AU2002239965B2 - Stimulation of bone growth and cartilage formation with thrombing peptide derivatives - Google Patents

Description

WO 03/061690 PCT/US02/01451 -1- STIMULATION OF BONE GROWTH AND CARTILAGE FORMATION WITH THROMBIN PEPTIDE DERIVATIVES GOVERNMENT SUPPORT The invention was supported, in whole or in part, by grant 1 R43 AR45508- 01 and 2 R44 AR45508-02 from the National Institutes of Health. The Government has certain rights in the invention.

BACKGROUND OF THE INVENTION Mammalian bone tissue has a remarkable ability to regenerate and thereby repair injuries and other defects. For example, bone growth is generally sufficient to bring about full recovery from most simple and hairline fractures. Unfortunately, however, there are many injuries, defects or conditions where bone growth is inadequate to achieve an acceptable outcome. For example, bone regeneration 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. In other instances, it may be desirable to stimulate bone growth where bone is not normally found, i.e., ectopically. Spine fusion to relieve lower back pain where two or more vertebrae are induced to fuse is one example of desirable ectopic bone formation. Currently, such gaps or segmental defects require bone grafts for successful repair or gap filling. The development of effective bone graft substitutes would eliminate the need to harvest bone from a second surgical site for a graft procedure, thereby significantly reducing the discomfort experienced by the patient and risk of donor site healing complications.

WO 03/061690 PCT/US02/01451 -2- Compounds which stimulate or induce bone growth at sites where such growth would not normally occur if left untreated are said to be "osteoinductive".

An osteoinductive compound would have great value as a drug to treat the conditions described above. A number of 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. However, 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.

Furthermore, unlike most tissues, 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 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. In addition, the cost for cartilage repair from acute joint injury (meniscal lesions, patellar surface damage and chondromalacia) exceeds $1 billion annually. Therefore, new therapeutic approaches are needed to heal lesions of cartilage caused by degeneration or acute trauma.

SUMMARY OF THE INVENTION It has now been found that compounds which activate the non-proteolytic thrombin receptor are osteoinductive. For example, the compound TP508, an agonist of the non-proteolytic thrombin receptor, stimulates bone growth in segmental critical size defects created in the ulna of male New Zealand rabbits (Example As shown by x-ray and confirmed by histology and mechanical testing, there was a significant increase in bone formation induced by TP508 at doses of 100 pig and 200 P.AOPERWIMSPECI 12481 1 3 -IV. d.-I 0 -3p.g compared with untreated controls. Based on these results, novel methods of stimulating bone growth in a subject and novel implantable pharmaceutical compositions are disclosed herein.

It has now also been found that chondrocytes isolated from articular cartilage respond to compounds which activate the non-proteolytic thrombin cell surface receptor (hereinafter "NPAR"). For example, chondrocytes express approximately 233,000 ri thrombin binding sites per cell with apparent affinities of approximately 0.1 nM (3000

O

Ssites) and 27 nM (230,000 sites) (Example In addition, 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) and 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). These in vitro experiments demonstrate that NPAR agonists can stimulate proliferation and matrix production in chondrocytes isolated from articular cartilage. Additional in vivo experiments demonstrate that delivering TP508 in a sustained release formulation to rabbit trochlear grove cartilage defects which extend into the subchondral bone results in repair of the cartilage defect, including repair of subchondral bone, restoration of a normal cartilage surface and integration of the newly formed cartilage with uninjured cartilage outside of the defect area (Example 7).

Based on the results reported in the prior paragraph, 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.

According to one embodiment of the present invention there is provided a method of stimulating bone growth at a site in a subject in need of bone growth and at which bone growth would not occur if said site is left untreated, said method comprising the step of administering to the site a therapeutically effective amount of a physiologically functional P XOPERWjKRWSPECI\I 2487130- I sp. mod-dc. IS VD6 0 -4equivalent of a thrombin derivative peptide consisting of the amino acid sequence Alatt Gly-Tyr-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).

SAccording to another embodiment of the present invention there is provided a method of stimulating bone growth at a site in a subject in need of bone growth and at which bone growth would not occur if said site is left untreated, said method comprising C,1 the step of administering to the site a therapeutically effective amount of a physiologically Sfunctional equivalent of a thrombin derivative peptide consisting of the amino acid sequence Ac-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 7).

According to another embodiment of the present invention there is provided a method of stimulating bone growth at a site in a subject in need of bone growth and at which bone growth would not occur if said site is left untreated, said method comprising the step of administering to the site a therapeutically effective amount of a physiologically functional equivalent of a thrombin derivative peptide consisting of the amino acid sequence Ac-Ala-Gly-Tyr-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 8).

In another embodiment of the present invention there is provided a pharmaceutical composition comprising an implantable, biocompatible carrier and a physiologically functional equivalent thrombin derivative peptide 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-NH 2 (SEQ ID NO 6).

In another embodiment of the present invention there is provided a pharmaceutical composition comprising an implantable, biocompatible carrier and a physiologically functional equivalent thrombin derivative peptide comprising the amino acid sequence Ac- 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 7).

P \OPERNMKRISPECl 2487130. I pa mmendm is doc-159 6 -4A- In another embodiment of the present invention there is provided a pharmaceutical composition comprising an implantable, biocompatible carrier and a physiologically functional equivalent thrombin derivative peptide comprising the amino acid sequence Ac- Ala-Gly-Tyr-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 8).

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. Such 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. In addition, their lyophilized form is stable for at least thirty months when stored at 50 C and at 60% relative humidity.

In another aspect, the present invention is directed to a method of stimulating cartilage growth or repair at a site in a subject in need of such growth or repair and at which cartilage growth or repair would not occur if the site is left untreated, said method comprising the step of administering to the site a therapeutically effective amount of a physiologically functional equivalent thrombin derivative peptide consisting of 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-NH 2 (SEQ ID NO 6).

In another aspect, the present invention is directed to a method of stimulating cartilage growth or repair at a site in a subject in need of such growth or repair and at which cartilage growth or repair would not occur if the site is left untreated, said method comprising the step of administering to the site a therapeutically effective amount of a physiologically functional equivalent thrombin derivative peptide consisting of the amino acid sequence Ac-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 7).

In another aspect, the present invention is directed to a method of stimulating cartilage growth or repair at a site in a subject in need of such growth or repair and at which cartilage growth or repair would not occur if the site is left untreated, said method comprising the step of administering to the site a therapeutically effective amount of a PAOPERWIKR'.SPECI\I2487130-I.p. d.tdm4.5/09tD6 4B physiologically functional equivalent thrombin derivative peptide consisting of the amino acid sequence Ac-Ala-Gly-Tyr-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 8).

A further embodiment of the present invention is directed to a method of stimulating the proliferation and expansion of chondrocytes in vitro. The method comprises culturing chondrocytes in the presence of a stimulating amount of an NPAR agonist.

DETAILED DESCRIPTION OF THE INVENTION "Osteoinduction" 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".

WO 03/061690 PCT/US02/01451 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 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 Glowacki et al., Lancet 1: 959 (1981)); perfonning 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 Sigurdsson et al., J. Periodontol., 66: 511 (1995)).

In addition, sites in need of cartilage growth, repair or regeneration are found in subjects with osteoarthritis. 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. As osteoarthritis progresses, 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.

WO 03/061690 PCT/US02/01451 -6- 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, are osteoinductive. Applicants have further discovered that 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.

Applicants have also found that compounds which stimulate or activate NPAR stimulate proteoglycan synthesis in chondrocytes. Proteoglycan is a major cartilage component. Based on these results, Applicants delivered the NPAR agonist TP508, prepared in a sustained release formulation, to defects in rabbit trochlear grove cartilage and discovered that the peptide stimulated repair of the defect that included formation of new cartilage with a normal cartilage surface. The peptide also stimulated layering and integration of this new cartilage into adjacent, uninjured cartilage and restoration of the subchondral bone. It is concluded that 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)).

WO 03/061690 PCT/US02/01451 -7- 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 25 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 PAR1, PAR2, PAR3 and PAR4. PAR1 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, Vu, 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.

267:16975 (1992)). 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 NPAR requires no proteolytic cleavage.

Several lines of evidence indicate that NPAR is distinct from PAR receptors: a population of cells has been isolated that express fully functional PAR1 receptors, but are non-responsive to thrombin due to a defect in the NPAR signal transduction pathway (see, Kim, et al., J. Cell. Physiol. 160:573 (1994)); (2) neutrophils bind 125I thrombin with high affinity and their chemotaxis is stimulated by proteolytically inactivated thrombin or NPAR agonists (see, Ramakrishnan and Carney, Mol. Biol. Cell 4:1993 (1993)), yet they do not express PAR1 (see Jenkins, et al., J Cell Sci. 108:3059 (1995)); IIC9 fibroblasts over-express PAR1, but do not bind thrombin with high affinity (see, Kim, D. Ph.D. Dissertation. The University of Texas Medical Branch at Galveston, 1995; and Low, et al., "Cancer Cells 3/Growth Factors and Transformation", Cold Spring Harbor Laboratory, New WO 03/061690 PCT/US02/01451 -8- York); and 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).

One example of an NPAR agonist is a thrombin peptide derivative, 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. One example of a thrombin peptide derivative comprises a moiety represented by Structural Formula Asp-Ala-R

(I)

R is a serine esterase conserved domain. Serine esterases, 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.

In one embodiment, the serine esterase conserved sequence has the amino acid sequence of SEQ ID NO. 1 (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 1. 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 1. Preferably, the amino acids in the serine esterase conserved sequence which differ from the corresponding amino acid in SEQ ID NO 1 are conservative substitutions, and are more preferably highly conservative susbstitutions. 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-XI-Gly-Asp-Ser-Gly-Gly-Pro-X 2 -Val; X, is Glu or Gin and X, 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.

In a preferred embodiment, the thrombin pepride derivative comprises a serine esterase conserved sequence and a polypeptide having a more specific t 5 thirombin amino acid sequence Arg-Gly-Asp-Ala (SEQ ID NO One example of a thrombin peptide derivative of this type comprises Arg-Gly-Asp-Ala-Cys-X,-Gly- Asp-Ser-Gly-Gly-Pro-X 2 -Val (SEQ ID NO X, and X 2 are as defined above.

When the thrombin peptide derivative comprises SEQ ID NO 4, it preferably has the Samino acid sequence of SEQ ID NO 5 (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-termLinal 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 lD NO 5. Preferably, 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 susbstitutions. An "N-terminal truncated fragment" refers to a fragment rernauung 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 thrombmi derivative peptide encompasses molecules which differ from thrombin derivatives in particulars which do not affect the function of the peptide as an NPAR 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 carn-ier molecule, or sequence alterations in accordance with the serine esterase conserved sequences.

Physiologically functional equivalents of the thrombin derivative peptides are also within the scope of the invention. For example, such peptides ca.n be amidated at the carboxyl terminus, acylated at the amino terminus or both. In particular embodiments, the amino acid sequence of SEQ ID NO.: 3 is represented as the following physiologically functional equivalents: Ala-Gly-Tyr-Lys-Pro-Asp- G1 Ilu-Gly-Lys-Axg-Gly-Asp -A la-Cys-G] u-Gly-Asp-S er-Gly-Gly-Pro-Phe-V al-NB (SEQ ID NO.: Ac-Al a-G ly -Tyr-Lys-Pro -Asp- Glu- Gly- Lys-Akrg-G ly-A sp-Al a- Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Pbe-Val (SEQ ID NO.: 7) or Ac-Ala-Gly-Tyr- Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro- Phe-Val-N}1 2 (SEQ ID NO.: 8) is an acetyl group).

IND ~Amidation of the carboxyl term11ius can be accoinaplished by any mnethod known in the art. Thus the C-terminal amnino acid is represented in Structu-ral c-i Formula (I1): -N}1-CHIR-C(O)-N\RR 2

(II)

\vheretni R, and R 2 individually are selected from the groups of H, Cr- C 6 aLk-y] and, R, and R, together with the nitrogen to whijch they are bound form a nonaromatic heterocyclic ring such as pyrrolidinyl, piperazinyl, morphulinyl or piperdinyl. R, and R 2 are preferably H. "-Val-N-H2 means -N}{-CH[-CH-(CH 3 2 1- CON142.

Acylation of the amino ter-inus can be accomplished by any methaod knowo in the art. Thus, the N-terminial amnino is represented in the Structur-al Formnula (III):

R,-C(O)-NH-CHR-C(O)-

wherein R is the amrino acid side chain and R, is a C 1

C

6 aLkyl branched and straight chained. R is preferably mnethyl (-CH 3 TP508 is an example of a physiologically functional equivalent of a th-rombin peptide derivative and has the amino acid sequence of SEQ ID NO 6.

A "conservative substitution" is the replacemnent of an amino acid with another amino acid that has the same net electronic charge and approximately the WO 03/061690 PCT/US02/01451 -11same 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 numnber 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 C 1-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 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" 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.

WO 03/061690 PCT/US02/01451 -12- Other NPAR agonists include small organic molecules which bind and activate NPAR. Agonists of this type can be conveniently identified with high through-put screening, 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.

The term "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-thrombin and the combination of DIP-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-inflaimmatory, 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.

Examples of suitable osteoconductive carriers include collagen bovine dermal collagen), fibrin, calcium phosphate ceramics hydroxyapatite and tricalcium phosphate), calcium sulfate, guanidine-extracted allogenic bone and combinations thereof. A number of suitable carriers are commercially available, such as COLLOGRAFT (Collagen Corporation, Palo Alto, CA), which is a mixture WO 03/061690 PCT/US02/01451 -13of hydroxyapatite, tricalcium phosphate and fibrillar collagen, and INTERPORE (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:S118 (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 ahydroxy 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 hyroxyapatite 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 (IV):

R

N=P--

R'

WO 03/061690 PCT/US02/01451 -14-

(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. 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., JBiomed 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 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 1,3bis(p-carboxyphenoxy)propane (see Leong et al., J. Biomed. Mater. Res. 19:941 (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-l,6bis(carboxyphenoxy)hexane and pyromellityimidoalanine: 1,6-bis(pcarboxyphenoxy)hexane copolymers.

0 0 0-C-R-Cn

(V)

Poly(propylene fumnarates) (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 WO 03/061690 PCT/US02/01451 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. 10:363 (1999) and Yaszemski et al., Tissue Eng. 1:41 (1995), the entire teachings of which are incorporated herein by reference). The addition of solid phase components such as P-tricalcium phosphate and sodium chloride can improve the mechanical properties of PPF polymers (see Peter et al., J. Biomed. Mater. Res. 44:314 (1999), the entire teachings of which are incorporated herein by reference).

The pharmaceutical 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 bone growth in the presence of the NPAR agonist but little or no growth in its absence) at the site when the NPAR 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 preferred 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. The 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.

In yet another alternative, the pharmaceutical composition can be partially enclosed in a supporting physical structure such as a mesh, wire matrix, stainless WO 03/061690 PCT/US02/01451 -16steel cage, threaded interbody fusion cage and the like before administering to the site in need of osteoinduction.

Another alternative for applying the pharmaceutical composition of the present invention is by 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 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.

For example, 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. Alternatively, 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.

Thus 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 WO 03/061690 PCT/US02/01451 -17prosthetic 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 a greater amount of cartilage growth or better quality of cartilage growth in the presence of the NPAR agonist than in its absence).

In one means of administration, 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, Easton, PA. 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.

In another means of administration, 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 ahydroxy esters such as polylactic acid/polyglycolic acid copolymers and WO 03/061690 PCT/US02/01451 -18polyanhydrides.

The 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. Similarly, the polyanhydrides as shown in Structural Formula 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. After implantation, the material is slowly absorbed by the body and is replaced by cartilage in the shape of or very nearly the shape of the implant.

In one aspect, 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.

In another aspect, 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 ORTHOVISC developed by Anika, SYNVISC, developed by Biomatrix, HYALGAN, 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. 98:843 (196), the entire teachings of which are incorporated herein by reference).

NPAR agonists can be used to accelerate the growth or to maintain the WO 03/061690 PCT/US02/01451 -19functionality of isolated chondrocytes. In one embodiment, 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. In another embodiment, because the NPAR agonists appear to stimulate matrix production, such 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, a "stimulatory amount". Typically, between about 0.1 pig/ml and about 100 |tg/ml of NPAR agonist is used.

With respect to bone growth, 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. Typically, the agonist is administered for a sufficient period of time to achieve the desired therapeutic or cosmetic effect, 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.

Typically, between about 1 pg per day and about 1 mg per day of NPAR agonist (preferably between about 5 jig per day and about 100 [ig per day) is administered by continuous release or by direct application to the site in need of bone growth.

With respect to cartilage 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.

Alternatively or addition, 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. Typically, the agonist (or chondrocytes) is administered for a sufficient period of time to achieve the desired therapeutic or effect. The amount administered will depend on the amount of cartilage growth that is desired, the health, size, weight, age and sex of the subject WO 03/061690 PCT/US02/01451 and the release characteristics of the pharmaceutical formulation. Typically, between about 0.1 [tg per day and about 1 mg per day ofNPAR agonist (preferably between about 5 [tg per day and about 100 Ipg 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. With respect to chondrocytes, "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, companion animals dogs, cats, and the like), farm animals cows, pigs, horses and the like) and laboratory animals rats, mice, guinea pigs and the like).

Thrombin peptide derivatives can be synthesized by solid phase peptide synthesis 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. 88:2149 (1963); Meienhofer, Hormonal Proteins and Peptides, C.H. 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, J. Org. Chem., 37: 3404 (1972); and Gauspohl, H. et al., Synthesis, 5: 315 (1992)). The teachings of these six articles are incorporated herein by reference in their entirety.

The invention is illustrated by the following examples which are not intended to be limiting in any way.

EXEMPLIFICATION

Example 1 Preparation of Polylactic Acid/Polyglycolic Acid Copolvmer Microspheres of TP508 WO 03/061690 PCT/US02/01451 -21- A double emulsion technique was used to prepare microspheres of polylactic acidlpolyglycolic 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 emulsion. Polyvinyl alcohol in water) was added to the emulsion with further vortexing to form the second emulsion 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. 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 1).

Table 1: Composition of different microsphere formulations Formu- PLGA Polymer polylation M. Wt. TP508 ethylene glycol A 50:50 46,700 5 0 B 50:50 7,200 5 0 C 50:50 46,700 5 D 50:50 46,700 5 0 E 75:25 120,000 5 0 The mean diameter of the microspheres was measured in a Coulter counter and the drug entrapment efficiency was measured by WO 03/061690 PCT/US02/01451 -22spectrophotometric 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).

Table 2: Formulation diameter and drug entrapment efficiency Formulation Diameter, jtm TP508 Entrapment, A 26.0 53.8 B 16.2 27.1 C 17.6 58.9 D 23.9 42.6 E 25.8 36.2 To measure 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. The remaining formulations all released detectable amounts of TP508 although in all cases the amount of drug released fell below detectable limits [ig/mg matrix/day) within 3-4 days. Formulations A and C showed the greatest release of TP508, releasing 60-80% of the entrapped drug over 3-4 days.

The formulation with the fastest release kinetics, C was chosen for further testing in in vivo studies.

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 WO 03/061690 PCT/US02/01451 -23oscillating 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 pg TP508 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 ig 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 pg of TP508. These different doses were achieved by mixing TP508-containing and TP508-devoid microspheres in different proportions. Animals were xrayed at two week intervals, beginning at week three, and sacrificed at nine weeks.

100 pLg of TP508 stimulated mineralization in the defect at 3 and weeks post-surgery. X-rays at 7 and 9 weeks appeared similar to those obtained at 5 weeks. Animals were sacrificed at 9 weeks post-surgery and the ulna-radius was removed and photographed. In most cases a large defect is still visible in ulnas from the control limbs, in contrast with the TP508-treated limbs, in which most of the defects have successfully closed.

After sacrifice at 9 weeks post-surgery, repair strength was measured by torsion testing (MTS-858 Minibionix machine). The results are shown in Tables 3 and 4.

Table 3: Torsion testing of segmental defects treated with 100 jpg TP508.

Parameter Control SEM TP508, 100 [ig SEM Ultimate torque 0.107 0.034 0.255+ 0.041 Failure torque 0.103 0.032 0.239+ 0.042 Ultimate energy 0.815 0.365 1.916" 0.398 Failure energy 0.940 0.436 2.064A 0.421 Stiffness coeff. 0.013 0.004 0.028 0.006 ^p 0.05, +p 0.01 Table 4: Torsion testing of segmental defects treated with 200 pig TP508.

WO 03/061690 PCT/US02/01451 Parameter Control SEM TP508, 200 [ig SEM Ultimate torque 0.095 0.042 0.322* 0.046 Failure torque 0.093 0.041 0.306* 0.046 Ultimate energy 0.534 0.355 2.947* 0.543 Failure energy 0.641 0.374 3.433* 0.701 Stiffness coeff. 0.016 0.006 0.033A 0.004 Ap 0.05, *p 0.005 At 100 gig, TP508 more than doubled the mechanical strength of the healing defect as measured by all the parameters tested (Table Even stronger repairs were noted in the 200-p|g group (Table with most parameters being approximately higher than those seen in the low dose treatment group.

Example 3 Thrombin Binding to Rat Chondrocytes Primary cultures of rat articular chondrocytes were isolated and prepared for in vitro analysis using established methods (see Kuettner, K 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 370 C in an atmosphere of 5% CO 2 The specific binding of '2'I thrombin to chondrocytes was carried out using established thrombin receptor binding assays as disclosed in US Patent 5,352,664 and Camey, 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.

WO 03/061690 PCT/US02/01451 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).

Example 4A NPAR Agonist Stimulation of Bovine Chondrocyte Proliferation 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 uig/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.

Example 4B NPAR Agonist Stimulation of Bovine Chondrocyte Proteoglycan Synthesis To determine the effect of NPAR agonists on proteoglycan synthesis, 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 ig per ml (Table After 6 days in culture with daily changes of culture medium with or without TP508, "S sulfate was added to the medium and incubation continued for an additonal 24 hours. As shown in Table 5, treatment with high concentrations of TP508 (100 tig per ml) increased "S sulfate incorporation relative to untreated cells by more than WO 03/061690 PCT/US02/01451 -26- Table 5. Effect of the NPAR agonist TP508 on "S sulfate incorporation in bovine chondrocyte cultures.

Treatment Mean CPM Std. Dev of Mean 1% Serum Control 4975 3552 TP508 (1pg/ml) 4701 2692 TP508 (0g/ml) 6960 3265 TP508 (100pg/ml) 81946 13783 Example 5A NPAR Agonist Stimulation of Proliferation Synthesis in Cultured Rat Articular Chondrocytes 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 w/v) and slowly expressed through a 22 gauge needle in a dropwise fashion into 102 mM CaCl 2 As the drops contact the CaCl 2 there is a nearly instantaneous polymerization of the alginate to create a gel bead. The beads were then washed three times in DMEM culture medium and transferred to 35mm dishes and maintained in culture at 37 C in a 5% C02 atmosphere by feeding with culture medium every two days.

The effect of 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 370 C for minutes. Cell number was determined by diluting the 1 ml of dissolved beads 1:10 WO 03/061690 PCT/US02/01451 -27with 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.

WO 03/061690 PCT/US02/01451 -28- Table 6. Effect of the NPAR agonist TP508 on Proliferation of Rat Chondrocytes in 3-D Bead Culture.

Treatment Cells/bead After 3 Std. dev Increase over days Control Control 6238 688 TP508 30nM 7463 167 19.7 TP508 300 nM 8882 148 42.4 TP508 3 iM 8866 4 42.1 TP508 30 jiM 7772 258 24.6 Example 5B NPAR Agonist Stimulation of Proteoglycan Synthesis in Cultured Rat Articular Chondrocytes To determine the effectos of the NPAR agonist TP508 on proteoglycan synthesis, 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 5 S]-sulfate (20 pCi/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 ml of 55 mM sodium citrate, 0.15 M NaCi at 37 C for 10 minutes as described above, and counted in a liquid scintillation counter. ["S]-sulfate incorporation was normalized in each sample for number of beads added. As shown in Table 7, TP508 treatment alone at a concentration of 300 nM (about 0.7 pg per ml), stimulated sulfate incorporation about 50% over controls. There was also a large stimulation by 30 [pM TP508 (about 70 pg per ml), however, there was a large relative standard deviation in measurements at this concentration.

WO 03/061690 PCT/US02/01451 -29- Table 7. Effect of the NPAR agonist TP508 on 3 sS]-sulfate incorporation into proteoglycans.

Treatment CPM/bead Std. dev Increase over Control Control 665 24 TP508 30nM 829 87 24.7 TP508 300 nM 1008 29 51.6 TP508 3 VM 827 9 24.1 TP508 30 [iM 1153 519 73.3 Example 6 Preparation of Polylactic Acid/Polyglycolic Acid Copolymer Microspheres of TP508 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-inoil emulsion. Polyvinyl alcohol in water) was added to the emulsion with further vortexing to form the second emulsion 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. 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).

WO 03/061690 PCT/US02/01451 Table 8. Composition of different microsphere formulations Formu-lation PLA:PGA Polymer poly-ethylen M. Wt. TP508 glycol A 50:50 46,700 5 0 B 50:50 7,200 5 0 C 50:50 46,700 5 D 50:50 46,700 5 0 E 75:25 120,000 5 0 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 ofa weighed sample of microspheres in methylene chloride and extraction of the released drug into water (Table 9).

Table 9. Formulation diameter and drug entrapment efficiency Formulation Diameter, pm TP508 Entrapment, A 26.0 53.8 B 16.2 27.1 C 17.6 58.9 D 23.9 42.6 E 25.8 36.2 To measure 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 0 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 WO 03/061690 PCT/US02/01451 -31release during 28 days of incubation at 37°C. The remaining formulations all released detectable amounts of TP508 although in all cases the amount of drug released fell below detectable limits pig/mg matrix/day) within 3-4 days.

Formulations A and C showed the greatest release of TP508, releasing 60-80% of the entrapped drug over 3-4 days. Formulation C showed the fastest release kinetics and was chosen for testing in the rabbit cartilage defect model described in Example 7.

Example 7 The NPAR Agonist TP508 Stimulates Cartilage Growth in Rabbit Models Young, male New Zealand rabbits (2-3 kilograms) (n=1 5) were anesthetized and given bilateral, medial longitudinal parapatellar arthrotomies. The skin, subcutaneous tissue and joint capsule were incised, using electrocautery to minimize bleeding. The joint surface was exposed by lateral dislocation of the patella. A 3mm diameter, 1-2-mm deep full-thickness defect was made in the trochlear groove of the femur using a surgical drill and pointed stainless steel drill bit. The aim was to extend the defect into the subchondral plate without piercing the subchondral bone.

The rabbits were divided into three groups. For each rabbit, both right and left trochlear groove defects were filled with the same treatment. For this study, TP508 was formulated into PLGA controlled release microspheres, prepared as described in Example 6 (Formulation The microspheres were mixed with sufficient Pluronic F68 gel 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. Samples were fixed and processed for histological analysis.

WO 03/061690 PCT/US02/01451 -32- At the time of sacrifice, there appeared to be considerable fibrous granulation tissue and no evidence of white cartilage-like material in the control defects. In contrast, the defect had a nearly uniform, dense, white material filling in the defects from the 10 [ig treated group and 50 ig group. By 6 weeks post-surgery, the macroscopic differences between treated and control defects were not so pronounced.

Histology of the four week samples showed that indeed the control defects were filled with what appeared to represent early granulation tissue including inflammatory and fibroblastic cells. In contrast, the 10 and 50 microgram treated defects appeared to have a large number of chondrocytes and early signs of cartilage formation. This effect was seen more dramatically at week six. Controls had a small amount of connective tissue, yet little evidence of cartilage repair. In contrast, in both the 10 jig and 50 [tg treated defects, there appeared to be good integration with hyaline cartilage forming at the top of the defect and extensive subchondral bone repair.

Nine-week TP508 treated defects exhibited a predominantly hyaline matrix with evidence of significant aggrecan content as shown by positive safranin-O staining. In most instances there was no difference in aggrecan content between the repair site and native tissue. Histological results were quantitatively assessed using a grading system adapted by Freed, et al., J. Biomed. Materials Res. 28:891-899 (1944) from the scheme of O'Driscoll, et al., J. Bone Joint Surg. 126:1448-1452 (2000) with a maximum score of 25 for normal articular cartilage. Experimental TP508 treated defects scored mean averages that were significantly higher than control defects (Table WO 03/061690 PCT/US02/01451 -33- Table 10. Histology Scoring For Articular Defect Study Milligrams of TP508 Repair Score SE 0 9.4 1.6 18.6 1.4 50 19.8 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. Overall, 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.

WO 03/061690 PCT/US02/01451 -34- In summation, 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 significant 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.

P kOPER04KRISPECRI 2487130. I p. ,-dmis doc. I S)A While this invention has been particularly shown and described with references to preferred 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.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

Claims (24)

1. A method of stimulating bone growth at a site in a subject in need of bone growth and at which bone growth would not occur if said site is left untreated, said method comprising the step of administering to the site a therapeutically effective amount of a physiologically functional equivalent of a thrombin derivative peptide consisting of 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-NH 2 (SEQ ID NO 6).

2. A method of stimulating bone growth at a site in a subject in need of bone growth and at which bone growth would not occur if said site is left untreated, said method comprising the step of administering to the site a therapeutically effective amount of a physiologically functional equivalent of a thrombin derivative peptide consisting of the amino acid sequence Ac-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 7).

3. A method of stimulating bone growth at a site in a subject in need of bone growth and at which bone growth would not occur if said site is left untreated, said method comprising the step of administering to the site a therapeutically effective amount of a physiologically functional equivalent of a thrombin derivative peptide consisting of the amino acid sequence Ac-Ala-Gly-Tyr-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 8).

4. The method of any one of claims 1 to 3 wherein the site is in need of a bone graft. The method of any one of claims 1 to 3 wherein the site is a segmental gap in a bone, a bone void or at a non-union fracture.

6. The method of any one of claims 1 to 3 wherein the thrombin peptide derivative is administered in a pharmaceutical composition additionally comprising an implantable, biocompatible carrier. P.%OPER2WKRISPECJI I 2487130O.I sp. d-d-1I510910 -37-

7. The method of claim 6 wherein the implantable, biocompatible carrier is an osteoconductive matrix.

8. The method of claim 6 wherein the carrier comprises a polylactic acid/polyglycolic acid homopolymer or copolymer.

9. A pharmaceutical composition comprising an implantable, biocompatible carrier and a physiologically functional equivalent thrombin derivative peptide 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-NH 2 (SEQ ID NO 6). A pharmaceutical composition comprising an implantable, biocompatible carrier and a physiologically functional equivalent thrombin derivative peptide comprising the amino acid sequence Ac-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 7).

11. A pharmaceutical composition comprising an implantable, biocompatible carrier and a physiologically functional equivalent thrombin derivative peptide comprising the amino acid sequence Ac-Ala-Gly-Tyr-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 8).

12. The pharmaceutical composition of any one of claims 9 to 11 wherein the carrier is osteoconductive.

13. The pharmaceutical composition of any one of claims 9 to 11 wherein the carrier is a biodegradable synthetic polymer.

14. The pharmaceutical composition of claim 13 wherein the biodegradable synthetic polymer is a polylactic acid/polyglycolic acid homopolymer or copolymer. P WOERVAKRWSECINI24S7 130.1I Spa a,,cdmwoi. doc-l5Al09 -38- The pharmaceutical composition of any one of claims 9 to 11 wherein the carrier comprises collagen, fibrin, calcium phosphate salts, calcium sulfate, guanidine-extracted allogenic bone or a combination thereof.

16. The pharmaceutical composition of any one of claims 9 to 11 wherein the carrier is injectable.

17. The pharmaceutical composition of any one of claims 9 to 11 wherein the carrier is a poly(propylene fumarate) solution or a calcium phosphate ceramic paste.

18. The pharmaceutical composition of any one of claims 9 to 11 wherein the pharmaceutical composition is administered as microparticles.

19. The pharmaceutical composition of any one of claims 9 to 11 wherein the pharmaceutical composition is pre-shaped before applying to the site in need of osteoinduction. A method of stimulating cartilage growth or repair at a site in a subject in need of such growth or repair and at which cartilage growth or repair would not occur if the site is left untreated, said method comprising the step of administering to the site a therapeutically effective amount of a physiologically functional equivalent thrombin derivative peptide consisting of 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-NH 2 (SEQ ID NO 6).

21. A method of stimulating cartilage growth or repair at a site in a subject in need of such growth or repair and at which cartilage growth or repair would not occur if the site is left untreated, said method comprising the step of administering to the site a therapeutically effective amount of a physiologically functional equivalent thrombin P:AOPERWMSPECflI247130. I spa wv~d,,ws do-3/09 -39- derivative peptide consisting of the amino acid sequence Ac-Ala-Gly-Tyr-Lys-Pro-Asp- SGlu-Gly-Lys-Arg-Gly-Asp-Ala-Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val (SEQ ID NO 7).

22. A method of stimulating cartilage growth or repair at a site in a subject in need of such growth or repair and at which cartilage growth or repair would not occur if the site is C, left untreated, said method comprising the step of administering to the site a Stherapeutically effective amount of a physiologically functional equivalent thrombin derivative peptide consisting of the amino acid sequence Ac-Ala-Gly-Tyr-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 8).

23. The method of any one of claims 20 to 22 wherein the site is an arthritic joint.

24. The method of any one of claims 20 to 22 wherein the site is being treated for cartilage damage or loss. The method of any one of claims 20 to 22 wherein the site is being treated for cartilage damage or loss due to traumatic injury.

26. The method of any one of claims 20 to 22 wherein the physiologically functional equivalent thrombin peptide derivative is administered in a pharmaceutical composition additionally comprising an implantable, biocompatible carrier.

27. The method of any one of claims 20 to 22 wherein the carrier comprises a polylactic acid/polyglycolic acid homopolymer or copolymer.

28. The method of any one of claims I to 3, substantially as hereinbefore described with reference to the examples and/or sequence listings. P:\OPER\MKRlSPECRI 2487130. I pa I dmidm ol doc-15 /A 40

29. The method of any one of claims 20 to 22, substantially as hereinbefore described with reference to the examples and/or sequence listings. The composition of any one of claims 22 to 24, substantially as hereinbefore described with reference to the examples and/or sequence listings.