Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
2001-07-19
2004-11-09
Carlson, Karen Cochrane (Department: 1653)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C514S002600, C514S013800, C424S422000, C424S094640, C424S078080, C530S350000, C530S399000, C530S326000
Reexamination Certificate
active
06815416
ABSTRACT:
BACKGROUND OF THE INVENTION
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 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. 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 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 thrombin binding sites per cell with apparent affinities of approximately 0.1 nM (3000 sites) and 27 nM (230,000 sites) (Example 1). 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 2A) and stimulates by itself the proliferation of rat chondrocytes cultured in three dimensional matrix culture (Example 3A). This same TP508 compound also stimulates proteoglycan synthesis as measured by the incorporation of
35
S sulfate in both bovine chondrocytes (Example 2B) and 3-dimensional cultures of rat chondrocytes (Example 3B). 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 5).
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.
The present invention is 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 agonist of the non-proteolytically activated thrombin receptor to the site of injury.
Another embodiment of the present invention is a method of stimulating the proliferation and expansion of chrondrocytes in vitro. The method comprises culturing chrondrocytes in the presence of a stimulating amount of an NPAR agonist.
DETAILED DESCRIPTION OF THE INVENTION
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. 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 non-proteolytically activated thrombin receptor (hereinafter “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.
Compounds which stimulate or activate NPAR are said to be NPAR agonists. NPAR is a high-affinity thrombin receptor present on the surface of most cells. NPAR is largely responsible for high-affinity binding of thrombin, proteolytically inactivated thrombin, and thrombin derived peptides to cells. NPAR agonists and antagonists can compete for the affinity binding with thrombin to cells (see, e.g., Glenn et al.,
J. Peptide Research
1:65 (1988)). 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 as disclosed in U.S. Pat. Nos. 5,352,664 and 5,500,412.
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
.
Bergmann John
Carney Darrell H.
Crowther Roger S.
Stiernberg Janet
Carlson Karen Cochrane
Hamilton Brook Smith & Reynolds P.C.
Robinson Hope A.
The Board of Regents The University of Texas System
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