Stimulation of cartilage growth with agonists of the non-proteolytically activated thrombin receptor

Abstract

Disclosed is a method of stimulating cartilage growth, repair or regeneration at a site in a subject in need of such growth, repair or regeneration. The method comprises the step of administering a therapeutically effective amount of an agonist of the non-proteolytically activated thrombin receptor to the site. Also disclosed is a method of stimulating the proliferation and expansion of chrondrocytes in vitro. The method comprises culturing chrondrocytes in the presence of a stimulating amount of an NPAR agonist.

1 . A method of stimulating cartilage growth or repair at a site in a subject in need of such growth or repair, said method comprising administering to the site a therapeutically effective amount of an agonist of the non-proteolytically activated thrombin receptor.  
     
     
         2 . The method of  claim 1  wherein the site is an arthritic joint.  
     
     
         3 . The method of  claim 1  wherein the site is being treated for cartilage damage or loss.  
     
     
         4 . The method of  claim 1  wherein the site is being treated for cartilage damage or loss due to traumatic injury.  
     
     
         5 . The method of  claim 1  said agonist comprises a thrombin receptor binding domain and a serine esterase conserved sequence.  
     
     
         6 . The method of  claim 5  wherein said agonist has between about 12 and about 23 amino acids.  
     
     
         7 . The method of  claim 5  wherein said agonist comprises a C-terminus amide.  
     
     
         8 . The method of  claim 5  wherein said agonist is unsubstituted at the C-terminus.  
     
     
         9 . The method of  claim 7  wherein the serine esterase conserved sequence comprises the amino acid sequence Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val (SEQ ID NO: 1), or a C-terminus 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: 1.  
     
     
         10 . The method of  claim 7  wherein the serine esterase conserved sequence comprises the amino acid sequence Cys-X 1 -Gly-Asp-Ser-Gly-Gly-Pro-X 2 -Val (SEQ ID NO: 2), wherein X 1  is Glu or Gln and X 2  is Phe, Met, Leu, His or Val), or a C-terminus truncated fragment of SEQ ID NO: 2, said fragment having at least six amino acids.  
     
     
         11 . The method of  claim 7  wherein said agonist is a thrombin peptide derivative comprising the amino acid sequence Arg-Gly-Asp-Ala (SEQ ID NO: 3).  
     
     
         12 . The method of  claim 11  wherein the thrombin peptide derivative comprises the amino acid sequence Arg-Gly-Asp-Ala-Cys-X 1 -Gly-Asp-Ser-Gly-Gly-Pro-X 2 -Val (SEQ ID NO: 4), wherein X 1  is Glu or Gln and X 2  is Phe, Met, Leu, His or Val.  
     
     
         13 . The method of  claim 7  wherein said agonist 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), or an N-terminal truncated fragment thereof, provided that zero, one, two or three amino acids at positions 1-9 in said agonist differ from the amino acid at the corresponding position of SEQ ID NO: 5.  
     
     
         14 . The method of  claim 7  wherein said agonist 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), or an N-terminal truncated fragment thereof, provided that zero, one or two amino acids at positions 1-9 in the thrombin peptide derivative are conservative substitutions of the amino acid at the corresponding position of SEQ ID NO: 6.  
     
     
         15 . The method of  claim 1  wherein said agonist is administered in a pharmaceutical composition additionally comprising an implantable, biocompatible carrier.  
     
     
         16 . The method of  claim 15  wherein the carrier comprises a polylactic acid/polyglycolic acid homopolymer or copolymer.  
     
     
         17 . A method of stimulating cartilage growth or repair at a site in a subject in need there such growth or repair, said method comprising administering to the site a therapeutically effective amount of a peptide comprising the 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: 5).  
     
     
         18 . A method of stimulating cartilage growth at an arthritic joint in a subject, said method comprising administering to the arthritic joint a therapeutically effective amount of a peptide comprising the 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: 5).  
     
     
         19 . A method of stimulating cartilage growth in a subject at a site being treated for cartilage loss, said method comprising administering to the site a therapeutically effective amount of a peptide comprising the 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: 5).  
     
     
         20 . A method of stimulating cartilage growth in a subject at a site being treated for cartilage loss due to traumatic injury, said method comprising administering to the site a therapeutically effective amount of a peptide comprising the 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: 5).  
     
     
         21 . A method of stimulating cartilage growth or repair at a site in a subject in need of such growth or repair, said method comprising administering to the site a therapeutically effective amount of a C-terminus amidated peptide comprising the 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).  
     
     
         22 . A method of stimulating cartilage growth at an arthritic joint in a subject, said method comprising to the arthritic joint a therapeutically effective amount of a C-terminus amidated peptide comprising the 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).  
     
     
         23 . A method of stimulating cartilage growth in a subject at a site being treated for cartilage loss, said method comprising administering to the site a therapeutically effective amount of a C-terminus amidated peptide comprising the 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).  
     
     
         24 . A method of stimulating cartilage growth in a subject at a site being treated for cartilage loss due to traumatic injury, said method comprising the step of administering to the site a therapeutically effective amount of a peptide comprising the 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).