ser- Gly-Gly-Pro-X2-Val, or a fragment thereof comprising amino acids 10-18 of SEQ ID NO:2. In another embodiment of the invention, the thrombin peptide derivative of the dimer is the polypeptide H-Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly- Asp- Ala-Cys-Glu-Gly- Asp-Ser-Gly-Gly-Pro-Phe- VaI-NH2 (SEQ ID NO:3). In another specific embodiment, the thrombin peptide derivative dimer is represented by the structural formula (IV).
In further embodiments, the NPAR agonist is an antibody or antigen-binding fragment thereof that binds to and activates the non-proteolytically activated thrombin receptor or binds to a complementary peptide, wherein the complementary peptide is encoded by the complement of a nucleotide sequence encoding a portion of thrombin.
The thrombin referred to above can be a mammalian thrombin, and in particular, a human thrombin. The portion of thrombin can be a thrombin receptor binding domain or a portion thereof. In one embodiment, the thrombin receptor binding domain or portion thereof comprises the amino acid sequence Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg- Gly-Asp-Ala-Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val (SEQ ID NO:6). Another portion of a thrombin receptor binding domain comprises the amino acid sequence Glu-Gly-Lys- Arg-Gly-Asp-Ala-Cys-Glu-Gly (SEQ ID NO:7). The complementary peptide to which the antibody or the antigen-binding fragment thereof binds can be encoded by the 5 '-3' sequence of the antisense RNA strand or encoded by the 3 '-5' sequence of the antisense RNA strand.
In specific embodiments, the complementary peptide comprises the amino acid sequence Lys-Gly-Ser-Pro-Thr-Val-Thr-Phe-Thr-Gly-Ile-Pro-Cys-Phe-Pro-Phe-Ile-Arg-Leu- Val-Thr-Ser (SEQ ID NO:8) or Thr-Phe-Thr-Gly-Ile-Pro-Ser-Phe-Pro-Phe (SEQ ID NO:9) or Arg-Pro-Met-Phe-Gly-Leu-Leu-Pro-Phe-Ala-Pro-Leu-Arg-Thr-Leu-Pro-Leu-Ser-Pro-Pro- Gly-Lys-Gln (SEQ ID NO: 10) or Lys-Pro-Phe- Ala-Pro-Leu- Arg-Thr-Leu-Pro (SEQ ID NO:11). The NPAR agonist to be used in the methods of the invention can be a polyclonal antibody, or a monoclonal antibody or antigen-binding fragment thereof. In particular embodiments, these are human antibodies. Monoclonal antibodies to be used as NPAR agonists in methods of therapy can be humanized antibodies, chimeric antibodies or antigen- binding fragments of any of the foregoing, which can include Fab fragments, Fab' fragments, F(ab')2 fragments and Fv fragments.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a graph showing time to immobilization removal for osteopenic patients treated with TP508 and patients treated with placebo.
FIG. 2 is a graph showing time to overall radiographic healing for osteopenic patients treated with TP508 and patients treated with placebo.
FIG. 3 is a graph showing time for 30 degree or greater in flexion for osteopenic patients treated with TP508 and patients treated with placebo.
FIG. 4 is graph showing time for 30 degree extension in osteopenic patients treated with TP508 and patients treated with placebo. FIG. 5 is a graph showing time to immobilization removal for osteopenic patients and normal patients treated with placebo.
FIG. 6 is a graph showing time to clinical evaluation of fracture healing for osteopenic patients and normal patient treated with placebo.
FIG. 7 is a graph showing time to radial cortical bridging for osteopenic patients and normal patients treated with placebo, in the female patient population that was also the subject of Figures 5, 6, 8 and 9. Logrank test: p = 0.40. FIG. 8 is a graph showing time to overall radiographic healing for osteopenic patients and normal patients treated with placebo, in the female patient population that was also the subject of Figures 5, 6, 7 and 9. Logrank test: p = 0.15.
FIG. 9 is a graph showing time to trabeculae bridging for osteopenic patients and normal patients treated with placebo, in the female patient population that was also the subject of figures 5-8. Logrank test: p = 0.45.
DETAILED DESCRIPTION OF THE INVENTION
The invention includes methods of promoting healing of a bone fracture in an osteopenic human, comprising administering to the patient a therapeutically effective amount of an agonist of the non-proteolytically activated thrombin receptor (NPAR).
Compounds which stimulate NPAR are said to be NPAR agonists. NPAR is a high- affinity thrombin receptor present on the surface of most cells. This NPAR component is largely responsible for high-affinity binding of thrombin, proteolytically inactivated thrombin, and thrombin derived peptides to cells. NPAR appears to mediate a number of cellular signals that are initiated by thrombin independent of its proteolytic activity. An example of one such signal is the upregulation of annexin V and other molecules identified by subtractive hybridization (see Sower, et. al, Experimental Cell Research 247:422 (1999)). NPAR is therefore characterized by its high affinity interaction with thrombin at cell surfaces and its activation by proteolytically inactive derivatives of thrombin and thrombin derived peptide agonists as described below. NPAR activation can be assayed based on the ability of molecules to stimulate cell proliferation when added to fibroblasts in the presence of submitogenic concentrations of thrombin or molecules that activate protein kinase C, as disclosed in U.S. Patent Nos. 5,352,664 and 5,500,412. The entire teachings of these patents are incorporated herein by reference. NPAR agonists can be identified by this activation or by their ability to compete with 125I-thrombin binding to cells.
A thrombin receptor binding domain is defined as a polypeptide or portion of a polypeptide which directly binds to the thrombin receptor and/or competitively inhibits binding between high-affinity thrombin receptors and alpha-thrombin. NPAR agonists of the present invention include thrombin derivative peptides, modified thrombin derivative peptides, thrombin derivative peptide dimers and NPAR agonist antibodies to complementary peptides of thrombin as disclosed herein. Thrombin Peptide Derivative
Among NPAR agonists are thrombin peptide derivatives (also: "thrombin derivative peptides"), which are analogs of thrombin that have an amino acid sequence derived at least in part from that of thrombin and are active at the non-proteolytically activated thrombin receptor. Thrombin peptide derivatives include, for example, peptides that are produced by recombinant DNA methods, peptides produced by enzymatic digestion of thrombin, and peptides produced synthetically, which can comprise amino acid substitutions compared to thrombin and/or modified amino acids, especially at the termini.
NPAR agonists of the present invention include thrombin derivative peptides described in U.S. Patent Nos. 5,352,664 and 5,500,412. In one embodiment, the NPAR agonist of the present invention is a thrombin peptide derivative or a physiologically functional equivalent, i.e., a polypeptide with no more than about fifty amino acids, preferably no more than about thirty amino acids and having sufficient homology to the fragment of human thrombin corresponding to thrombin amino acids 508-530 (Ala-Gly-Tyr- Lys-Pro- Asp-Glu-Gly-Lys- Arg-Gly- Asp- Ala-Cys-Glu-Gly- Asp-Ser-Gly-Gly-Pro-Phe- VaI ; SEQ ID NO:6) that the polypeptide activates NPAR. The thrombin peptide derivatives or modified thrombin peptide derivatives described herein preferably have from about 12 to about 23 amino acid residues, more preferably from about 19 to about 23 amino acid residues. In another embodiment, the NPAR agonist of the present invention is a thrombin peptide derivative comprising a moiety represented by Structural Formula (I):
Asp-Ala-R (I).
R is a serine esterase conserved domain. Serine esterases, e.g., trypsin, thrombin, chymotrypsin and the like, have a region that is highly conserved. "Serine esterase conserved domain" refers to a polypeptide having the amino acid sequence of one of these conserved regions or is sufficiently homologous to one of these conserved regions such that the thrombin peptide derivative retains NPAR activating ability. A physiologically functional equivalent of a thrombin derivative encompasses molecules which differ from thrombin derivatives in particulars which do not affect the function of the thrombin receptor binding domain or the serine esterase conserved amino acid sequence. Such particulars may include, but are not limited to, conservative amino acid substitutions and modifications, for example, amidation of the carboxyl terminus, acetylation of the amino terminus, conjugation of the polypeptide to a physiologically inert carrier molecule, or sequence alterations in accordance with the serine esterase conserved sequences.
A domain having a serine esterase conserved sequence can comprise a polypeptide sequence containing at least 4-12 of the N-terminal amino acids of the dodecapeptide previously shown to be highly conserved among serine proteases (Asp-Xi -CyS-X2-GIy-ASp- Ser-Gly-Gly-Pro-X3-Val; SEQ ID NO: 13); wherein X1 is either Ala or Ser; X2 is either GIu or GIn; and X3 is Phe, Met, Leu, His, or VaI).
In one embodiment, the serine esterase conserved sequence comprises the amino acid sequence of SEQ ID NO: 14 (Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val) or a C-terminal truncated fragment of a polypeptide having the amino acid sequence of SEQ ID NO: 14. It is understood, however, that zero, one, two or three amino acids in the serine esterase conserved sequence can differ from the corresponding amino acid in SEQ ID NO: 14.
Preferably, the amino acids in the serine esterase conserved sequence which differ from the corresponding amino acid in SEQ ID NO: 14 are conservative substitutions, and are more preferably highly conservative substitutions. A "C-terminal truncated fragment" refers to a fragment remaining after removing an amino acid or block of amino acids from the C- terminus, said fragment having at least six and more preferably at least nine amino acids.
In another embodiment, the serine esterase conserved sequence comprises the amino acid sequence of SEQ ID NO: 15 (Cys-Xi-Gly-Asp-Ser-Gly-Gly-Pro-XrVal; Xi is GIu or GIn and X2 is Phe, Met, Leu, His or VaI) or a C-terminal truncated fragment thereof having at least six amino acids, preferably at least nine amino acids. In a preferred embodiment, the thrombin peptide derivative comprises a serine esterase conserved sequence and a polypeptide having a more specific thrombin amino acid sequence Arg-Gly-Asp-Ala (SEQ ID NO: 16). One example of a thrombin peptide derivative of this type comprises Arg-Gly-Asp-Ala-Cys-Xi-Gly-Asp-Ser-Gly-Gly-Pro-X2-Val (SEQ ID NO:1). Xi and X2 are as defined above. The thrombin peptide derivative can comprise the amino acid sequence of SEQ ID NO:6 (Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly- Asp-Ala-Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val) or an N-terminal truncated fragment thereof, provided that zero, one, two or three amino acids at positions 1-9 in the thrombin peptide derivative differ from the amino acid at the corresponding position of SEQ ID NO:6. Preferably, the amino acid residues in the thrombin peptide derivative which differ from the corresponding amino acid residues in SEQ ID NO: 6 are conservative substitutions, and are more preferably highly conservative substitutions. An 'W-terminal truncated fragment" refers to a fragment remaining after removing an amino acid or block of amino acids from the TV-terminus, preferably a block of no more than six amino acids, more preferably a block of no more than three amino acids.
Optionally, the thrombin peptide derivatives described herein can be amidated at the C-terminus and/or acylated at the N-terminus. In a specific embodiment, the thrombin peptide derivatives comprise a C-terminal amide and optionally comprise an acylated N- terminus, wherein said C-terminal amide is represented by -C(O)NRaRb, wherein Ra and Rb are independently hydrogen, a C1- C10 substituted or unsubstituted aliphatic group, or Ra and Rb, taken together with the nitrogen to which they are bonded, form a C1-C10 non-aromatic heterocyclic group, and said N-terminal acyl group is represented by RcC(O)-, wherein Rc is hydrogen, a C1-C10 substituted or unsubstituted aliphatic group, or a C1-C io substituted or unsubstituted aromatic group. In another specific embodiment, the N-terminus of the thrombin peptide derivative is free (i.e., unsubstituted) and the C-terminus is free (i.e., unsubstituted) or amidated, preferably as a carboxamide (i.e., -C(O)NH2). In a specific embodiment, the thrombin peptide derivative comprises the following amino acid sequence: Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Cys-Glu-Gly-Asp-Ser-Gly-Gly- Pro-Phe-Val (SEQ ID NO:6). In another specific embodiment, the thrombin peptide derivative comprises the amino sequence of Arg-Gly-Asp-Ala-Cys-Glu-Gly-Asp-Ser-Gly- Gly-Pro-Phe-Val (SEQ ID NO: 17). Alternatively, the thrombin peptide derivative comprises the amino acid sequence of SEQ ID NO: 18: Asp-Asn-Met-Phe-Cys-Ala-Gly-Tyr- Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val- Met-Lys-Ser-Pro-Phe. The thrombin peptide derivatives comprising the amino acids of SEQ ID NO: 6, 17, or 18 can optionally be amidated at the C-terminus and/or acylated at the N- terminus. Preferably, the N-terminus is free (i.e., unsubstituted) and the C-terminus is free (i.e., unsubstituted) or amidated, preferably a carboxamide (i.e., -C(O)NH2). It is understood, however, that zero, one, two or three amino acids at positions 1-9 and 14-23 in the thrombin peptide derivative can differ from the corresponding amino acid in SEQ ID NO:6. It is also understood that zero, one, two or three amino acids at positions 1-14 and 19-33 in the thrombin peptide derivative can differ from the corresponding amino acid in SEQ ID NO: 18. Preferably, the amino acids in the thrombin peptide derivative which differ from the corresponding amino acid in SEQ ID NO: 6 or SEQ ID NO: 18 are conservative substitutions, and are more preferably highly conservative substitutions. Alternatively, an N- terminal truncated fragment of the thrombin peptide derivative having at least fourteen amino acids or a C-terminal truncated fragment of the thrombin peptide derivative having at least eighteen amino acids is a thrombin peptide derivative to be used as an NP AR agonist. A "C-terminal truncated fragment" refers to a fragment remaining after removing an amino acid or block of amino acids from the C-terminus. An "N-terminal truncated fragment" refers to a fragment remaining after removing an amino acid or block of amino acids from the N-terminus. It is to be understood that the terms "C-terminal truncated fragment" and 'W-terminal truncated fragment" encompass acylation at the N-terminus and/or amidation at the C-terminus, as described above.
A preferred thrombin peptide derivative for use in the disclosed method comprises the amino acid sequence SEQ ID ΝO:2: Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly- Asp-Ala-Cys-Xi-Gly-Asp-Ser-Gly-Gly-Pro-X2-Val. Another preferred thrombin peptide derivative for use in the disclosed method comprises the amino acid sequence of SEQ ID NO: 19: Asp- Asn-Met-Phe-Cys-Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly- Asp- AIa- CyS-X1 -GIy- Asp-Ser-Gly-Gly-Pro-X2-Val-Met-Lys-Ser-Pro-Phe. Xi is GIu or GIn; X2 is Phe, Met, Leu, His or VaI. The thrombin peptide derivatives of SEQ ID NO:2 and SEQ ID NO: 19 can optionally comprise a C-terminal amide and/or acylated N-terminus, as defined above. Preferably, the N-terminus is free (i.e., unsubstituted) and the C-terminus is free (i.e., unsubstituted) or amidated, preferably as a carboxamide (i.e., -C(O)NH2). Alternatively, N- terminal truncated fragments of these preferred thrombin peptide derivatives, the N-terminal truncated fragments having at least fourteen amino acids, or C-terminal truncated fragments of these preferred thrombin peptide derivatives, the C-terminal truncated fragments having at least eighteen amino acids, can also be used in the disclosed method.
TP508 is an example of a thrombin peptide derivative and is 23 amino acid residues long, wherein the N-terminal amino acid residue Ala is unsubstituted and the COOH of the C-terminal amino acid VaI is modified to an amide represented by
-C(O)NH2 (SEQ ID NO:3). Another example of a thrombin peptide derivative comprises the amino acid sequence of SEQ ID NO: 6, wherein both N- and C-termini are unsubstituted ("deamide TP508"). Other examples of thrombin peptide derivatives which can be used in the disclosed method include N-terminal truncated fragments of TP508 (or deamide TP508), the N-terminal truncated fragments having at least fourteen amino acids, or C-terminal truncated fragments of TP5O8 (or deamide TP508), the C-terminal truncated fragments having at least eighteen amino acids.
As used herein, a "conservative substitution" in a polypeptide is the replacement of an amino acid with another amino acid that has the same net electronic charge and approximately the same size and shape. Amino acids with aliphatic or substituted aliphatic amino acid side chains have approximately the same size when the total number of carbon and heteroatoms in their side chains differs by no more than about four. They have approximately the same shape when the number of branches in their side chains differs by no more than one. Amino acids with phenyl or substituted phenyl groups in their side chains are considered to have about the same size and shape. Listed below are five groups of amino acids. Replacing an amino acid in a polypeptide with another amino acid from the same group results in a conservative substitution:
Group I: glycine, alanine, valine, leucine, isoleucine, serine, threonine, cysteine, and non-naturally occurring amino acids with C 1 -C4 aliphatic or C 1 -C4 hydroxyl substituted aliphatic side chains (straight chained or monobranched).
Group II: glutamic acid, aspartic acid and non-naturally occurring amino acids with carboxylic acid substituted C1-C4 aliphatic side chains (unbranched or one branch point).
Group III: lysine, ornithine, arginine and non-naturally occurring amino acids with amine or guanidino substituted C1-C4 aliphatic side chains (unbranched or one branch point).
Group IV: glutamine, asparagine and non-naturally occurring amino acids with amide substituted C1-C4 aliphatic side chains (unbranched or one branch point).
Group V: phenylalanine, phenylglycine, tyrosine and tryptophan. As used herein, a "highly conservative substitution" in a polypeptide is the replacement of an amino acid with another amino acid that has the same functional group in the side chain and nearly the same size and shape. Amino acids with aliphatic or substituted aliphatic amino acid side chains have nearly the same size when the total number of carbon and heteroatoms in their side chains differs by no more than two. They have nearly the same shape when they have the same number of branches in the their side chains. Examples of highly conservative substitutions include valine for leucine, threonine for serine, aspartic acid for glutamic acid and phenylglycine for phenylalanine. Examples of substitutions which are not highly conservative include alanine for valine, alanine for serine and aspartic acid for serine.
Modified Thrombin Peptide Derivatives
In one embodiment of the invention, the NPAR agonists are modified relative to the thrombin peptide derivatives described above, wherein cysteine residues of aforementioned thrombin peptide derivatives are replaced with amino acids having similar size and charge properties to minimize dimerization of the peptides. Examples of suitable amino acids include alanine, glycine, serine, or an 5-protected cysteine. Preferably, cysteine is replaced with alanine. The modified thrombin peptide derivatives have about the same biological activity as the unmodified thrombin peptide derivatives.
It will be understood that the modified thrombin peptide derivatives disclosed herein can optionally comprise C-terminal amides and/or iV-terminal acyl groups, as described above. Preferably, the N-terminus of a thrombin peptide derivative is free (i.e., unsubstituted) and the C-terminus is free (i.e., unsubstituted) or amidated, preferably as a carboxamide (i.e., -C(O)NH2).
In a specific embodiment, the modified thrombin peptide derivative comprises a polypeptide having the amino acid sequence of SEQ ID NO:4: Arg-Gly-Asp-Ala-Xaa-Xj- Gly-Asp-Ser-Gly-Gly-Pro-X2-Val, or a C-terminal truncated fragment thereof having at least six amino acids. More specifically, the thrombin peptide derivative comprises the amino acid sequence of SEQ ID NO:20: Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp- Ala-Xaa-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val or a fragment thereof comprising amino acids 10-18 of SEQ ID NO:20. Even more specifically, the thrombin peptide derivative comprises the amino acid sequence SEQ ID NO:5: Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys- Arg-Gly-Asp-Ala-Xaa-Xi-Gly-Asp-Ser-Gly-Gly-Pro-X2-Val, or a fragment thereof comprising amino acids 10-18 of SEQ ID NO:5. Xaa is alanine, glycine, serine or an S- protected cysteine. Xi is GIu or GIn and X2 is Phe, Met, Leu, His or VaI. Preferably Xi is GIu, X2 is Phe, and Xaa is alanine. One example of a thrombin peptide derivative of this type is a polypeptide having the amino acid sequence Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly- Lys-Arg-Gly- Asp-Ala- Ala-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe- VaI (SEQ ID NO:21). A further example of a thrombin peptide derivative of this type is the polypeptide H-AIa-GIy- Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Ala-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe- VaI-NH2 (SEQ ID NO:22). Zero, one, two or three amino acids in the thrombin peptide derivative differ from the amino acid at the corresponding position of SEQ ID NO:4, 20, 5, 21 or 22, provided that Xaa is alanine, glycine, serine or an ^-protected cysteine. Preferably, the difference is conservative.
In another specific embodiment, the thrombin peptide derivative comprises a polypeptide having the amino acid sequence SEQ ID NO:23: Asp-Asn-Met-Phe-Xbb-Ala- Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Xaa-Glu-Gly-Asp-Ser-Gly-Gly-Pro- Phe-Val-Met-Lys-Ser-Pro-Phe, or a fragment thereof comprising amino acids 6-28. More preferably, the thrombin peptide derivative comprises a polypeptide having the amino acid sequence SEQ ID NO: 24: Asp-Asn-Met-Phe-Xbb-Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys- Arg-Gly-Asp-Ala-Xaa-Xi-Gly-Asp-Ser-Gly-Gly-Pro-X2-Val-Met-Lys-Ser-Pro-Phe, or a fragment thereof comprising amino acids 6-28. Xaa and Xbb are independently alanine, glycine, serine or an S-protected cysteine. Xi is GIu or GIn and X2 is Phe, Met, Leu, His or VaI. Preferably Xi is GIu, X2 is Phe, and Xaa and Xbb are alanine. One example of a thrombin peptide derivative of this type is a polypeptide comprising the amino acid sequence Asp-Asn-Met-Phe-Ala-Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Ala-Glu- Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val-Met-Lys-Ser-Pro-Phe (SEQ ID NO:25). A further example of a thrombin peptide derivative of this type is the polypeptide H-Asp-Asn-Met- Phe-Ala-Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Ala-Glu-Gly-Asp-Ser- Gly-Gly-Pro-Phe-Val-Met-Lys-Ser-Pro-Phe-NH2 (SEQ ID NO:26). Zero, one, two or three amino acids in the thrombin peptide derivative can differ from the amino acid at the corresponding position of SEQ ID NO:23, 24, 25 or 26. Xaa and Xbb are independently alanine, glycine, serine or an S-protected cysteine. Preferably, the difference is conservative. An "S-protected cysteine" is a cysteine residue in which the reactivity of the thiol moiety, -SH, is blocked with a protecting group. Suitable protecting groups are are known in the art and are disclosed, for example, in T. W. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 3rd Edition, John Wiley & Sons, (1999), pp. 454-493, the teachings of which are incorporated herein by reference in their entirety. Suitable protecting groups should be non-toxic, stable in pharmaceutical formulations and have minimum additional functionality to maintain the activity of the thrombin peptide derivative. A free thiol can be protected as a thioether, a thioester, or can be oxidized to an unsymmetrical disulfide. Preferably the thiol is protected as a thioether. Suitable thioethers include, but are not limited to, S-alkyl thioethers (e.g., C1-C5 alkyl), and S-benzyl thioethers (e.g, cysteine-S- S-t-Bu). Preferably the protective group is an alkyl thioether. More preferably, the S- protected cysteine is an S-methyl cysteine. Alternatively, the protecting group can be: 1) a cysteine or a cysteine-containing peptide (the "protecting peptide") attached to the cysteine thiol group of the thrombin peptide derivative by a disulfide bond; or 2) an amino acid or peptide ("protecting peptide") attached by a thioamide bond between the cysteine thiol group of the thrombin peptide derivative and a carboxylic acid in the protecting peptide (e.g., at the C-terminus or side chain of aspartic acid or glutamic acid). The protecting peptide can be physiologically inert (e.g., a polyglycine or polyalanine of no more than about fifty amino acids optionally interrupted by a cysteine), or can have a desirable biological activity. The thrombin peptide derivatives or the modified thrombin peptide derivatives of the present invention can be mixed with a dimerization inhibitor for the preparation of a pharmaceutical composition comprising the thrombin peptide derivatives or the modified thrombin peptide derivatives of the present invention. Dimerization inhibitors can include chelating agents and/or thiol-containing compounds. An antioxidant can also be used in combination with the chelating agent and/or the thiol-containing compound.
A "chelating agent," as used herein, is a compound having multiple sites (two, three, four or more) which can simultaneously bind to a metal ion or metal ions such as, for example, lead, cobalt, iron or copper ions. The binding sites typically comprise oxygen, nitrogen, sulfur or phosphorus. For example, salts of EDTA (ethylenediaminetetraacetic acid) can form at least four to six bonds with a metal ion or metal ions via the oxygen atoms of four acetic acid moieties (-CH2C(O)O') and the nitrogen atoms of ethylenediamine moieties (>N-CH2-CH2-N<) of EDTA. It is understood that a chelating agent also includes a polymer which has multiple binding sites to a metal or metal ions. Preferably, a chelating agent of the invention is non-toxic and does not cause unacceptable side effects at the dosages being administered. As a chelating agent of the invention, a copper-chelating agent is preferable. A "copper-chelating agent" refers to a chelating agent which can bind to a copper ion or copper ions. Examples of the copper-chelating agent include ethylenediaminetetraacetic acid (EDTA), penicillamine, trientine, N, TV- diethyldithiocarbamate (DDC), 2,3,2'-tetraamine (2,3,2'-tet), neocuproine, N.N.N'.N- tetrakis(2-pyridylmethyl)ethylenediamine (TPEΝ), 1,10-phenanthroline (PHE), tetraethylenepentamine (TEPA), triethylenetetraamine and tris(2-carboxyethyl) phosphine (TCEP). Additional chelating agents are diethylenetriaminepentacetic acid (DTPA) and bathophenanthroline disulfonic acid (BPADA). EDTA is a preferred chelating agent. Typical amounts of a chelating agent present in the pharmaceutical compositions of the instant invention is in a range of between about 0.00001 % and about 0.1 % by weight, preferably between about 0.0001 % and about 0.05 % by weight. A "pharmaceutically acceptable thiol-containing compound," as used herein, is a compound which comprises at least one thiol (-SH) group and which does not cause unacceptable side effects at the dosages which are being administered. Examples of the pharmaceutically acceptable thiol-containing compound include thioglycerol, mercaptoethanol, thioglycol, thiodiglycol, cysteine, thioglucose, dithiothreitol (DTT) and dithio-bis-maleimidoethane (DTME). Typically, between about 0.001% and about 5% by weight, preferably between about 0.05% and about 1.0 % by weight of a pharmaceutically acceptable thiol-containing compound is present in the pharmaceutical compositions of the invention.
An "antioxidant," as used herein, is a compound which is used to prevent or reduce an oxidation reaction caused by an oxidizing agent such as oxygen. Examples of the antioxidant include tocopherol, cystine, methionine, glutathione, tocotrienol, dimethyl glycine, betaine, butylated hydroxyanisole, butylated hydroxytoluene, vitamin E, ascorbic acid, ascorbyl palmitate, thioglycolic acid and antioxidant peptides such as, for example, turmerin. Typically, between about 0.001% and about 10% by weight, preferably between about 0.01% and about 5%, more preferably between about 0.05% and about 2.0% by weight of an antioxidant is present in the pharmaceutical compositions of the invention. It is understood that certain chelating agents or thiol-containing compounds may also function as an antioxidant, for example, tris(2-carboxyethyl) phosphine, cysteine or dithiothreitol. Other types of commonly used antioxidants, however, do not contain a thiol group. It is also understood that certain thiol-containing compounds may also function as a chelating agent, for example, dithiothreitol. Other types of commonly used chelating agents, however, do not contain a thiol group. It is also understood that the pharmaceutical compositions of the instant invention can comprise more than one chelating agent, thiol- containing compound or antioxidant. That is, for example, a chelating agent can be used either alone or in combination with one or more other suitable chelating agents.
Thrombin Peptide Derivative Dimers
In some aspects of the present invention, the NPAR agonists of the methods are thrombin peptide derivative dimers. The dimers essentially do not revert to monomers and still have about the same biological activity as the thrombin peptide derivatives monomer described above. A "thrombin peptide derivative dimer" is a molecule comprising two thrombin peptide derivatives linked by a covalent bond, preferably a disulfide bond between cysteine residues. Thrombin peptide derivative dimers are typically essentially free of the corresponding monomer, e.g., greater than 95% free by weight and preferably greater than 99% free by weight. Preferably the polypeptides are the same and covalently linked through a disulfide bond.
The thrombin peptide derivative dimers of the present invention comprises the thrombin peptide derivatives described above. Specifically, thrombin peptide derivatives have less than about fifty amino acids, preferably less than about thirty-three amino acids. Thrombin peptide derivatives also have sufficient homology to the fragment of human thrombin corresponding to thrombin amino acid residues 508-530: Ala-Gly-Tyr-Lys-Pro- Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val (SEQ ID NO: 6) so that the polypeptide activates NPAR. The thrombin peptide derivative dimers described herein are formed from polypeptides typically having at least six amino acids and preferably from about 12 to about 33 amino acid residues, and more preferably from about 12 to about 23 amino acid residues.
In a specific embodiment, each thrombin peptide derivative comprising a dimer comprises a polypeptide having the amino acid sequence SEQ ID NO: 1 : Arg-Gly-Asp-Ala- Cys-Xi-Gly-Asp-Ser-Gly-Gly-Pro-X2-Val, or a C-terminal truncated fragment thereof comprising at least six amino acids. More specifically, each thrombin peptide derivative comprises the amino acid sequence of SEQ ID NO:6: Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly- Lys-Arg-Gly-Asp-Ala-Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val, or a fragment thereof comprising amino acids 10-18 of SEQ ID NO: 5. Even more specifically, the thrombin peptide derivative comprises the amino acid sequence SEQ ID NO:2: Ala-Gly-Tyr-Lys-Pro- Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Cys-Xi-Gly-Asp-Ser-Gly-Gly-Pro-X2-Val, or a fragment thereof comprising amino acids 10-18 of SEQ ID NO:2. X1 is GIu or GIn and X2 is Phe, Met, Leu, His or VaI. Preferably X1 is GIu, and X2 is Phe. One example of a thrombin peptide derivative of this type is a polypeptide comprising the amino acid sequence Ala-Gly- Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe- VaI (SEQ ID NO:6). A further example of a thrombin peptide derivative of this type is a polypeptide having the amino acid sequence H-Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys- Arg-GIy-ASp-AIa-CyS-GIu-GIy-ASp-Ser-GIy-GIy-PrO-Phe-VaI-NH2 (SEQ ID NO:3). Zero, one, two or three amino acids in the thrombin peptide derivative differ from the amino acid at the corresponding position of SEQ ID NO:6, 1, 2, or 3. Preferably, the difference is conservative.
One example of a thrombin peptide derivative dimer of the present invention is represented by Formula (IV):
Figure imgf000017_0001
In another specific embodiment, each thrombin peptide derivative comprising a dimer comprises a polypeptide comprising the amino acid sequence SEQ ID NO:27: AIa- Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro- Phe-Val-Met-Lys-Ser-Pro-Phe-Asn-Asn-Arg-Trp-Tyr, or a C-terminal truncated fragment thereof having at least twenty-three amino acids. More preferably, each thrombin peptide derivative comprises the amino acid sequence SEQ ID NO:28: Ala-Gly-Tyr-Lys-Pro-Asp- Glu-Gly-Lys-Arg-Gly-Asp-Ala-Cys-X,-Gly-Asp-Ser-Gly-Gly-Pro-X2-Val-Met-Lys-Ser- Pro-Phe-Asn-Asn- Arg-Trp-Tyr, or a C-terminal truncated fragment thereof comprising at least twenty-three amino acids. Xi is GIu or GIn and X2 is Phe, Met, Leu, His or VaI. Preferably Xi is GIu, and X2 is Phe. One example of a thrombin peptide derivative of this type is a polypeptide comprising the amino acid sequence Ala-Gly-Tyr-Lys-Pro-Asp-Glu- Gly-Lys-Arg-Gly-Asp-Ala-Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val-Met-Lys-Ser-Pro- Phe-Asn-Asn- Arg-Trp-Tyr (SEQ ID NO:27). A further example of a thrombin peptide derivative of this type is a polypeptide comprising the amino acid sequence H-Ala-Gly-Tyr- Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val- Met-Lys-Ser-Pro-Phe-Asn-Asn-Arg-Trp-Tyr-NH2 (SEQ ID NO:29). Zero, one, two or three amino acids in the thrombin peptide derivative differ from the amino acid at the corresponding position of SEQ ID NO:27, 28 or 29. Preferably, the difference is conservative.
NPAR Agonist Antibodies
A particular class of NPAR agonists includes antibodies and antigen-binding fragments that can both bind to and activate the non-proteolytically activated thrombin receptor (NPAR). Agonist antibodies that bind to thrombin receptors have been described in the art. For example, Frost et al. teach that a monoclonal antibody, TR-9, can mimic the effects of thrombin's high affinity interaction with the high affinity thrombin receptor (Frost, G.H., et al., J. Cell Biol. 105 (6 PT. 1):2551-58(1987)).
Antibodies or antigen-binding fragments thereof that are NPAR agonists can be found by their binding to a complementary peptide that is encoded by the complement of a nucleotide sequence encoding a portion of thrombin. See Molecular Recognition Theory below. The NPAR agonist antibody or antigen-binding fragment binds to a complementary peptide that is encoded by the complement of a nucleotide sequence encoding a thrombin. An NPAR agonist antibody or antigen-binding fragment can be found by its binding to a complementary peptide that is encoded by the complement of a nucleotide sequence encoding a portion of thrombin. In one embodiment, the thrombin or portion thereof (which is encoded by the sense or +RNA strand and is the complement of the RNA strand encoding the complementary peptide to which the antibody or antigen-binding fragment binds) is a mammalian thrombin or a portion of a mammalian thrombin. In another embodiment, the thrombin or portion thereof is a human thrombin or a portion of a human thrombin.
Antibodies or antigen-binding fragments thereof that bind to a complementary peptide, wherein the complementary peptide is encoded by the complement of a nucleotide sequence encoding thrombin or a portion thereof, can be NPAR agonists. In one embodiment, the portion of thrombin (which is encoded by the sense or +RNA strand and is the complement of the RNA strand encoding the complementary peptide to which the antibody or antigen-binding fragment binds) is a thrombin receptor binding domain or a portion thereof. As used herein, a thrombin receptor binding domain or a portion thereof is a segment of thrombin that is capable of selectively binding to the high-affinity non- proteolytically activated thrombin receptor (NPAR). Such thrombin receptor binding domains contain a portion of a domain (represented by amino acid residues 517-520 of human thrombin. The amino acid sequence of human prothrombin includes a sequence homologous to the tripeptide cell binding domain of fibronectin, Arg-Gly- Asp. In a particular embodiment, the thrombin receptor binding domain or portion thereof comprises the amino acid sequence AGYKPDEGKRGDACEGDSGGPFV (i.e., amino acids 508-530 of human thrombin (SEQ ID NO:6)). In another embodiment, the thrombin receptor binding domain or portion thereof is a portion of the thrombin receptor binding domain and comprises the amino acid sequence EGKRGDACEG (SEQ ID NO:7). As described herein, complementary peptides of domains of thrombin that are encoded by both the 5'-3' sequence of the antisense RNA strand and the 3'-5' sequence of the antisense RNA strand can be used to produce the NPAR agonist antibodies and antigen- binding domains of the invention. Therefore, in one embodiment, the complementary peptide (to which the antibodies and antigen-binding fragments bind) is encoded by the 5 '-3' sequence of the antisense RNA strand. In another embodiment, the complementary peptide is encoded by the 3 '-5' sequence of the antisense RNA strand.
In one example, a complementary peptide (to which the NPAR agonist antibodies and antigen-binding fragments of the invention bind) comprises the amino acid sequence KGSPTVTFTGIPCFPFIRLVTS (AC-23; SEQ ID NO:30). In another example, the complementary peptide comprises the amino acid sequence KGSPTVTFTGIPSFPFIRLVTS (23C53; SEQ ID NO:31). In yet another example, the complementary peptide comprises the amino acid sequence TFTGIPSFPF (C1053; SEQ ID NO:32). In still another example, the complementary peptide comprises the amino acid sequence RPMFGLLPFAPLRTLPLSPPGKQ [AC-23rev (SEQ ID NO:33), which is the complementary 5 '-3' peptide corresponding to AC-23]. In still a further example, the complementary peptide comprises the amino acid sequence LPF APLRTLP [C1053rev (SEQ ID NO: 12), which is the complementary 5 '-3' peptide corresponding to C 1053].
One example of an NPAR agonist antibody or an antigen-binding fragment thereof binds to a cysteine-altering complementary peptide comprising the amino acid sequence KGSPTVTFTGIPSFPFIRLVTS (23C53; SEQ ID NO:31). 23C53, which differs from AC- 23 by a single amino acid, is the complementary peptide of TP508, except that it possesses a single amino acid alteration from Cys to Ser.
In binding experiments using biotin-conjugated thrombin, thrombin was found to bind specifically to AC23 and 23C53. Half maximal binding of biotin-labeled thrombin to AC-23 was 4.8 ± 0.2 nM (n=2 ± SD).
Addition of TP508 inhibited specific binding of biotin-labeled thrombin to AC-23. Up to 60% of the binding of thrombin to AC-23 can be inhibited by the addition of TP508. Therefore, both thrombin and TP508 bind to the complementary peptide, AC-23. This suggests that AC-23 has a three-dimensional structure that is similar to the thrombin-TP508 receptor on cells. Antibodies to AC-23 and other complementary peptides of thrombin can therefore be used to characterize the thrombin binding site that is activated by TP5O8, and can be used in the therapeutic and other methods described herein.
In addition to the thrombin receptor binding domain, the stimulatory (agonistic) thrombin polypeptide derivatives possess a domain (represented by amino acid residues 519- 530 of human thrombin) with a high degree of homology to a number of serine esterases. However, the inhibitory (antagonistic) thrombin polypeptide derivatives do not include the serine esterase domain.
Thrombin peptide derivatives from amino acid residues 508-530 of human thrombin have been described for promoting thrombin receptor mediated cell stimulation. In addition, stimulatory (agonistic) thrombin polypeptide derivatives containing both fibronectin- and serine protease-homologous domains (residues 508 to 530 of human thrombin) bind to thrombin receptors with high-affinity and substitute for DIP-alpha-thrombin as an initiator of receptor occupancy-related mitogenic signals. (DIP-alpha-thrombin is a proteolytically inactive derivative of thrombin that retains receptor binding activity.) In contrast, inhibitory (antagonistic) thrombin polypeptide derivatives containing only the fibronectin-homologous domain (p517-520) (but not the serine protease-homologous domain) bind to the thrombin receptor without inducing mitogenesis. An intermediate thrombin peptide derivative (p519- 530) retains the ability to mediate mitogenesis but to a much lesser degree than p508-530.
Molecular Recognition Theory
Blalock and Smith (1984) observed that the hydropathic character of an amino acid residue is related to the identity of the middle letter of the triplet codon from which it is transcribed (Blalock, J.E., and Smith, E.M., Biochem. Biophys. Res. Commun. 12: 203-07 (1984)). Specifically, a triplet codon with thymine (T) as its middle base codes for a hydrophobic residue while adenine (A) codes for a hydrophilic residue. A triplet codon with middle bases cytosine (C) or guanine (G) encode residues that are relatively neutral and with similar hydropathy scores. Hydropathy is an index of the affinity of an amino acid for a polar environment; hydrophilic residues yielding a more negative score, while hydrophobic residues exhibit more positive scores. Kyte and Doolittle (1982) conceived a hydropathy scale that is widely used (Kyte, J., and Doolittle, R.F., J. MoI. Biol. 5:105-32 (1982)). The observed relationship between the middle base of a triplet codon and residue hydropathy entails that peptides encoded by complementary DNA will exhibit complementary, or inverted, hydropathic profiles. It was proposed that because two peptide sequences encoded in complementary DNA strands display inverted hydropathic profiles, they may form amphipathic secondary structures, and bind to one another (Bost, K.L., et al., Proc. Natl. Acad. Sci. USA 82: 1372-75 (1985)). Complementary peptides have been reported to form binding complexes with their "sense" peptide counterparts for a number of different systems (Root-Bernstein, R.S., and Holsworthy, D.D., J. Theor. Biol. 190: 107-19 (1988)). For example, Gho and Chae describe peptide antagonists of human angiogenin that are complementary peptides encoded by the antisense RNA sequence corresponding to the receptor binding site of angiogenin (Gho, Y. S. and Chae, CB. J. Biol. Chem. 272(39):24294-99 (1997)). Ghiso et al. describe a peptide complementary to a region of cystatin C that exhibits inhibitory activity (Ghiso, J., et al., Proc. Natl. Acad. Sci. USA 87(4): 1288-91 (1990)), and Bost and Blalock describe the production of anti-idiotypic antibodies by immunization with a pair of complementary peptides (Bost. K.L., and Blalock, J.E., J. Molec. Recognit. 1 :179-83 (1989)). The scope of this analysis for explaining the interactions between proteins was further developed by Blalock to propose a Molecular Recognition Theory (MRT) (Bost, K.L., et al., Proc. Natl. Acad. Sci. USA 82:1372-75 (1985); Blalock, J.E., Nature Med. 1 : 876-78 (1995)). This theory suggests that a "molecular recognition" code of interaction exists between peptides that are encoded by complementary strands of DNA, based on the observation that such peptides will exhibit inverted hydropathic profiles. MRT has proved successful for predicting particular binding interactors.
Blalock suggested that it is the linear pattern of amino acid hydropathy scores in a sequence (rather than the combination of specific residue identities), that defines the secondary structure environment. Furthermore, he suggested that sequences with inverted hydropathic profiles are complementary in shape by virtue of inverse forces that determine their steric relationships.
Deriving a Complementary Peptide in the 3 '-5' Reading Frame As a corollary to his original work, Blalock contended that as well as reading a complementary codon in the usual 5'-3' direction, reading a complementary codon in the 3'-5' direction would also yield amino acid sequences that displayed opposite hydropathic profiles (Bost, K.L., et al., Proc. Natl. Acad. Sci. USA 82: 1372-75 (1985)). This follows from the observation that the middle base of a triplet codon determines the hydropathy index of the residue it codes for, and therefore reading a codon in the reverse direction may change the identity, but not the hydropathic nature of the coded amino acid (Table 1).
TABLE 1 : The relationships between amino acids and the residues encoded in the complementary strand
Figure imgf000023_0001
Antibodies and Antibody Producing Cells
NPAR agonists as referred to herein encompass antibodies and antigen-binding fragments thereof that bind to the complementary peptides described herein and activate the non-proteolytically activated thrombin receptor. The antibodies as referred to herein can be polyclonal or monoclonal, and the term "antibody" is intended to encompass both polyclonal and monoclonal antibodies. The terms polyclonal and monoclonal refer to the degree of homogeneity of an antibody preparation, and are not intended to be limited to particular methods of production. In one embodiment, the antibody or antigen-binding fragment is a monoclonal antibody or antigen-binding fragment thereof. The term "monoclonal antibody" or "monoclonal antibody composition" as used herein, refers to a population of antibody molecules that contain only one species of an antigen binding site capable of immunoreacting with a particular epitope of a polypeptide of the invention. A monoclonal antibody composition thus typically displays a single binding affinity for a particular polypeptide of the invention with which it immunoreacts. The term "antibody" as used herein also encompasses functional fragments of antibodies, including fragments of chimeric, humanized, primatized, veneered or single chain antibodies. Functional fragments include antigen-binding fragments of antibodies that bind to the complementary peptides, wherein complementary peptides are encoded by the complement of a nucleotide sequence encoding thrombin or a portion thereof. For example, antibody fragments capable of binding to a complementary peptide, include, but are not limited to Fv, Fab, Fab' and F(ab')2 fragments. Such fragments can be produced by enzymatic cleavage or by recombinant techniques. For example, papain or pepsin cleavage can generate Fab or F(ab')2 fragments, respectively. Other proteases with the requisite substrate specificity can also be used to generate Fab or F(ab')2 fragments. Antibodies can also be produced in a variety of truncated forms using antibody genes in which one or more stop codons have been introduced upstream of the natural stop site. For example, a chimeric gene encoding a F(ab')2 heavy chain portion can be designed to include DNA sequences encoding the CHi domain and hinge region of the heavy chain.
Single chain antibodies, and chimeric, humanized or primatized (CDR-grafted), or veneered antibodies, as well as chimeric, CDR-grafted or veneered single chain antibodies, comprising portions derived from different species, are also encompassed by the term antibody. The various portions of these antibodies can be joined together chemically by conventional techniques, or can be prepared as a contiguous protein using genetic engineering techniques. For example, nucleic acids encoding a chimeric or humanized chain can be expressed to produce a contiguous protein. See, e.g., Cabilly et al., U.S. Patent No. 4,816,567; Cabilly et al., European Patent No. 0, 125,023 B 1 ; Boss et al., U.S. Patent No. 4,816,397; Boss et al., European Patent No. 0,120,694 B 1 ; Neuberger, M.S. et al., WO 86/01533; Neuberger, M.S. et al., European Patent No. 0,194,276 Bl; Winter, U.S. Patent No. 5,225,539; Winter, European Patent No. 0,239,400 Bl; Queen et al., European Patent No. 0 451 216 Bl; and Padlan, E.A. et al., EP 0 519 596 Al . See also, Newman, R. et al., BioTechnology, 10: 1455-1460 (1992), regarding primatized antibody, and Ladner et al., U.S. Patent No. 4,946,778 and Bird, R.E. et al., Science, 242: 423-426 (1988)) regarding single chain antibodies.
Humanized antibodies can be produced using synthetic or recombinant DNA technology using standard methods or other suitable techniques. Nucleic acid (e.g., cDNA) sequences coding for humanized variable regions can also be constructed using PCR mutagenesis methods to alter DNA sequences encoding a human or humanized chain, such as a DNA template from a previously humanized variable region (see e.g., Kamman, M., et al., Nucl. Acids Res., 17: 5404 (1989)); Sato, K., et al., Cancer Research, 53: 851-856 (1993); Daugherty, B.L. et al., Nucleic Acids Res., 19(9): 2471-2476 (1991); and Lewis, A.P. and J.S. Crowe, Gene, 101 : 297-302 (1991)). Using these or other suitable methods, variants can also be readily produced. In one embodiment, cloned variable regions can be mutated, and sequences encoding variants with the desired specificity can be selected (e.g., from a phage library; see e.g., Krebber et al., U.S. 5,514,548; Hoogenboom et al., WO 93/06213).
The antibody can be a humanized antibody comprising one or more immunoglobulin chains [e.g., an antibody comprising a complementarity-determining region (CDR) of nonhuman origin (e.g., one or more CDRs derived from an antibody of nonhuman origin)] and a framework region derived from a light and/or heavy chain of human origin (e.g., CDR- grafted antibodies with or without framework changes)]. In one embodiment, the antibody or antigen-binding fragment thereof comprises the light chain CDRs (CDRl, CDR2 and CDR3) and heavy chain CDRs (CDRl , CDR2 and CDR3) of a particular immunoglobulin. In another embodiment, the antibody or antigen-binding fragment further comprises a human framework region. Antibodies that are specific for a complementary peptide, wherein the complementary peptide is encoded by the complement of a nucleotide sequence encoding thrombin or a portion thereof, can be raised against an appropriate immunogen, such as a synthetic or recombinant complementary peptide or a portion thereof. Antibodies can also be raised by immunizing a suitable host (e.g., mouse) with transfected cells that express a complementary peptide. Such cells can also be used in a screen for an antibody that binds thereto (See e.g., Chuntharapai et al., J. Immunol., 152: 1783-1789 (1994); Chuntharapai et al., U.S. Patent No. 5,440,021).
Preparation of immunizing antigen, and polyclonal and monoclonal antibody production can be performed using any suitable technique (e.g., as exemplified herein). A variety of methods have been described (see e.g., Kohler et al., Nature, 256: 495-497 (1975) and Eur. J. Immunol. 6: 511-519 (1976); Milstein et al., Nature 266: 550-552 (1977); Koprowski et al., U.S. Patent No. 4,172,124; Harlow, E. and D. Lane, 1988, Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory: Cold Spring Harbor, NY); Current Protocols In Molecular Biology, Vol. 2 (Supplement 27, Summer '94), Ausubel, F. M. et al., Eds., (John Wiley & Sons: New York, NY), Chapter 11, (1991)). Generally, a hybridoma is produced by fusing a suitable immortal cell line (e.g., a myeloma cell line, such as SP2/0, P3X63Ag8.653 or a heteromyeloma) with antibody-producing cells. Antibody-producing cells can be obtained from the peripheral blood or, preferably the spleen or lymph nodes, of humans or other suitable animals immunized with a complementary peptide. The fused cells (hybridomas) can be isolated using selective culture conditions, and cloned by limiting dilution. Cells that produce antibodies with the desired specificity can be selected by a suitable assay (e.g., ELISA).
Other suitable methods of producing or isolating antibodies of the requisite specificity (e.g., human antibodies or antigen-binding fragments) can be used, including, for example, methods that select recombinant antibody from a library (e.g., a phage display library). Transgenic animals capable of producing a repertoire of human antibodies (e.g., Xenomouse® (Abgenix, Fremont, CA)) can be produced using suitable methods (see e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90: 2551-2555 (1993); Jakobovits et al., Nature, 362: 255-258 (1993)). Additional methods that are suitable for production of transgenic animals capable of producing a repertoire of human antibodies have been described (e.g., Lonberg et al., U.S. Patent No. 5,545,806; Surani et al., U.S. Patent No. 5,545,807; Lonberg et al., WO 97/13852).
The invention also encompasses bispecific antibodies, or functional fragments thereof (e.g., F(ab')2), which bind to a complementary peptide as described herein and at least one other antigen (e.g., a tumor antigen, a viral antigen). Bispecific antibodies can be secreted by triomas and hybrid hybridomas. Generally, triomas are formed by fusion of a hybridoma and a lymphocyte (e.g., antibody-secreting B cell) and hybrid hybridomas are formed by fusion of two hybridomas. Each of the fused cells (i.e., hybridomas, lymphocytes) produces a monospecific antibody. However, triomas and hybrid hybridomas can produce an antibody containing antigen-binding sites that recognize different antigens. The supernatants of triomas and hybrid hybridomas can be assayed for bispecific antibody using a suitable assay (e.g., ELISA), and bispecific antibodies can be purified using conventional methods, (see, e.g., U.S. Patent No. 5,959,084 (Ring et al.), U.S. Patent No. 5,141,736 (Iwasa et al.), U.S. Patent Nos. 4,444,878, 5,292,668, 5,523,210 (all to Paulus et al.) and U.S. Patent No. 5,496,549 (Yamazaki et al.)).
Methods of Treatment With NPAR Agonists
The present invention is directed to methods of promoting, stimulating or accelerating healing of a bone fracture in an osteopenic human comprising administering to the human a therapeutically effective amount of an NPAR agonist.
"Promoting, stimulating or accelerating healing of a bone fracture" means causing, after the administration of the NPAR agonist or composition comprising an NPAR agonist, compared to fractures not treated with the NPAR agonist, a decreased time to any one or more of the following: (1) trabecular bridging (e.g., at least 1%, 2%, 3%, 4%, 5%, 6%, 7% or 2-50%, or 2-40%, or 2-20% or 2-15%, or 2-10%, or 5-10% shorter in time for fractures treated with the NPAR agonist compared to that for untreated fractures); (2) overall radiographic healing (e.g., at least 1%, 2%, 5%, 7%, 10%, 11%, 12%, 13%, or 2-50%, or 5- 40%, or 5-30%, or 10-25%, or 10-20% shorter in time for fractures treated with the healing agent and untreated fractures); (3) cortical bridging (e.g., at least 1%, 2%, 5%, 7%, 10%, 12%, 13%, 14%, 15%, 16%, 17%, or 2-50%, or 5-40%, or 5-30%, or 10-25%, or 10-20% shorter in time for fractures treated with the NPAR agonist compared to that for untreated fractures); (4) clinical evaluation of fracture healing (e.g., at least 1%, 2%, 3%, 5%, 7%, 9%, 10%, 1 1%, 12% or 2-50%, or 5-40%, or 5-30%, or 10-25%, or 10-20% shorter in time for fractures treated with the NPAR agonist compared to that for untreated fractures); and (5) removal of immobilization devices (e.g., at least 1%, 2%, 3%, 4%, 5% or 2-50%, or 2-40%, or 2-20% or 2-15%, or 2-10%, or 2-8%, or 2-6% shorter in time for fractures treated with the NPAR agonist compared to that for untreated fractures).
As used herein, a "bone fracture" refers to a fracture in a bone where healing would occur without treatment, where healing would occur after immobilization or where healing would occur after bone fragments have been aligned (or reduction). As such, "bone fracture" includes hairline fractures, simple non-displaced fractures, and non-displaced complex fractures. It also includes displaced simple, displaced complex and non-union fractures, in which the bone fragments have been aligned. "Bone fracture" does not include gaps between bones where bone healing would not spontaneously occur (e.g. gaps typically greater than 0.5, 0.6, 0.7, 0.8, 0.9, 1.0 or 1.5 cm). Such gaps occur, for example, after removal of a bone tumor or cyst or before alignment of a non-union fracture. (See, for example, U. S. Patent No. 6.914,050, incorporated by reference by its entirety).
The disclosed methods can be used as a primary treatment method, where the method is used shortly after the occurrence of the bone fracture. The disclosed methods can also be used as a secondary treatment method, where the bone fractures have been previously treated, but have not properly healed. The disclosed methods are not limited to any particular kind of bone(s) or bone location(s). Examples of fractures which can be treated by the disclosed methods include fractures of the humerus, radius, ulna, femur, tibia, fibula, spine, pelvis, carpus, metacarpus, phalanx (hand), talus, calcaneus, navicular, cuneiform, cuboid, metatarsus, phalanx (foot), clavicle, scapula, or patella. Bone fractures at different bone segments can also be treated by the present methods. For example, the bone segments include proximal, diaphyseal, or distal for long bone. For the tibia and fibula, a fourth segment is known as malleolar segment. For the pelvis, the two segments are pelvic ring and acetabulum.
As used herein, "osteopenic human" or "human with osteopenia" refers to a human having decreased density of bone. Osteopenia is classified based on bone mineral density (BMD). The BMD is reported in units called "T-scores." In accordance with the present invention, an osteopenic human is a human with a T-score between and including -1.1 and -2.4 in the hip and/or spine. Osteopenic humans can be males or females. A "therapeutically effective amount" is the quantity of the NPAR agonist that results in a decreased time to any one or more of the following: (1) trabecular bridging; (2) overall radiographic healing; (3) cortical bridging; (4) clinical evaluation of fracture healing; and (5) removal of immobilization devices. The amount of the NPAR agonist administered will depend on the degree, severity, and type of the bone fracture, and the release characteristics of the pharmaceutical formulation. It will also depend on the human's health, size, weight, age, sex and tolerance to drugs. The NPAR agonists can be administered to the bone fracture site for 1, 2, 3, 4, 5, 6, or 7 or more times during the period in which the fracture is healing or daily during the period in which the fracture is healing. When administered more than once, the NPAR agonists are preferably administered at evenly spaced intervals; each dose can be the same or different, but is preferably the same. A dose delivered to the bone fracture site can be, for example, 0.1-500 μg, preferably 1- 50 μg of NPAR agonist, and is commonly 3, 5, 10, 30 or 50 μg. In one embodiment, the first administration of NPAR agonist is shortly (less than 11 days) after the occurrence of the bone fracture. In another embodiment, the NPAR agonist is administered after an closed or open reduction surgery (within 11 days). In another embodiment, the NPAR agonist is administered during an open reduction surgery. In another embodiment, the NPAR agonist is administered conincident with or before immobilization (within 3, 2, or 1 day).
The disclosed NPAR agonists can be administered by any suitable route, including, for example, by local introduction to the bone fracture by, for example, percutaneous injection. The NPAR agonist can be advantageously administered to the fracture in a sustained release formulation, or can be delivered by a pump or an implantable device, or implantable carrier, such as the polymers discussed below. "Administered to the bone fracture" means delivered between the fractured ends or on to the surfaces of the bones at the fracture, as seen, for example, by fluoroscopy. Alternatively, the point of delivery of the
NPAR agonist can be in sufficient proximity to the fractured ends or surfaces of the bones so that the agonist can diffuse and contact the fractured ends or surfaces, for example, within 1 cm of one or both fractured ends or surfaces of the bone.
In one embodiment, the disclosed method can be used in combination with immobilization devices such as a cast. In another embodiment, the disclosed method can be used in combination with measures that align the bone fracture, for example, reduction surgery. In another embodiment, the disclosed method can be used in combination with measures that fasten the bone fracture together, for example, through the use of a wire, a pin, a rod or a plate.
In another embodiment, the disclosed method can used to treat displaced simple, displaced complex or non-union fractures in combination with measures that re-align the bone fractures, typically through immobilization, reduction surgery or a combination thereof. Optionally, the fracture are held together through the use of a wire, a pin, a rod or a plate.
The NPAR agonists can be administered to the human in conjunction with an acceptable pharmaceutical carrier as part of a pharmaceutical composition. The formulation of the pharmaceutical composition will vary according to the mode of administration selected. Suitable pharmaceutical carriers may contain inert ingredients which do not interact with the compound. The carriers should be biocompatible, i.e., non-toxic, noninflammatory, non-immunogenic and devoid of other undesired reactions at the administration site. Examples of pharmaceutically acceptable carriers include, for example, saline, commercially available inert gels, or liquids supplemented with albumin, methyl cellulose or a collagen matrix. Further examples include sterile water, physiological saline, bacteriostatic saline (saline containing about 0.9% mg/ml benzyl alcohol), phosphate-buffered saline, Hank's solution, Ringer's-lactate and the like. Standard pharmaceutical formulation techniques can be employed, such as those described in Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. Pharmaceutical composition may include gels. Gels are comprising a base selected from an oleaginous base, water, or an emulsion-suspension base. To the base is added a gelling agent which forms a matrix in the base, increasing its viscosity to a semisolid consistency. Examples of gelling agents are hydroxypropyl cellulose, acrylic acid polymers, and the like. The active ingredients are added to the formulation at the desired concentration at a point preceding addition of the gelling agent or can be mixed after the gelation process.
In one embodiment, the NPAR agonists are administered in a sustained release formulation. Polymers are often used to form sustained release formulations. Examples of these polymers include poly α-hydroxy esters such as polylactic acid/polyglycolic acid homopolymers and copolymers, polyphosphazenes (PPHOS), polyanhydrides and poly(propylene fumarates).
Polylactic acid/polyglycolic acid (PLGA) homo and copolymers are well known in the art as sustained release vehicles. The rate of release can be adjusted by the skilled artisan by variation of polylactic acid to polyglycolic acid ratio and the molecular weight of the polymer (see Anderson, et al, Adv. Drug Deliv. Rev. 28:5 (1997), the entire teachings of which are incorporated herein by reference). The incorporation of poly(ethylene glycol) into the polymer as a blend to form microparticle carriers allows further alteration of the release profile of the active ingredient (see Cleek et al, J. Control Release 48:259 (1997), the entire teachings of which are incorporated herein by reference). Ceramics such as calcium phosphate and hydroxyapatite can also be incorporated into the formulation to improve mechanical qualities.
PPHOS polymers contain alternating nitrogen and phosphorous with no carbon in the polymer backbone, as shown below in Structural Formula (II):
Figure imgf000031_0001
The properties of the polymer can be adjusted by suitable variation of side groups R and R' that are bonded to the polymer backbone. For example, the degradation of and drug release by PPHOS can be controlled by varying the amount of hydrolytically unstable side groups. With greater incorporation of either imidazolyl or ethylglycol substituted PPHOS, for example, an increase in degradation rate is observed (see Laurencin et al., J Biomed Mater. Res. 27:963 (1993), the entire teachings of which are incorporated herein by reference), thereby increasing the rate of drug release.
Polyanhydrides, shown in Structural Formula (III), have well defined degradation and release characteristics that can be controlled by including varying amounts of hydrophobic or hydrophilic monomers such as sebacic acid and l,3-bis(p- carboxyphenoxy)propane (see Leong et al, J. Biomed. Mater. Res. /9:941 (1985), the entire teachings of which are incorporated herein by reference). To improve mechanical strength, anhydrides are often copolymerized with imides to form polyanhydride-co-imides. Examples of polyanhydride-co-imides that are suitable for orthopaedic applications are poly(trimellitylimido-glycine-co- 1 ,6-bis(carboxyphenoxy)hexane and pyromellityimidoalanine: 1 ,6-bis(p-carboxyphenoxy)hexane copolymers.
Figure imgf000032_0001
Poly(propylene fumarates) (PPF) are highly desirable biocompatible implantable carriers because they are an injectable, in situ polymerizable, biodegradable material. "Injectable" means that the material can be injected by syringe through a standard needle used for injecting pastes and gels. PPF, combined with a vinyl monomer (N-vinyl pyrrolidinone) and an initiator (benzoyl peroxide), forms an injectable solution that can be polymerized in situ. It is particularly suited for filling skeletal defects of a wide variety of sizes and shapes (see Suggs et al., Macromolecules 50.4318 (1997), Peter et al., J. Biomater. ScL Poly,. Ed. 10:363 (1999) and Yaszemski et al., Tissue Eng. 7:41 (1995), the entire teachings of which are incorporated herein by reference). The addition of solid phase components such as β- tricalcium phosphate and sodium chloride can improve the mechanical properties of PPF polymers (see Peter et al, J. Biomed. Mater. Res. 44:3\4 (1999), the entire teachings of which are incorporated herein by reference).
Alternatively, the pharmaceutical compositions can be administered to the site in the form of microparticles or microspheres. The microparticles are placed in contact or in close proximity to the bone fracture site either by surgically exposing the site and applying the microparticles on or in close proximity to the site by painting, pipetting, spraying, injecting or the like. Microparticles can also be delivered to the site by laparoscopy or by percutaneous injection. Compositions which are injectable include the solutions of poly(propylene fumarate) copolymers described above and pastes of calcium phosphate ceramics (see Schmitz et al. , J. Oral Maxillofacial Surgery 57:1122 (1999), the entire teachings of which are incorporated herein by reference). Injectable compositions can be injected directly to the bone fracture site.
Bone fractures are often accompanied by symptoms and infirmities such as pain and infection. In certain instances it may be advantageous to co-administer one or more additional pharmacologically active agents along with an NPAR agonist to address such issues. For example, managing pain and inflammation may require co-administration with analgesic or an anti-inflammatory agents. Managing infection may require co-administration with antimicrobial, antibiotic or disinfectant agents. Thrombin peptide derivatives and modified thrombin peptide derivatives can be synthesized by solid phase peptide synthesis (e.g., BOC or FMOC) method, by solution phase synthesis, or by other suitable techniques including combinations of the foregoing methods. The BOC and FMOC methods, which are established and widely used, are described in Merrifield, J. Am. Chem. Soc. #5:2149 (1963); Meienhofer, Hormonal Proteins and Peptides, CH. Li, Ed., Academic Press, 1983, pp. 48-267; and Barany and Merrifield, in The Peptides, E. Gross and J. Meienhofer, Eds., Academic Press, New York, 1980, pp. 3- 285. Methods of solid phase peptide synthesis are described in Merrifield, R.B., Science, 232: 341 (1986); Carpino, L.A. and Han, G. Y., J. Org. Chem., 37: 3404 (1972); and Gauspohl, H. et ah, Synthesis, 5: 315 (1992)). The teachings of these six articles are incorporated herein by reference in their entirety.
Thrombin peptide derivative dimers can be prepared by oxidation of the monomer. Thrombin peptide derivative dimers can be prepared by reacting the thrombin peptide derivative with an excess of oxidizing agent. A well-known suitable oxidizing agent is iodine.
A "non-aromatic heterocyclic group" as used herein, is a non-aromatic carbocyclic ring system that has 3 to 10 atoms and includes at least one heteroatom, such as nitrogen, oxygen, or sulfur. Examples of non-aromatic heterocyclic groups include piperazinyl, piperidinyl, pyrrolidinyl, morpholinyl, thiomorpholinyl. The term "aryl group" includes both carbocyclic and heterocyclic aromatic ring systems. Examples of aryl groups include phenyl, indolyl, furanyl and imidazolyl.
An "aliphatic group" is a straight chain, branched or cyclic non-aromatic hydrocarbon. An aliphatic group can be completely saturated or contain one or more units of unsaturation (e.g., double and/or triple bonds), but is preferably saturated, i.e., an alkyl group. Typically, a straight chained or branched aliphatic group has from 1 to about 10 carbon atoms, preferably from 1 to about 4, and a cyclic aliphatic group has from 3 to about 10 carbon atoms, preferably from 3 to about 8. Aliphatic groups include, for example, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, tert-butyl, pentyl, cyclopentyl, hexyl, cyclohexyl, octyl and cyclooctyl. Suitable substituents for an aliphatic group, an aryl group or a non-aromatic heterocyclic group are those which do not significantly lower therapeutic activity of the NPAR agonist, for example, those found on naturally occurring amino acids. Examples include -OH, a halogen (-Br, -Cl, -I and -F), -0(Re), -0-CO-(Re), -CN, -NO2, -COOH, =0, -NH2 -NH(Re), -N(Re)2. -COO(Re), -CONH2, -CONH(Re),-CON(Re)2, -SH, -S(Re), an aliphatic group, an aryl group and a non-aromatic heterocyclic group. Each Re is independently an alkyl group or an aryl group. A substituted aliphatic group can have more than one substituent.
The invention is illustrated by the following examples which are not intended to be limiting in any way.
EXAMPLE 1 : A PHASE 3 STUDY SHOWS THAT TP508 HAS BENEFICIAL EFFECTS
IN BONE FRACTURE HEALING FOR OSTEOPENIC WOMEN Study Design
A double blind, randomized, placebo-controlled Phase 3 clinical trial was carried out to evaluate the efficacy and safety of a single percutaneous injection of TP508 at 10 μg formulated in 20 mg/ml mannitol and 10 μM EDTA on the rate of healing in distal radius fractures. Adult females with unstable and/or displaced intra- or extra-articular fractures of the distal radius requiring closed reduction and stabilization with both cast and K-wires or with external fixation with or without K-wires were enrolled into two parallel study arms. One group of patients received a single percutaneous injection of TP508 at 10 μg/ml and the other group received a single percutaneous injection of placebo control. Qualified patients underwent closed reduction surgery and were treated with the study agent under fluoroscopic guidance at the time of surgery after final fracture reduction and immobilization. The randomization scheme was stratified by fracture classification: intra-articular vs. extraarticular. Patients were followed at weeks 1-8, 10, 12, and 26 weeks with an additional 52- week safety timepoint. Safety was assessed based on the incidence of treatment-emergent adverse events and laboratory changes. Time to immobilization removal was the primary efficacy endpoint in this trial. Secondary efficacy endpoints included clinical healing, blinded longitudinal radiographic evaluation, and functional outcome parameters of grip strength, range of motion, and the Patient-Rated Wrist Evaluation.
DXA Analysis
After enrollment in the Phase 3 trial began, a protocol amendment was prepared requiring patients to have a single bone mineral density (BMD) measurement of the proximal femur, lumbar spine, and contralateral forearm using dual-energy x-ray absorptiometry (DXA) not later than 6 months after study enrollment. Specifically, regions of interest (ROI) were: femoral neck, trochanter, total hip, Ll-4, ultradistal radius, one-third radius, and total radius. Investigators were not restricted with respect to the type of DXA system used. Quality assurance records were obtained from each center to document stability of the DXA system used. DXA systems were not cross-calibrated across all study centers.
Usable DXA scan data were obtained on 394 patients in this study. Among them, a total of 302 female patients had DXA scans. The coordination of scans, assessment of scan quality and accuracy, interface with DXA technicians, and DXA data analysis were conducted. T-scores, reflecting BMD relative to normative values, were tabulated for each patient.
To allow pooling of data for analyses, only T-scores from total hip and spine were used if generated from the NHANESIII standardized database for total hip and the manufacturer's reference database for spine. The basis of this decision was that previous work has documented > 90% diagnostic agreement across DXA systems from different manufacturers when using the NHANESIII standardized database for total hip and the manufacturer's reference database for spine (Kiebzak GM, Binkley N, Lewiecki EM, Miller PD (2007) Journal of Clinical Densitometry, in press; Binkley N, Kiebzak GM, Lewiecki EM, Kreuger D, Gangnon RE, Miller PD, Shepherd JA, Drezner MK (2005) Journal of Bone and Mineral Research 20(2): 195-201). T-scores from the radius were not used to classify patients due to substantial inter-manufacturer discrepancies and lack of a standardized database for radius (Kiebzak et al., (2004) Journal of Clinical Densitometry 7(2): 143-152). Classification of bone status was based on the lowest T-score from total hip or spine and defined as: normal, T-score > - 1.0; osteopenia, T-score between and including -1.1 and -2.4; osteoporosis, T-score < -2.5 (Assessment of Fracture Risk and its Application to Screening for Postmenopausal Osteoporosis (1994) WHO Technical Report Series 843; Hamdy RC, Petak SA, Lenchik L (2002) Journal of Clinical Densitometry 5(Suppl 1): Sl 1-17).
Statistical Methods
The study's primary, protocol-specified outcome was time to removal of immobilization. Baseline comparability of the two study groups, osteopenic patients treated with TP508 and osteopenic patients treated with placebo, was tested using t-tests and chi-squares. The primary analysis of the time-to-removal outcome was performed using a Cox proportional hazards model, with terms for treatment, type of fixation (cast or external fixator), dominance (injured hand was or was not dominant), intra- vs. extra-articular fracture, smoking status (current vs. not current), body mass index (BMI), age, and gender. Secondary analyses of time-to-healing outcomes (time to radiographic healing and time to clinical evaluation of healing) were performed using similar Cox models. A second model for each outcome added a term for the interaction of treatment group with intra- vs. extra- articular fracture. The assumption of proportional hazards was validated graphically. Analyses were performed with SAS statistical software, version 8.1.
Repeatedly measured, continuous outcomes (PRWE, grip strength, range of motion) were described by t-tests at each time point. Other exploratory analyses of these secondary outcomes were by repeated measures, random effects regression models (SAS/STAT PROC MIXED). Covariates for these models included the terms cited above plus a time term. For grip strength outcomes, additional covariates were the outcome values in the contralateral (uninjured) hand.
Results The efficacy of TP508 in the female osteopenic subset as well as comparisons among women in the three BMD categories was investigated.
Based on patients' DXA scans, analyses were stratified by BMD T-score (cutpoints defined in the section on DXA Analysis), thereby classifying the female pateints as normal, osteopenic, or osteoporotic (Table 1). In this study, 157 women (57% of 302 women) were classified as osteopenic, while 90 (30%) and 55 (18%) were classified as normal and osteoporotic, respectively. In this analysis, patients from both treatment groups were pooled.
There are large, highly significant differences among the three subsets of female patients; age, weight, and BMI are the most striking. Osteopenic women were older and smaller
(lighter, shorter, lower BMD) than women with normal BMD, and osteoporotic women differed in the same way compared to osteopenic women, with the latter group in an intermediate position (regarding age and size) between women with normal BMD and osteoporotic women.
Figure imgf000037_0001
Table 1. Descriptive Statistics of Demographic Outcomes Female Evaluable Patients with DXA Data (n = 302)
Table 2 displays demographic variables in osteopenic women by treatment, TP508 or placebo. In general, there was good balance between the TP5O8-treated and placebo- treated osteopenic females. Variables that might affect injury dynamics such as weight, BMI, and age, appear to have been well balanced between the randomization groups. The only significant difference between treatment groups is seen in the percentage of patients who were treated with external fixation (42% in the placebo group vs. 24% in the TP508 group). Additionally, an imbalance in the percentage of extra-articular fractures (42% in the placebo group vs. 54% in TP508 treated) was observed.
Figure imgf000038_0001
Table 2. Baseline Comparison of TP508 and Placebo Groups among Osteopenic Women (n = 157)
Time to Removal of Immobilization
Time to immobilization removal is defined as the time elapsed between the date of final fracture reduction (date of surgery) and the first study visit at which the investigator, based on clinical and radiographic assessments of healing, removes all rigid immobilization hardware used to stabilize the fracture.
As shown in Figure 1 and Table 3, among osteopenic women, statistically significant treatment effect was observed for the primary endpoint, time to immobilization removal (log-rank test, p-value 0.033; Cox model adjusted p = 0.033, hazard ratio 1.44).
Time to Clinical Evaluation of Healing Global clinical assessment of bone healing is based on the patient's assessment of pain at the fracture site using a 10cm pain Visual Analog Scale (VAS), the investigator's palpation of the fracture to evaluate the degree of motion, inflammation, and extent of edema, and a confirmation of clinical findings by a brief review of the radiographs.
In Table 3, time to clinical evaluation of healing (absence of any pain or motion at the fracture site upon palpation), another clinically meaningful measure of fracture healing, was significantly decreased in favor of TP508 treatment.
Figure imgf000039_0001
Table 3. Summary of exploratory analysis of time to healing outcomes in the female osteopenic subset (n = 157)
(1) Cox model estimate of the placebo-to-TP508 hazard ratio, adjusted for reduction method (Cast/Ex-fix vs. splint), injured hand dominant, fracture classification (extra-articular), body mass index, smoking status (current smoker vs. non-smoker/ex-smoker), and age.
Radiographic outcomes
A longitudinal radiographic assessment of fracture healing was performed after each enrolled study patient had completed the 26-week (6 month) timepoint. Radiographic progression of healing at each evaluable time point was based on:
• the presence or absence of radial/ulnar cortical bridging on the posteroanterior view
• the degree of trabecular bridging on the posteroanterior view rated as: o NONE: no radio-opacity visible o MINIMAL: blunting of the fracture line o MODERATE: small to moderate trabeculae present across fracture line o COMPLETE: continuous individual trabeculae present
• for intra-articular fractures, the degree of healing at the articular surface rated as: o NONE: visible gap o MINIMAL: increased radio-opacity visible o MODERATE: visible or no continuous line present o COMPLETE: continuous line present • Global assessment of radiographical healing rated as: o NONE: less than 25% healed o MINIMAL: approximately 25% to 50% healed o MODERATE: approximately 50% to 75% healed o COMPLETE: healed at approximately 75% or greater
In the osteopenic female population, strong, statistically significant differences in radiographic outcomes were observed between the TP508-treated and placebo-treated patients (Table 3). There were statistically significant treatment effects for two radiographic parameters: time to radial cortical bridging and time to overall radiographic healing.
For time to radial cortical bridging, the TP508-treated osteopenic women had a 17.5 percent shorter median healing time than placebo recipients (log-rank p- value 0.021, Cox model p-value 0.022, hazard ratio 1.48). For time to overall radiographic healing, TP508- treated osteopenic women showed a very statistically significant decrease compared to the placebo group (log-rank p-value 0.0024, Cox model p-value 0.0028, hazard ratio 1.64), as shown in FIG. 2. A 13% decrease in median time for overall radiographic healing was observed for TP508-treated osteopenic women compared to the placebo group.
Range of motion outcomes
Range of motion of the affected and contralateral limbs was evaluated by measurements of wrist extension, flexion, ulnar and radial deviation, pronation and supination commencing at the time of rigid immobilization removal and continuing at all subsequent postoperative evaluations. A goniometer, a protractor with movable arms that can measure an arc of movement around a joint, was used.
For osteopenic women, the data suggest earlier return to improved flexion and extension among TP508 treated patients, which may be due to earlier time to immobilization removal and earlier initiation of hand therapy. With this in mind, time to improved ROM was examined among osteopenic women using targets of 30 degrees and 45 degrees (roughly the 1st quartile and median in the distributions of either flexion or extension) to define improvement. The flexion result for 30 degrees, (Figure 3, log-rank p-value 0.0090, Cox model p- value 0.0096, hazard ratio 1.54) showed a significantly better outcome for TP508 treated patients. For 45 degrees, the same comparison was not significant (log-rank p-value 0.49, Cox model p-value 0.55, hazard ratio 1.11). Switching from flexion to extension, the 30- degree result was again significant (Figure 4, log-rank p-value 0.029, Cox model p-value 0.027, hazard ratio 1.45) and the 45-degree result was not (log-rank p-values 0.14, Cox model p-value 0.15, hazard ratio 1.28).
Grip strength
Grip strength in the affected and contralateral limb was assessed using a calibrated Jamar hand dynamometer, which is a hand instrument with high instrument stability. Grip strength was evaluated by recording the results of 3 grip strength trials for each of the hands with the dynamometer set at the appropriate grip setting for the patient. Upon the selection of the initial appropriate dynamometer setting, the same setting was used for the duration of the study for an individual patient. In addition, for each patient, the dynamometer setting was the same for both the affected and the contralateral limbs for the duration of the study. The measurement was performed for all patients commencing at the time of rigid immobilization removal and continuing at all subsequent postoperative evaluations. Grip strength was adjusted to balance for non-dominant versus dominant hand fractures.
Analysis of time to grip strength improvement (re-attainment) showed no significant or nearly significant effect for TP508 treated group compared with the placebo group.
Patient Rated Wrist Evaluation (PRWE) outcomes PRWE data were collected baseline and post-baseline at weeks 4, 6, 8, 10, 12 and 26.
No clear evidence of a TP508 effect was found.
In conclusion, statistically significant effects in the primary endpoint as well as certain secondary endpoints were observed for TP508 treatment in osteopenic females.
EXAMPLE 2: A PHASE 3 STUDY SHOWS THAT OSTEOPENIC WOMEN HAVE
SLOWER FRACTURE HEALING THAN NORMAL WOMEN In the same clinical study described in Example 1, 123 women received a single percutaneous injection of placebo control. Among these women, 50 women had BMD within the normal range and 73 women were characterized as osteopenic. As shown in FIG. 5, being osteopenic clearly slowed down fracture healing based on the primary endpoint with a very significant p value of 0.008.
In addition, a statistically significant effect with a p value of 0.012 was observed for osteopenia on fracture healing based on time to clinical evaluation of fracture healing, shown in FIG. 6.
The radiographic endpoints, such as time to radial cortical bridging, time to overall radiographic healing, time to trabeculae bridging, however, did not show this same statistically significant effect. However, there is a trend suggesting that osteopenic women require longer time to reach radiographic endpoints than normal women (FIGs 7-9).
While this invention has been particularly shown and described with references to example embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

Claims

CLAIMSWhat is claimed is:
1. A method of promoting healing of a bone fracture in an osteopenic human comprising administering to the patient a therapeutically effective amount of an agonist of the non-proteolytically activated thrombin receptor.
2. The method of Claim 1, wherein the agonist is administered directly to the bone fracture.
3. The method of Claim 1, wherein the agonist is administered alone, directly to the bone fracture.
4. The method of Claim 1 , wherein the agonist is a thrombin peptide derivative comprising the amino acid sequence Asp-Ala-R, wherein R is a serine esterase conserved sequence.
5. The method of Claim 4, wherein the thrombin peptide derivative comprises from about 12 to about 23 amino acids.
6. The method of Claim 5, wherein the serine conserved sequence comprises the amino acid sequence of Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val (SEQ ID NO: 14), or a C-terminal truncated fragment thereof having at least six amino acids, provided that zero, one, two or three amino acids in the serine esterase conserved sequence differ from the corresponding position of SEQ ID NO: 14.
7. The method of Claim 5, wherein the serine esterase conserved sequence comprises the amino acid sequence of Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val (SEQ ID NO: 14), or a C-terminal truncated fragment thereof having at least nine amino acids, provided that zero, one or two of the amino acids in the serine esterase conserved region are conservative substitutions of the corresponding amino acid in SEQ ID NO:14.
8. The method of Claim 5, wherein the serine esterase conserved sequence comprises the amino acid sequence of Cys-Xi -GIy- Asp-Ser-Gly-Gly-Pro-X2- VaI (SEQ ID NO: 15), or a C-terminus truncated fragment of SEQ ID NO: 15 having at least six amino acids, wherein Xi is GIu or GIn and X2 is Phe, Met, Leu, His or VaI.
9. The method of Claim 8, wherein the thrombin peptide derivative comprises the amino acid sequence Arg-Gly-Asp-Ala (SEQ ID NO: 16).
10. The method of Claim 5, wherein the thrombin peptide derivative comprises the amino acid sequence of Arg-Gly-Asp-Ala-Cys-Glu-Gly- Asp-Ser-Gly-Gly-Pro-Phe- VaI (SEQ ID NO: 17), or a C-terminal truncated fragment thereof having at least six amino acids, wherein zero, one, two, or three amino acids in the peptide differ from the corresponding position of SEQ ID NO: 17.
11. The method of Claim 10, wherein the thrombin derivative comprises a C-terminal amide and optionally comprises an acylated N-terminus, wherein said C-terminal amide is represented by -C(O)NR3Rb, wherein Ra and Rb are independently hydrogen, a C1-C]0 substituted or unsubstituted aliphatic group, or R3 and Rb, taken together with the nitrogen to which they are bonded, form a C1-C10 non-aromatic heterocyclic group, and said N-terminal acyl group is represented by RcC(O)-, where
Rc is hydrogen, a C1-C10 substituted or unsubstituted aliphatic group, or a C1-C10 substituted or unsubstituted aromatic group.
12. The method of Claim 10, wherein the thrombin peptide derivative comprises an N- terminus which is unsubstituted and a C-terminus which is unsubstituted or a C- terminal amide represented by -C(O)NH2.
13. The method of Claim 12, wherein the thrombin peptide derivative comprises a polypeptide comprising the amino acid sequence of Arg-Gly-Asp-Ala-Cys-Glu-Gly- Asp-Ser-Gly-Gly-Pro-Phe-Val (SEQ ID NO: 17), or a C-terminal truncated fragment thereof having at least six amino acids, wherein zero, one, or two of the amino acids in the peptide are conservative substitutions of the corresponding amino acid in SEQ
ID NO: 17.
14. The method of Claim 13, wherein the thrombin peptide derivative comprises a polypeptide having the amino sequence of Arg-Gly-Asp-Ala-Cys-Xi-Gly-Asp-Ser- Gly-Gly-Pro-X2-Val (SEQ ID NO:1), wherein X1 is GIu or GIn and X2 is Phe, Met, Leu, His or VaI.
15. The method of Claim 14, wherein Xi is GIu and X2 is Phe.
16. The method of Claim 12, wherein the thrombin peptide derivative comprises the amino acid sequence of Ala-Gly-Tyr-Lys-Pro- Asp-Glu-Gly-Lys-Arg-Gly- Asp- AIa-
Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val (SEQ ID NO: 6), an N-terminal truncated fragment of the thrombin peptide derivative having at least fourteen amino acids, or a C-terminal truncated fragment of the thrombin peptide derivative having at least eighteen amino acids, provided that zero, one, two or three amino acids at positions 1-9 and 14-23 in the thrombin derivative differ from the amino acid at the corresponding position of SEQ ID NO:6.
17. The method of Claim 12, wherein the thrombin peptide derivative comprises the amino acid sequence of Ala-Gly-Tyr-Lys-Pro- Asp-Glu-Gly-Lys-Arg-Gly- Asp- AIa- Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val (SEQ ID NO: 6), an N-terminal truncated fragment of the thrombin peptide derivative having at least fourteen amino acids, or a C-terminal truncated fragment of the thrombin peptide derivative having at least eighteen amino acids, provided that zero, one, or two of the amino acids at positions 1-9 and 14-23 in the thrombin derivative are conservative substitutions of the amino acid at the corresponding position of SEQ ID NO:6.
18. The method of Claim 17, wherein the thrombin peptide derivative comprises the amino acid sequence of Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly- Asp- AIa- Cys-X]-Gly-Asp-Ser-Gly-Gly-Pro-X2-Val (SEQ ID NO:2), an N-terminal truncated fragment of the thrombin peptide derivative having at least fourteen amino acids, or a C-terminal truncated fragment of the thrombin peptide derivative having at least eighteen amino acids, wherein Xi is GIu or GIn and X2 is Phe, Met, Leu, His or VaI.
19. A method of promoting healing of a bone fracture in an osteopenic human comprising administering to the patient a therapeutically effective amount of an agonist of the non-proteolytically activated thrombin receptor, wherein the agonist is the polypeptide H-Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Cys- Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe- VaI-NH2 (SEQ ID NO:3).
20. The method of Claim 19, wherein the bone fracture is a simple bone fracture.
21. The method of Claim 5, wherein the thrombin peptide derivative comprises the amino acid sequence of Arg-Gly-Asp-Ala-Xaa-Glu-Gly- Asp-Ser-Gly-Gly-Pro-Phe-
VaI (SEQ ID NO:39), or a C-terminal truncated fragment thereof having at least six amino acids, wherein zero, one, two, or three amino acids in the peptide differ from the corresponding position of SEQ ID NO: 39, provided that Xaa is alanine, glycine, serine, or an S-protected cysteine.
22. The method of Claim 21, wherein the thrombin peptide derivative comprises a C- terminal amide and optionally comprises and an acylated N-terminus, wherein said C-terminal amide is represented by -C(O)NR3Rb, wherein R3 and Rb are independently hydrogen, a C1-C10 substituted or unsubstituted aliphatic group, or R3 and Rb, taken together with the nitrogen to which they are bonded, form a C1-C10 non-aromatic heterocyclic group, and said N-terminal acyl group is represented by
R0C(O)-, where Rς is hydrogen, a C1-C10 substituted or unsubstituted aliphatic group, or a C1-C1o substituted or unsubstituted aromatic group.
23. The method of Claim 21 , wherein the thrombin peptide derivative comprises an N- terminus which is unsubstituted and a C-terminus which is unsubstituted or a C- terminal amide represented by -C(O)NH2.
24. The method of Claim 3 wherein the thrombin peptide derivative comprises the amino acid sequence of Arg-Gly- Asp- Ala-Xaa-Glu-Gly- Asp-Ser-Gly-Gly-Pro-Phe- VaI (SEQ ID NO: 39), or a C-terminal truncated fragment thereof having at least six amino acids, provided that zero, one or two of the amino acids in the polypeptide are conservative substitutions of the corresponding amino acid in SEQ ID NO:39.
25. The method of Claim 24, wherein Xaa is alanine.
26. The method of Claim 23, wherein the thrombin peptide derivative comprises the amino acid sequence of Arg-Gly-Asp-Ala-Xaa-Xi-Gly-Asp-Ser-Gly-Gly-Pro-X2-Val (SEQ ID NO:4), wherein Xi is GIu or GIn and X2 is Phe, Met, Leu, His or VaI.
27. The method of Claim 23, wherein the thrombin peptide derivative comprises the amino acid sequence Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-
Xaa-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val (SEQ ID NO:20), or a fragment thereof comprising amino acids 10-18 of SEQ ID NO:20, provided that zero, one or two amino acids in the thrombin peptide derivative differ from the amino acid at the corresponding position of SEQ ID NO:20.
28. The method of Claim 23, wherein the thrombin peptide derivative comprises the amino acid sequence Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala- Xaa-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val (SEQ ID NO:20), or a fragment thereof comprising amino acids 10-18 of SEQ ID NO:20, provided that zero, one or two amino acids in the thrombin peptide derivative are conservative substitutions of the amino acid at the corresponding position of SEQ ID NO:20.
29. The method of Claim 28, wherein the thrombin peptide derivative comprises the amino acid sequence of Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly- Asp- AIa- Xaa-Xi-Gly-Asp-Ser-Gly-Gly-Pro-X2-Val (SEQ ID NO:5) or a fragment thereof comprising amino acids 10-18 of SEQ ID NO:5, wherein Xj is GIu or GIn and X2 is Phe, Met, Leu, His or VaI.
30. The method of Claim 29, wherein Xaa is alanine.
31. The method of Claim 23, wherein the thrombin peptide derivative comprises the amino acid sequence of Ala-Gly-Tyr-Lys-Pro- Asp-Glu-Gly-Lys-Arg-Gly- Asp-Ala- Xaa-Xi-Gly-Asp-Ser-Gly-Gly-Pro-X2-Val (SEQ ID NO:5), wherein X, is GIu or GIn and X2 is Phe, Met, Leu, His or VaI.
32. The method of Claim 31 , wherein Xaa is alanine.
33. The method of Claim 31, wherein Xi is GIu and X2 is Phe.
34. The method of Claim 23, wherein the thrombin peptide derivative is the polypeptide H-Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Ala-Glu-Gly-Asp-Ser- Gly-Gly-Pro-Phe-Val-NH2 (SEQ ID NO:22).
35. The method of Claim 1, wherein the agonist is a peptide dimer comprising two thrombin peptide derivatives which, independently, comprise the amino acid sequence of SEQ ID NO: 17 (Arg-Gly-Asp-Ala-Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro- Phe-Val) or a C-terminal truncated fragment thereof having at least six amino acids, provided that zero, one, two, or three amino acids in the polypeptide differ from the corresponding position of SEQ ID NO: 17; said thrombin peptide derivatives optionally comprising a C-terminal amide; and said thrombin peptide derivatives optionally comprising an acylated N-terminus.
36. The method of Claim 35, wherein the dimer is essentially free of monomer.
37. The method of Claim 36, wherein the thrombin peptide derivatives are the same.
38. The method of Claim 37, wherein the thrombin peptide derivatives are covalently linked through a disulfide bond.
39. The method of Claim 38, wherein the thrombin peptide derivatives consist of from about 12 to about 23 amino acids.
40. The method of Claim 39, wherein the thrombin peptide derivatives comprise a C- terminal amide and optionally comprise an acylated N-terminus, wherein said C- terminal amide is represented by -C(O)NR3Rb, Ra and Rb are independently hydrogen, a C1-C10 substituted or unsubstituted aliphatic group, or R3 and Rb, taken together with the nitrogen to which they are bonded, form a C1-C10 non-aromatic heterocyclic group, and said N-terminal acyl group is represented by RcC(O)-, wherein R0 is hydrogen, a C1-C10 substituted or unsubstituted aliphatic group, or a Cj- C1o substituted or unsubstituted aromatic group.
41. The method of Claim 39, wherein the thrombin peptide derivatives each comprise an N-terminus which is unsubstituted; and a C-terminus which is unsubstituted or a C- terminal amide represented by -C(O)NH2.
42. The method of Claim 41 , wherein the thrombin peptide derivatives comprise the amino acid sequence of Arg-Gly-Asp-Ala-Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe- VaI (SEQ ID NO: 17) or a C-terminal truncated fragment thereof having at least six amino acids, provided that zero, one or two of the amino acids in the thrombin peptide derivatives are conservative substitutions of the corresponding amino acid in
SEQ ID NO: 17.
43. The method of Claim 41 , wherein the thrombin peptide derivatives comprise the amino acid sequence of Arg-Gly-Asp-Ala-Cys-Xi-Gly-Asp-Ser-Gly-Gly-Pro^-Val (SEQ ID NO:1), wherein Xi is GIu or GIn and X2 is Phe, Met, Leu, His or VaI.
44. The method of Claim 41 , wherein the thrombin peptide derivatives comprise the amino acid sequence Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Cys- Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val (SEQ ID NO:6), or a fragment thereof comprising amino acids 10-18 of SEQ ID NO:6, provided that zero, one, two or three amino acids in the thrombin peptide derivatives differ from the amino acid at the corresponding position of SEQ ID NO:6.
45. The method of Claim 41, wherein the thrombin peptide derivatives comprise the amino acid sequence Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Cys- Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val (SEQ ID NO:6), or a fragment thereof comprising amino acids 10-18 of SEQ ID NO:6, provided that zero, one, or two amino acids in the thrombin peptide derivatives are conservative substitutions of the amino acid at the corresponding position of SEQ ID NO:6.
46. The method of Claim 41 , wherein the thrombin peptide derivatives comprise the amino acid sequence Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Cys- Xi-Gly-Asp-Ser-Gly-Gly-Pro^-Val (SEQ ID NO:2), wherein Xi is GIu or GIn and X2 is Phe, Met, Leu, His or VaI or a fragment thereof comprising amino acids 10-18 of SEQ ID NO:2.
47. The method of Claim 41 , wherein the thrombin peptide derivatives comprise the amino acid sequence Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Cys- Xi-Gly-Asp-Ser-Gly-Gly-Pro-X2-Val (SEQ ID NO:2), wherein Xj is GIu or GIn and X2 is Phe, Met, Leu, His or VaI.
48. The method of Claim 47, wherein Xi is GIu and X2 is Phe.
49. The method of Claim 40, the thrombin peptide derivatives comprise the amino acid sequence H-Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys- Arg-Gly- Asp-Ala-Cys-X i -GIy-
Asp-Ser-Gly-Gly-Pro-X2- VaI-NH2 (SEQ ID NO:40), wherein X1 is GIu or GIn and X2 is Phe, Met, Leu, His or VaI.
50. The method of Claim 49, wherein Xi is GIu and X2 is Phe.
51. The method of Claim 1 , wherein the agonist is a peptide dimer comprising two thrombin derivatives, each with the amino acid sequence Ala-Gly-Tyr-Lys-Pro-Asp-
Glu-Gly-Lys-Arg-Gly-Asp-Ala-Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val (SEQ ID NO:6), wherein the thrombin peptide derivatives are covalently linked by a disulfide bond.
52. The method of Claim 1, wherein the agonist is a peptide dimer represented by the following structural formula:
Figure imgf000050_0001
53. The method of Claim 1, wherein the agonist is an antibody or antigen-binding fragment thereof that binds to a complementary peptide, wherein said complementary peptide is encoded by the complement of a nucleotide sequence encoding a portion of thrombin.
54. The method of Claim 1, wherein the human is a female.
55. The method of Claim 1 , wherein the bone fracture occurs in a bone selected from the group consisting of humerus, radius, ulna, femur, tibia, fibula, spine, pelvis, carpus, metacarpus, phalanx (hand), talus, calcaneus, navicular, cuneiform, cuboid, metatarsus, phalanx (foot), clavicle, scapula, and patella.
56. The method of Claim 1 , wherein the agonist is administered in combination with another therapeutic agent.
57. The method of Claim 1, wherein the bone fracture is a simple bone fracture.
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