WO2003061690A1 - Stimulation de la croissance osseuse et de la formation du cartilage au moyen de derives peptidiques de la thrombine - Google Patents

Stimulation de la croissance osseuse et de la formation du cartilage au moyen de derives peptidiques de la thrombine Download PDF

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WO2003061690A1
WO2003061690A1 PCT/US2002/001451 US0201451W WO03061690A1 WO 2003061690 A1 WO2003061690 A1 WO 2003061690A1 US 0201451 W US0201451 W US 0201451W WO 03061690 A1 WO03061690 A1 WO 03061690A1
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asp
ala
glu
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PCT/US2002/001451
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Darrell H. Carney
Roger S. Crowther
David J. Simmons
Jinping Yang
William R. Redin
Janet Stiernberg
John Bergmann
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The Board Of Regents, The University Of Texas System
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Priority to CA002511257A priority Critical patent/CA2511257A1/fr
Priority to PCT/US2002/001451 priority patent/WO2003061690A1/fr
Priority to EP02705842A priority patent/EP1467748A1/fr
Priority to AU2002239965A priority patent/AU2002239965B2/en
Priority to CNA028285689A priority patent/CN1622826A/zh
Priority to JP2003561633A priority patent/JP2005519067A/ja
Publication of WO2003061690A1 publication Critical patent/WO2003061690A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
    • A61L27/3817—Cartilage-forming cells, e.g. pre-chondrocytes
    • 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
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/18—Macromolecular materials obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A61L27/227—Other specific proteins or polypeptides not covered by A61L27/222, A61L27/225 or A61L27/24
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3839—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by the site of application in the body
    • A61L27/3843—Connective tissue
    • A61L27/3852—Cartilage, e.g. meniscus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3895—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells using specific culture conditions, e.g. stimulating differentiation of stem cells, pulsatile flow conditions
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0655—Chondrocytes; Cartilage
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K2035/126—Immunoprotecting barriers, e.g. jackets, diffusion chambers
    • A61K2035/128—Immunoprotecting barriers, e.g. jackets, diffusion chambers capsules, e.g. microcapsules
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    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/70—Enzymes
    • 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
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/30—Synthetic polymers
    • C12N2533/40—Polyhydroxyacids, e.g. polymers of glycolic or lactic acid (PGA, PLA, PLGA); Bioresorbable polymers

Definitions

  • Mammalian bone tissue has a remarkable ability to regenerate and thereby repair injuries and other defects.
  • bone growth is generally sufficient to bring about full recovery from most simple and hairline fractures.
  • fractures generally does not occur throughout large voids or spaces. Therefore, fractures cannot heal unless the pieces are in close proximity. If a significant amount of bone tissue was lost as a result of the injury, the healing process may be incomplete, resulting in undesirable cosmetic and/or mechanical outcomes. This is often the case with non-union fractures or with bone injuries resulting from massive trauma.
  • Tissue growth is also generally inadequate in voids and segmental gaps in bone caused, for example, by surgical removal of tumors or cysts.
  • osteoinductive proteins have been identified, isolated and expressed using recombinant technology. Examples include the bone morphogenic proteins (BMPs) disclosed in U.S Patent No. 5,902,705 and WO 95/16035.
  • BMPs bone morphogenic proteins
  • the use of recombinant proteins as therapeutic agents generally has a number of drawbacks, including the cost of manufacture, in vivo biodegradation and short shelf lives. Consequently, scientists are continuing to search for new osteoinductive agents which do not have the aforementioned shortcomings.
  • cartilage does not self-repair following injury.
  • Cartilage is an avascular tissue made up largely of cartilage specific cells, the chondrocytes, special types of collagen, and proteoglycans.
  • the inability of cartilage to self-repair after injury, disease, or surgery is a major limiting factor in rehabilitation of degrading joint surfaces and injury to meniscal cartilage.
  • Osteoarthritis the major degenerative disease of weight bearing joint surfaces, is caused by eroding or damaged cartilage surfaces and is present in approximately 25% of the over 50-year-old population. In the US more than 20 million people suffer from osteoarthritis, with annual healthcare costs of more than $8.6 billion.
  • the cost for cartilage repair from acute joint injury meniscal lesions, patellar surface damage and chondromalacia
  • new therapeutic approaches are needed to heal lesions of cartilage caused by degeneration or acute trauma.
  • chondrocytes isolated from articular cartilage respond to compounds which activate the non-proteolytic thrombin cell surface receptor (hereinafter "NPAR").
  • NPAR non-proteolytic thrombin cell surface receptor
  • chondrocytes express approximately 233,000 thrombin binding sites per cell with apparent affinities of approximately 0.1 nM (3000 sites) and 27 nM (230,000 sites) (Example 3).
  • the compound TP508 an agonist of the non-proteolytic thrombin receptor, stimulates proliferation of bovine chondrocytes in culture in the presence of thrombin as a co-mitogen
  • Example 4A stimulates by itself the proliferation of rat chondrocytes cultured in three dimensional matrix culture (Example 5A).
  • This same TP508 compound also stimulates proteoglycan synthesis as measured by the incorporation of 35 S sulfate in both bovine chondrocytes (Example 4B) and 3 -dimensional cultures of rat chondrocytes (Example 5B).
  • NPAR agonists can stimulate proliferation and matrix production in chondrocytes isolated from articular cartilage.
  • novel methods of stimulating chondrocyte growth in vivo and cartilage repair in a subject and novel delivery methods for delivering pharmaceutical compositions to articular defects to aid in surface repair and to prevent articular degradation are disclosed herein.
  • One embodiment of the present invention is a method of stimulating bone growth at a site in a subject in need of osteoinduction.
  • the method comprises the step of administering a therapeutically effective amount of an agonist of the non- proteolytically activated thrombin receptor to the site.
  • Another embodiment of the present invention is a pharmaceutical composition comprising an implantable, biocompatible carrier and an NPAR agonist such as a physiologically functional equivalent of a thrombin peptide derivative.
  • These methods of the present invention are directed at stimulating bone growth in a subject and can be used at sites where bone growth would not occur, absent treatment with autologous bone grafts or administration of bone growth factors.
  • the method involves the administration of agonists of the non-proteolytic thrombin receptor.
  • agonists include small peptides having homology to the segment between amino acid 508 and 530 of human prothrombin. These small peptides are inexpensive to prepare in bulk quantities and are osteoinductive at low dose.
  • their lyophilized form is stable for at least thirty months when stored at 5° C and at 60% relative humidity.
  • the present invention is directed to a method of stimulating cartilage growth, regeneration or repair at a site in a subject where cartilage growth, repair or regeneration is needed.
  • the method comprises the step of administering a therapeutically effective amount of an NPAR agonist, such as a physiologically functional equivalent of a thrombin peptide derivative to the site of injury.
  • an NPAR agonist such as a physiologically functional equivalent of a thrombin peptide derivative
  • a further embodiment of the present invention is directed to 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.
  • Ostoinduction refers to stimulating bone growth at a site within a subject at which little or no bone growth would occur if the site were left untreated. Sites which could therapeutically benefit from the induction of bone growth are referred to as "in need of osteoinduction". Examples include non-union fractures or other severe or massive bone trauma. It is noted that bone growth normally occurs at bone injuries such as simple or hairline fractures and well opposed complex fractures with minimal gaps without the need for further treatment. Such injuries are not considered to be "in need of osteoinduction”. Simple fracture repair appears to be quite different from the induction of bone formation required to fill non-union fractures, segmental gaps or bone voids caused, for example, by removal of a bone tumor or cyst.
  • Segmental gaps larger than 0.5 cm generally are in need of osteoinduction, whereas segmented gaps larger than 0.6 cm, 0.7 cm, 0.8 cm, 0.9 cm, 1.0 cm or 1.5 cm typically are in need of osteoinduction.
  • These cases require bone grafting or induction of new bone growth generally employing some type of matrix or scaffolding to serve as a bone growth substitute.
  • Induced bone growth can also be therapeutically beneficial at certain sites within a subject (referred to as "ectopic" sites) where bone tissue would not normally be found, such as a site in need of a bone graft or bone fusion. Fusions are commonly used to treat lower back pain by physically coupling one or more vertebrae to its neighbor.
  • the bone created by such a fusion is located at a site not normally occupied by bone tissue. Osteoinduction at these ectopic sites can act as a "graft substitute" whereby induced bone growth between the vertebrae takes the place of a graft and obviates the need for a second operation to harvest bone for the grafting procedure.
  • Induction of bone growth is also needed for treating acquired and congenital craniofacial and other skeletal or dental anomalies (see e.g., Glowacki et al, Lancet 1: 959 (1981)); performing dental and periodontal reconstructions where lost bone replacement or bone augmentation is required such as in a jaw bone; and supplementing alveolar bone loss resulting from periodontal disease to delay or prevent tooth loss (see e.g., NASAdsson et al, J. Periodontol, 66: 511 (1995)).
  • osteoarthritis or degenerative joint disease is a slowly progressive, irreversible, often monoarticular disease characterized by pain and loss of function.
  • the underlying cause of the pain and debilitation is the cartilage degradation that is one of the major symptoms of the disease.
  • Hyaline cartilage is a flexible tissue that covers the ends of bones and lies between joints such as the knee. It is also found in between the bones along the spine.
  • Cartilage is smooth, allowing stable, flexible movement with minimal friction, but is also resistant to compression and able to distribute applied loads.
  • surfaces of cartilage and exposed underlying bone become irregular. Instead of gliding smoothly, boney joint surfaces rub against each other, resulting in stiffness and pain. Regeneration of damaged cartilage and the growth of new cartilage at these arthritic sites would relieve the pain and restore the loss of function associated with osteoarthritis.
  • Cartilage damage can also occur from trauma resulting from injury or surgery. Sports injuries are a common cause of cartilage damage, particularly to joints such as the knee. Traumatic injury to cartilage can result in the same type of functional impairment. Therefore, sites in a subject with cartilage that has been damaged by trauma or disease are in need of treatment to restore or promote the growth of cartilage. Applicants have discovered that compounds which stimulate or activate the
  • NPAR NPAR agonists
  • NPAR agonists are osteoinductive.
  • compounds which stimulate or activate NPAR can stimulate chondrocytes to proliferate.
  • Chondrocytes are cells which make up about 1% of the volume of cartilage and which replace degraded matrix molecules to maintain the correct volume and mechanical properties of the tissue.
  • NPAR agonists can induce cartilage growth and repair when administered to sites needing cartilage growth and/or repair.
  • 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 its agonists to stimulate cell proliferation when added to fibroblasts in the presence of submitogenic concentrations of thrombin or molecules that activate protein kinase C or compete with 125 I-thrombin for high affinity binding to thrombin receptors, as disclosed in US Patent Nos. 5,352,664 and 5,500,412 and in Glenn et al, J. Peptide Research 1:65 (1988).
  • NPAR is to be distinguished from other thrombin binding proteins and the cloned family of proteolytically-activated receptors for thrombin, including the receptors PARl, PAR2, PAR3 and PAR4.
  • PARl possesses a specific thrombin cleavage site that allows thrombin cleavage to expose a new amino-terminus domain that acts as a tethered ligand folding back onto itself inducing its activation (see, Nu, et al, Cell. 64:1057 (1991)).
  • PAR2 has a similar mechanism for activation, but is principally activated by trypsin-like enzymes (see, Zhong, et al, J. Biol Chem.
  • PAR3 also has a similar mechanism of activation and appears to function as a second thrombin receptor in platelets (see, Ishihara, et al, Nature. 386:502 (1997)).
  • PAR4 has been detected in mouse megakaryocytes and studies suggest that it also functions in human platelets (see, Kahn, et al, Nature 394:690 (1998)). In contrast with these PAR receptors, activation of ⁇ PAR requires no proteolytic cleavage.
  • ⁇ PAR is distinct from PAR receptors: (1) a population of cells has been isolated that express fully functional PARl receptors, but are non-responsive to thrombin due to a defect in the ⁇ PAR signal transduction pathway (see, Kim, et al, J. Cell. Physiol. 160:573 (1994)); (2) neutrophils bind 1 5 I thrombin with high affinity and their chemotaxis is stimulated by proteolytically inactivated thrombin or ⁇ PAR agonists (see, Ramakrishnan and Carney, Mol Biol. Cell ⁇ :1993 (1993)), yet they do not express PARl (see Jenkins, et al, J. Cell Sci. 108:3059 (1995)); (3) HC9 fibroblasts over-express PARl, but do not bind thrombin with high affinity (see, Kim, D. Ph.D. Dissertation. The
  • NPAR agonists have distinct effects on gene expression from those of the PAR receptor agonist peptides (see, Sower, et. al, Experimental Cell Research 247: 422 (1999).
  • an NPAR agonist is a thrombin peptide derivative, 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 prothrombin amino acids 508-530 (SEQ ID NO. 5) that the polypeptide activates NPAR.
  • the thrombin peptide derivatives described herein preferably have between about 12 and 23 amino acids, more preferably between about 19 and 23 amino acids.
  • a thrombin peptide derivative comprises 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.
  • the serine esterase conserved sequence has the amino acid sequence of SEQ ID NO.
  • 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. More preferably, the serine esterase conserved sequence has the amino acid sequence of SEQ ID NO 2 (Cys-X r Gly-Asp-Ser-Gly-Gly-Pro-X 2 -Val; X, is Glu or Gln and X 2 is Phe, Met, Leu, His or Val) 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 3).
  • a thrombin peptide derivative of this type comprises Arg-Gly-Asp-Ala-Cys-X r Gly- Asp-Ser-Gly-Gly-Pro-X 2 -Val (SEQ ID NO 4).
  • X ! and X 2 are as defined above.
  • the thrombin peptide derivative comprises SEQ ID NO 4, it preferably has the amino acid sequence of SEQ ID NO 5 (Ala-Gly-Try-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 5.
  • the amino acids in the thrombin peptide derivative which differ from the corresponding amino acid in SEQ ID NO 5 are conservative substitutions, and are more preferably highly conservative substitutions.
  • An "N-terminal truncated fragment" refers to a fragment remaining after removing an amino acid or block of amino acids from the N-terminus, preferably a block of no more than six amino acids, more preferably a block of no more than three amino acids.
  • a physiologically functional equivalent of a thrombin derivative peptide encompasses molecules which differ from thrombin derivatives in particulars which do not affect the function of the peptide as an ⁇ PAR agonist.
  • Such particulars may include, but are not limited to, amino acid substitutions, as described herein, and modifications, for example, amidation of the carboxyl terminus, acylation 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.
  • thrombin derivative peptides are also within the scope of the invention.
  • such peptides can be amidated at the carboxyl terminus, acylated at the amino terminus or both.
  • the amino acid sequence of SEQ ID NO.: 3 is represented as the following physiologically functional equivalents: Ala-Gly-Try-Lys-Pro-Asp- Glu-Gly-Lys-Arg-Gly-Asp-Ala-Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val-NH 2 (SEQ ID NO.: 6), Ac-Ala-Gly-Try-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala- Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val (SEQ ID NO.: 7) or Ac-Ala-Gly-
  • R, ⁇ and R 2 individually are selected from the groups of H, C C 6 alkyl and, R j and R 2 together with the nitrogen to which they are bound form a non- aromatic heterocyclic ring such as pyrrolidinyl, piperazinyl, morphilinyl or piperdinyl.
  • R x and R 2 are preferably H.
  • "-Val-NH 2 means -NH-CH[-CH-(CH 3 ) 2 ]- CONH 2 .
  • R is the amino acid side chain and R j is a C,- C 6 alkyl branched and straight chained. R is preferably methyl (-CH 3 ).
  • TP508 is an example of a physiologically functional equivalent of a thrombin peptide derivative and has the amino acid sequence of SEQ ID NO 6.
  • a “conservative substitution” 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 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 the 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 C1-C4 aliphatic or C1-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 HI 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 IN 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” 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 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.
  • Example 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.
  • NPAR agonists include small organic molecules which bind and activate NPAR.
  • Agonists of this type can be conveniently identified with high through-put screening, e.g., with assays that assess 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 US Patent Nos. 5,352,664 and 5,500,412. The entire teachings for US Patent Nos. 5,352,664 and 5,500,412 are incorporated herein by reference.
  • NPAR agonist also includes compounds and combinations of compounds known to activate NPAR. Examples are disclosed in US Patent Nos. 5,352,664 and 5,500,412 and include thrombin, DIP-alpha-throinbin and the combination of DlP-alpha-thrombin with phorbol myristate acetate.
  • An implantable biocompatible carrier for use in the pharmaceutical compositions described herein functions as a suitable delivery or support system for the NPAR agonist utilized to stimulate bone growth.
  • a biocompatible carrier should be non-toxic, non-inflammatory, non-immunogenic and devoid of other undesired reactions at the implantation site. Suitable carriers also provide for release of the active ingredient and preferably for a slow, sustained release over time at the implantation site.
  • Suitable carriers include porous matrices into which bone progenitor cells may migrate. Osteogenic cells can often attach to such porous matrices, which can then serve as a scaffolding for bone and tissue growth. For certain applications, the carrier should have sufficient mechanical strength to maintain its three dimensional structure and help support the immobilization of the bone segments being united or grafted together. Porous matrices which provide scaffolding for tissue growth can accelerate the rate of bone growth and are said to be "osteoconductive”. Osteoconductive carriers are highly preferred for use in the pharmaceutical compositions described herein.
  • suitable osteoconductive carriers include collagen (e.g., bovine dermal collagen), fibrin, calcium phosphate ceramics (e.g., hydroxyapatite and tricalcium phosphate), calcium sulfate, guanidine-extracted allogenic bone and combinations thereof.
  • suitable carriers are commercially available, such as COLLOGRAFT (Collagen Corporation, Palo Alto, CA), which is a mixture of hydroxyapatite, tricalcium phosphate and fibrillar collagen, and LNTERPORE (Interpore International, Irvine CA), which is a hydroxyapatite biomatrix formed by the conversion of marine coral calcium carbonate to crystalline hydroxyapatite.
  • a number of synthetic biodegradable polymers can serve as osteoconductive carriers with sustained release characteristics. Descriptions of these polymers can be found in Behravesh et al, Clinical Orthopaedics 367:3118 (1999) and Lichun et al, Polymeric Delivery Vehicles for Bone Growth Factors in "Controlled Drug Delivery - Designing Technologies for the Future" Park and Mrsny eds., American Chemical Society, Washington, DC (2000). The entire teachings of these references are incorporated herein by reference. Examples of these polymers include poly - hydroxy esters such as polylactic acid/polyglycolic acid homopolymers and copolymers, polyphosphazenes (PPHOS), polyanhydrides and polypropylene fumarates).
  • PHOS polyphosphazenes
  • 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. DrugDeliv. 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).
  • PPHOS polymers contain alternating nitrogen and phosphorous with no carbon in the polymer backbone, as shown below in Structural Formula (IV):
  • 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 (V), 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 1,3- bis(p-carboxyphenoxy)propane (see Leong et al, J. Biomed. Mater. Res. 19:941 (1985), the entire teachings of which are incorporated herein by reference).
  • hydrophobic or hydrophilic monomers such as sebacic acid and 1,3- bis(p-carboxyphenoxy)propane (see Leong et al, J. Biomed. Mater. Res. 19:941 (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-l,6- bis(carboxyphenoxy)hexane and pyromellityimidoalanine: l,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 30:4318 (1997), Peter et al, J. Biomater. Sci. Poly,. Ed.
  • compositions of the present invention can be administered by implantation at a site in need of osteoinduction.
  • Implantation or “administration at a site” means in sufficient proximity to the site in need of treatment so that osteoinduction occurs (e.g., bone growth in the presence of the ⁇ PAR agonist but little or no growth in its absence) at the site when the ⁇ PAR agonist is released from the pharmaceutical composition.
  • the pharmaceutical compositions can be shaped as desired in anticipation of surgery or shaped by the physician or technician during surgery. It is prefereed to shape the matrix to span a tissue defect and to take the desired form of the new tissue. In the case of bone repair of a non-union defect, for example, it is desirable to use dimensions that span the non-union. In bone formation procedures, the material is slowly absorbed by the body and is replaced by bone in the shape of or very nearly the shape of the implant. Alternatively, the pharmaceutical compositions can be administered to the site in the form of microparticles or microspheres.
  • microparticles are placed in contact or in close proximity to the site in need of osteoinduction 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 endoscopy or by laparoscopy. The preparation of PLGA microparticles and their use to stimulate bone growth are described in Examples 1 and 2.
  • the pharmaceutical composition can be partially enclosed in a supporting physical structure such as a mesh, wire matrix, stainless steel cage, threaded interbody fusion cage and the like before administering to the site in need of osteoinduction.
  • a supporting physical structure such as a mesh, wire matrix, stainless steel cage, threaded interbody fusion cage and the like before administering to the site in need of osteoinduction.
  • 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 site in need of osteoinduction and can conveniently be used to fill voids and fuse bones without the need for invasive surgery.
  • NPAR agonists can also be administered by means other than implantation, for example, by applying a solution comprising the NPAR agonist in an acceptable pharmaceutical carrier directly to or in near proximity to the site. Administration of a solution can be conveniently accomplished, for example, by syringe, either through a surgical opening or by parenteral administration to the desired site. Standard pharmaceutical formulation techniques may be employed such as those described in Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA.
  • Suitable pharmaceutical carriers for parenteral administration include, for example, 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.
  • a NPAR agonist or an implantable pharmaceutical composition of the present invention can be used in conjuction with an implantable prosthetic device.
  • a therapeutically effective amount of the pharmaceutical composition can be disposed on the prosthetic implant on a surface region that is implantable adjacent to a site in need of osteoinduction.
  • the prosthetic device is constructed so as to continuously release the implantable pharmaceutical composition or NPAR agonist at a pre-determined rate.
  • the prosthesis may be made from a material comprising metal or ceramic. Examples of prosthetic devices include a hip device, a screw, a rod and a titanium cage for spine fusion.
  • this invention also provides a method for stimulating bone growth by implanting a prosthetic device into a site in need of osteoinduction in a subject.
  • the prosthetic is at least partially coated with an implantable pharmaceutical composition described hereinabove and implanted at a site in need of osteoinduction and maintained at the site for a period of time sufficient to permit stimulation of bone growth.
  • NPAR agonists used in the method of the present invention directed to regeneration of cartilage are typically administered as one component in a pharmaceutical composition to the site in need of cartilage growth, repair or regeneration.
  • Administering to the site in need of treatment means that the pharmaceutical composition containing the NPAR agonist is administered in sufficient proximity to the site in need of treatment so that cartilage growth or cartilage regeneration occurs at the site (e.g., a greater amount of cartilage growth or better quality of cartilage growth in the presence of the NPAR agonist than in its absence).
  • the pharmaceutical composition is a solution comprising the NPAR agonist and a suitable carrier. The solution is applied directly to or in near proximity to the site in need of treatment. Administration of the solution can be conveniently accomplished, for example, intraarticularly by syringe, in close proximity to the damaged tissue by syringe or through a surgical opening. Standard pharmaceutical formulation techniques may be employed such as those described in Remington's Pharmaceutical Sciences, Mack Publishing Company,
  • Suitable pharmaceutical carriers for include, for example, 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.
  • the pharmaceutical composition comprises the NPAR agonist and an implantable biocompatible carrier.
  • a biocompatible carrier should be non-toxic, non-inflammatory, non-immunogenic and devoid of other undesired reactions at the implantation site. Suitable carriers also provide for release of the active ingredient and preferably for a slow, sustained release over time at the implantation site.
  • a number of synthetic biodegradable polymers can serve as carriers with sustained release characteristics. Examples of these polymers include poly - hydroxy esters such as polylactic acid/polyglycolic acid copolymers and polyanhydrides.
  • polylactic acid/polyglycolic acid (PLGA) homo and copolymers discussed with regard to regeneration of bone tissue are also suitable for use as sustained release vehicles for the compounds utilized to treat cartilage.
  • polyanhydrides as shown in Structural Formula (N) can be used in the methods of treating collagen.
  • the pharmaceutical compositions can be shaped as desired in anticipation of surgery or shaped by the physician or technician during surgery. It is preferred to shape the matrix to span a tissue defect and to take the desired form of the new tissue. In the case of cartilage repair of large defects, it is desirable to use dimensions that span the defect.
  • the carrier is a porous matrix into which progenitor cells may migrate. Cells can often attach to such porous matrices, which can then serve as a scaffolding for tissue growth and thereby accelerate the rate of bone growth.
  • Chondrocytes can be applied to such matrices prior to implant to further accelerate healing.
  • Collagen or a collagen gel is an example of a suitable porous matrix.
  • the carrier is a viscous solution or gel that is injectable intraarticuarly or at the site in need of treatment.
  • Hyaluronic acid is an example of a carrier of this type.
  • Hyaluronic acid products are commercially available and include ORTHONISC developed by Anika, SY ⁇ NISC, developed by Biomatrix, HYALGA ⁇ , developed by Fidia and ARTZ, developed by Seikagaku.
  • Pluronic gel is another example of this type of carrier.
  • Pluronic gels are nontxoic block copolymers of ethylene oxide and propylene oxide. They exhibit thermosetting properties that allow them to exist as viscous liquids at room temperatures, but as gels at body temperatures.
  • Injectable compositions can be applied directly to the site in need of treatment without the need for invasive surgery.
  • Polymers of poly(ethylene oxide) and copolymers of ethylene and propylene oxide are also suitable as injectable matrices (see Cao et al, J. Biomater. Sci 9:475 (1998) and Sims et al, Plast Reconstr.Surg. P5/843 (196), the entire teachings of which are incorporated herein by reference).
  • ⁇ PAR agonists can be used to accelerate the growth or to maintain the functionality of isolated chondrocytes.
  • NPAR agonists can be added to tissue culture medium to stimulate proliferation and provide for more rapid proliferation and/or to prevent apoptotic death or senescence of cells often encountered when primary cell isolates are place in culture.
  • tissue culture medium to stimulate proliferation and provide for more rapid proliferation and/or to prevent apoptotic death or senescence of cells often encountered when primary cell isolates are place in culture.
  • NPAR agonists could be used to maintain the differentiated functionality of chondrocytes in culture.
  • NPAR agonists can be used alone in standard defined tissue culture medium or as a supplement to tissue culture medium containing serum or other growth factor to provide additive or synergistic effects on the in vitro production or maintenance of chondrocytes.
  • a sufficient quantity of the NPAR agonist is added to the culture to provide more rapid growth or to maintain greater functionality of the chondrocytes than in the absence of the agonist, i.e., a "stimulatory amount".
  • a sufficient quantity of the NPAR agonist is added to the culture to provide more rapid growth or to maintain greater functionality of the chondrocytes than in the absence of the agonist, i.e., a "stimulatory amount”.
  • a sufficient quantity of the NPAR agonist is added to the culture to provide more rapid growth or to maintain greater functionality of the chondrocytes than in the absence of the agonist, i.e., a "stimulatory amount”.
  • a "therapeutically effective amount” is the quantity of NPAR agonist which results in bone growth where little or no bone growth would occur in the absence of the agonist.
  • the agonist is administered for a sufficient period of time to achieve the desired therapeutic or cosmetic effect, i.e., sufficient bone growth.
  • the amount administered will depend on the amount of bone growth that is desired, the health, size, weight, age and sex of the subject and the release characteristics of the pharmaceutical formulation.
  • between about 1 ⁇ g per day and about 1 mg per day of NPAR agonist is administered by continuous release or by direct application to the site in need of bone growth.
  • a “therapeutically effective amount” is the quantity of NPAR agonist (or chondrocytes) which results in greater cartilage growth or repair in the presence of the NPAR agonist than in its absence.
  • a “therapeutically effective amount” is the quantity of NPAR agonist (or chondrocytes) which results in alleviation of the pain and/or lack of function associated with the cartilage damage.
  • the agonist (or chondrocytes) is admimstered for a sufficient period of time to achieve the desired therapeutic or effect. The amount admimstered will depend on the amount of cartilage growth that is desired, the health, size, weight, age and sex of the subject and the release characteristics of the pharmaceutical formulation.
  • NPAR agonist typically between about 0.1 ⁇ g per day and about 1 mg per day of NPAR agonist (preferably between about 5 ⁇ g per day and about 100 ⁇ g per day) is administered by continuous release or by direct application to the site in need of carilage growth or repair.
  • Chondrocytes cultured in the presence of an NPAR agonists can also be used to treat cartilage damage by administering a therapeutically effective amount of the chondrocytes to the site in need of treatment.
  • "therapeutically effective” also means which results in greater cartilage growth or repair with the treatment than in its absence.
  • the administration of chondrocytes to treat cartilage damage is described in US Patent No. 4,846,835, the entire teachings of which are incorporated herein by reference.
  • a "subject” is preferably a human, but can also be an animal in need of treatment, e.g., companion animals (e.g., dogs, cats, and the like), farm animals (e.g., cows, pigs, horses and the like) and laboratory animals (e.g., rats, mice, guinea pigs and the like).
  • companion animals e.g., dogs, cats, and the like
  • farm animals e.g., cows, pigs, horses and the like
  • laboratory animals e.g., rats, mice, guinea pigs and the like.
  • 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. Chem. Soc. ⁇ ° ⁇ °.-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.
  • Polyvinyl alcohol (0.3% in water) was added to the emulsion with further vortexing to form the second emulsion (O/W), thereby forming a double emulsion: an O/W emulsion comprised of PLGA droplets, and within those droplets, a second disperse phase consisting of TP508 in water.
  • O/W emulsion comprised of PLGA droplets
  • a second disperse phase consisting of TP508 in water.
  • the PLGA droplets formed discrete microspheres containing cavities holding TP508.
  • a 2% isopropyl alcohol solution was added. The particles were collected by centrifugation, and then lyophilized to remove residual moisture.
  • the composition of the matrix was varied to form microspheres with different release kinetics (Table 1).
  • the mean diameter of the microspheres was measured in a Coulter counter and the drug entrapment efficiency was measured by spectrophotometric assay at 276 nm following dissolution of a weighed sample of microspheres in methylene chloride and extraction of the released drug into water (Table 2).
  • TP508 release from the different PLGA matrices 20 mg of microspheres were placed in 1.0 ml of PBS contained in 1.5 ml polypropylene microcentrifuge tubes. Tubes were incubated at 37°C and shaken at 60 rpm. At various times, the tubes were centrifuged and the supernatant containing released TP508 was removed and frozen for subsequent analysis. Fresh PBS was added to the microspheres and incubation was continued. TP508 in the supernatant was measured by absorbance at 276 nm. For each formulation, quadruplicate release determinations were performed. Formulations B and D showed no detectable drug release during 28 days of incubation at 37°C.
  • Example 2 - PLGA Microspheres Containing TP508 Induce Bone Formation in Large (1.5 cm) Defects in Rabbit Ulna A 1.5 cm segmental defect was created in each ulna of 20 male New Zealand rabbits. These bilateral ulnar osteotomies were created exactly the same size by using a small metal guide to direct the cutting blade of the oscillating microsaw. Each rabbit acted as its own control; thus the left defect was filled with microspheres that did not contain TP508, while the right defect was filled with microspheres containing 100 or 200 ⁇ g TP508 (10 animals/group). The microspheres were prepared as described in Example 1. Rabbits given bilateral ulnar osteotomies were randomly divided into two groups.
  • the first group received 100 ⁇ g of TP508 in microspheres (30 mg) in the right limb and microspheres alone in the left limb.
  • the second group was treated similarly, but received 200 ⁇ g of TP508.
  • These different doses were achieved by mixing TP508-containing and TP508-devoid microspheres in different proportions. Animals were x- rayed at two week intervals, beginning at week three, and sacrificed at nine weeks.
  • Table 3 Torsion testing of segmental defects treated with 100 ⁇ g TP508.
  • Table 4 Torsion testing of segmental defects treated with 200 ⁇ g TP508.
  • rat articular chondrocytes Primary cultures of rat articular chondrocytes were isolated and prepared for in vitro analysis using established methods (see Kuettner, K E., et.al.,J. Cell Biology 93: 743-750, 1982). Briefly, cartilage pieces were dissected from the shoulder of rats and the pieces were digested with trypsin for one hour and with collagenase for three hours in tissue culture medium (DMEM) at 37 C with stirring. The cells were plated in flasks at high density (50,000 cells/cm sq.) and were culture in DMEM containing antibiotics an ascorbic acid at 37° C in an atmosphere of 5% CO 2 .
  • DMEM tissue culture medium
  • thrombin receptor binding assays as disclosed in US Patent 5,352,664 and Carney, DH and Cunningham, DD, Cell 15:1341-1349, 1978. Briefly, highly purified human thrombin was iodinated and added to cultures of chondrocytes with or without unlabeled thrombin to correct for nonspecific binding. By incubating cells with different concentrations of labeled thrombin and measuring the amount of thrombin bound to cells and the amount of free thrombin in the medium it is possible to estimate the number of receptors per cell and the affinity of thrombin for that binding site. Scatchard analysis of the labeled thrombin binding from three separate experiments suggest that rat chondrocytes express an average of 3000 very high affinity binding sites (100 pM affinity) and 230,000 high affinity sites (27 nM).
  • bovine chondrocytes Primary cultures of bovine chondrocytes were prepared using the procedure described for rat chondrocytes in Example 1. The cultures were subcultured into 24 well plastic dishes at a low density and placed in 1% serum. Addition of the NPAR agonist TP508 to these cultures at concentrations of 1.0 or 10 ⁇ g/ml by itself did not stimulate cell proliferation. In contrast, addition of these concentrations of TP508 together with a small amount of thrombin co-mitogen, resulted in a small, but significant (p ⁇ 0.05) increase in cell number relative to that seen in thrombin alone after three days in culture.
  • bovine chondrocytes were seeded into 96 well plates at a density of 2 x 105 cells per well and cultured in DMEM with 10% fetal calf serum. After establishment of these multi-layer cultures, the medium was replaced daily with DMEM containing 1% serum with indicated concentrations of TP508 from 1 to 100 ⁇ g per ml (Table 5). After 6 days in culture with daily changes of culture medimn with or without TP508, 35 S sulfate was added to the medium and incubation continued for an additonal 24 hours.
  • Rat articular chondrocytes were isolated from slices of rat articualar shoulder cartilage utilizing trypsin and collagenase digestions as described in Example 3. Preparations of chondrocyte "3 -dimensional" alginate bead cultures were established using established techniques as described by Guo et. al., (Conn. Tiss. Res. 19:277- 297, 1998). Following removal of cells from tissue culture flasks with trypsin, the cells were suspended in an alginate gel (1.2% w/v) and slowly expressed through a 22 gauge needle in a dropwise fashion into 102 mM CaCl 2 .
  • NPAR agonist TP508 on chondrocyte cell proliferation after three days in 3 -dimensional alginate culture was determined by removing beads from 35 mm dishes, washing them with 0.9% saline, and dissolving the alginate beads by adding 1 ml of 55 mM sodium citrate, 0.15 M NaCl at 37° C for 10 minutes. Cell number was determined by diluting the 1 ml of dissolved beads 1:10 with phosphate buffered saline (PBS) and counting the cells with a Z-series Coulter Counter. As shown in Table 6, TP508 by itself stimulated proliferation of chondrocytes in 3 dimensional culture.
  • PBS phosphate buffered saline
  • 3-dimensional alginate cultures were prepared as described above and assayed for incorporation of [ 35 S]-sulfate. Bead cultures were exposed to indicated concentrations of TP508 as well as [ 35 S]-sulfate (20 ⁇ Ci/ml) and with daily medium changes and were harvested on days 7 for [ 35 S]-sulfate incorporation. At each time point 5 -10 beads were removed, washed 3x with 0.9% ⁇ saline, dissolved by adding 0.5 ml of 55 mM sodium citrate, 0.15 M NaCl at 37 C for 10 minutes as described above, and counted in a liquid scintillation counter.
  • a double emulsion technique was used to prepare microspheres of polylactic acid/polyglycolic acid copolymer (PLGA) containing TP508. Briefly, the matrix components were dissolved in methylene chloride and TP508 was dissolved in water. The two were gradually mixed together while vortexing to form a water-in- oil (W/O) emulsion. Polyvinyl alcohol (0.3% in water) was added to the emulsion with further vortexing to form the second emulsion (O/W), thereby forming a double emulsion: an O/W emulsion comprised of PLGA droplets, and within those droplets, a second disperse phase consisting of TP508 in water.
  • W/O water-in- oil
  • O/W second emulsion
  • the PLGA droplets Upon phase separation, the PLGA droplets formed discrete microspheres containing cavities holding TP508. To cause phase separation of the microspheres, a 2% isopropyl alcohol solution was added. The particles were collected by centrifugation, and then lyophilized to remove residual moisture. The composition of the matrix was varied to form microspheres with different release kinetics (Table 8). Table 8. Composition of different microsphere formulations
  • the mean diameter of the microspheres was measured in a Coulter counter and the drug entrapment efficiency was measured by spectrophotometric assay at 276 nm following dissolution of a weighed sample of microspheres in methylene chloride and extraction of the released drug into water (Table 9).
  • TP508 release from the different PLGA matrices 20 mg of microspheres were placed in 1.0 ml of PBS contained in 1.5 ml polypropylene microcentrifuge tubes. Tubes were incubated at 37°C and shaken at 60 rpm. At various times, the tubes were centrifuged and the supernatant containing released TP508 was removed and frozen for subsequent analysis. Fresh PBS was added to the microspheres and incubation was continued. TP508 in the supernatant was measured by absorbance at 276 nm. For each formulation, quadruplicate release determinations were performed. Formulations B and D showed no detectable drug release during 28 days of incubation at 37°C.
  • Example 7 The NPAR Agonist TP508 Stimulates Cartilage
  • the rabbits were divided into three groups. For each rabbit, both right and left trochlear groove defects were filled with the same treatment.
  • TP508 was formulated into PLGA controlled release microspheres, prepared as described in Example 6 (Formulation C). The microspheres were mixed with sufficient Pluronic F68 gel (5% w/v) to bind the spheres together into a paste-like consistency that could easily be packed into the defect.
  • the control group received PLGA microspheres without TP508 in both defects.
  • the treated groups received microspheres containing either 10 or 50 mg of TP508/defect.
  • One rabbit from each group was sacrificed at 4 weeks, 2 from each group were sacrificed at 6 weeks and the remaining animals were sacrificed at 9 weeks.
  • Peptide treated defects repaired with smooth articular surfaces and were typically well bonded at the junction between repair and native tissue.
  • the quality of control repair tissue was characterized as mostly fibrocartilage with poor quality joint surfaces. Integration at the junction between repair and native tissue was usually poor.
  • the quality of cartilage repaired with TP508 was significantly enhanced over control non-treated defects. This improved quality of repair tissue should lead to more durable and functional restoration of joint biomechanics and reduction in the incidence of osteoarthritis in patients suffering from traumatic cartilage injuries.
  • ulnar osteotomies treated with microspheres containing the NPAR agonist TP508 showed evidence of bone mineralization and growth whereas in most control osteotomies that received osteoconductive microspheres, there was no bone growth and/or failure to fill the voided region.
  • Mechanical testing for mechanical strength and stiffness confirmed sigmficant effects of TP508 on bone formation in this model. Because TP508 induced bone formation in sites where it did not occur without TP508, this discovery of osteoinduction is distinct from prior studies, in which TP508 accelerated the rate of normal fracture healing in fracture or small gap defects that would heal without TP508.

Abstract

L'invention concerne une méthode pour stimuler la croissance osseuse sur un site d'un patient nécessitant une ostéoinduction ou une réparation du cartilage. Cette méthode consiste à administrer sur ledit site une quantité thérapeutiquement efficace d'un agoniste du récepteur de la thrombine activé de manière non-protéolytique. La présente invention porte également sur une méthode pour stimuler la prolifération et l'expansion de chondrocytes in vitro, cette méthode consistant à cultiver des chondrocytes en présence d'une quantité stimulante d'un agoniste NPAR.
PCT/US2002/001451 2000-07-19 2002-01-17 Stimulation de la croissance osseuse et de la formation du cartilage au moyen de derives peptidiques de la thrombine WO2003061690A1 (fr)

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AU2002239965A AU2002239965B2 (en) 2000-07-19 2002-01-17 Stimulation of bone growth and cartilage formation with thrombing peptide derivatives
CNA028285689A CN1622826A (zh) 2002-01-17 2002-01-17 用凝血酶肽衍生物刺激骨生长和软骨形成
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EP1807018A2 (fr) * 2004-10-21 2007-07-18 University of Iowa Research Foundation Systeme d'administration in situ de medicaments a liberation controlee
US7291596B2 (en) 2003-12-31 2007-11-06 Orthologic Corp. Pharmaceutical composition for thrombin peptide derivatives
WO2008100567A2 (fr) * 2007-02-15 2008-08-21 Orthologic Corp. Dérivés peptidiques de la thrombine destinés à traiter les fractures chez les patients ostéopéniques
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US8940311B2 (en) 2004-10-21 2015-01-27 Tae-Hong Lim In situ controlled release drug delivery system
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