WO1994009825A1

WO1994009825A1 - DIAGNOSIS AND TREATMENT OF OSTEOARTHRITIS INVOLVING A 92 kD GELATINASE

Info

Publication number: WO1994009825A1
Application number: PCT/US1993/010217
Authority: WO
Inventors: R. Lane Smith; Masaaki Mohtai; David J. Schurman; Kuniyoshi Tsuchiya
Original assignee: The Board Of Trustees Of The Leland Stanford Junior University
Priority date: 1992-10-23
Filing date: 1993-10-22
Publication date: 1994-05-11
Also published as: AU6280494A

Abstract

A 92 kD gelatinase is uniquely found in the chondrocytes or synovial fluid of subjects suffering from degenerative diseases of the cartilage. It is believed that the gelatinase has an active role in the etiology of the disease. Therefore, detection of the presence or absence of the 92 kD gelatinase may be used for diagnosis and treatment with compounds or protocols which inhibit its function or production are useful in therapy.

Description

Diagnosis and treatment of osteoarthritis involving a 92 KD gelatinase

Technical Field

The invention is related to the diagnosis and treatment of osteoarthritis based on the presence of a 92 kD gelatinase in cartilage or synovial fluid.

Background Art

Osteoarthritis is a degenerative disease that results in the breakdown of articular cartilage. It is notoriously difficult to diagnose early in its course. Generally, techniques available include general observation of the patient, examination of X-rays of the joints, and the use of magnetic resonance imaging (MRI) . All of these techniques are subjective and may result in false positives and negatives.

The ability to diagnose the presence of osteoarthritis is significant because various treatment and risk avoidance protocols are available, and such preventive and prophylactic care can yield important results. Thus, the ability to render an accurate diagnosis is of more than intellectual interest. It is presently thought that enzymatic pathways are involved in the loss of cartilage matrix associated with this disease. Of the various proteases that are capable of degrading cartilage, the matrix metalloproteinases, which operate at neutral pH in the extracellular matrix, are considered to be particularly important (Dean, D.D., Seminars in Arthritis and Rheumatism (1991) 20:2-11) . The metalloproteinase family of enzymes contains at least two distinct interstitial collagenases, two types of stromelysin (transin) , a small proteinase (PUMP-1) and the type IV collagenases/gelatinases. Both a 72 kD and 92 kD collagenase/gelatinase have been found.

These metalloproteinase enzymes vary in substrate specificity but all have a zinc-binding site and are secreted as proenzymes that can be activated in vitro by the action of various proteinases such as plasmin, kallikrein, mast cell tryptase, and trypsin. They can also be activated by non-proteolytic agents such as organo-mercurial compounds, N-ethyl-maleimide, oxidized glutathione, and hypochlorous acid. It is postulated that activation occurs by perturbing the interaction of the critical cysteine residue in the highly conserved propeptide domain of the metalloproteinases with the catalytic zinc atom, so that the active site becomes accessible to the substrates. ( oessner, J.F. FASEB) (1991) 5:2145-2154).

The Type IV collagenase/gelatinase which has a 92 kD precursor form and 82 kD mature species has been detected in a variety of cell lines, including H-ras oncogene-transformed human bronchial cells (Collier, I.E. et al., J Biol Che (1988)) 263:6579-6587; cultured human alveolar macrophages (Hibbs, M.S. et al., J Clin Invest (1987)) 8 :1644-1650; human neutrophils (Hibbs, M.S. et al., J Biol Chem (1985) 260:2493-2500: normal human macrophages (Wilhelm, S.M. et al., J Biol Chem (1989) 2S :17213-17221; HT1080 tumor cells (Moll, U.M. et al., Cancer Res (1990) ^0:6162-6170); HL-60 leukemia cells (Davis, G.E. et al., Cancer Res (1990) 150:1113-1120; mammary carcinoma cells (Lyons, I.G. et al., Biochemistry (1991) 3^:1449-1456). The gene for the 92 kD precursor of the type IV gelatinase/collagenase from fetal lung fibroblasts has been cloned and sequenced (U.S. Patent 4,992,537). In addition, antibodies have been prepared using peptide fragments which are i munoreactive with this protein (Corcoran, M.L. et al., J Biol Chem (1992) 267:515-519) .

It has now been found that the type IV gelatinase/collagenase represented by the 92 kD precursor or its mature form is found in chondrocytes from subjects with osteoarthritis or other cartilage degenerative diseases but not in normal chondrocytes; the protein is also found in the synovial fluid of osteoarthritic patients but not in the synovial fluid of normal subjects. Thus, this protein serves as a marker for aid in diagnosis of this condition.

Disclosure of the Invention

The invention provides methods for diagnosis and treatment of degenerative diseases of the cartilage, including osteoarthritis, in human subjects. Correct diagnosis permits the subject to employ a variety of preventive and remedial protocols with respect to this condition.

Thus in one aspect, the invention is directed to a method to assess the condition of a subject suspected of having a degenerative disease of the cartilage, which method comprises detecting the presence or absence of the 92 kD gelatinase or its activated products in the chondrocytes or synovial fluid of said subject; wherein the presence of said gelatinase or its activated forms indicates the presence of a degenerative condition of the cartilage in the subject.

The production in 92 kD gelatinase can also be indicated by the presence of its corresponding RNA. Accordingly, in another aspect, the invention is directed to a method to diagnose these conditions by detecting the presence or absence of RNA corresponding to the 92 kD gelatinase in the chondrocytes of the subject. As the gelatinase is considered instrumental in the development of the disease, the condition may also be treated by administering inhibitors to this enzyme. A number of such small molecule inhibitors are known. Other inhibitors include, of course, antibodies immunoreactive with the 92 kD gelatinase or with its activated forms.

Brief Description of the Drawings

Figure 1 is a photocopied photograph of the results of gel electrophoresis conducted on conditioned media from cartilage slices on a gelatin substrate gel. Zones of clearing represent gelatinolytic activity. Figure 2 is a photocopied photograph of a gelatin substrate gel electrophoresis similar to that of Figure 1 but in the presence and absence of gelatinase inhibitors.

Figure 3 is a photocopied photograph of a similar gel in the presence and absence of an activator for this enzyme.

Figure 4 is a photocopy of a nitrocellulose replica of conditioned media from chondrocytes or keratinocytes and developed with antibody to 92 kD gelatinase.

Figure 5 is a photocopy of a Northern blot of RNA extracted from chondrocytes and keratinocytes under various conditions.

Figure 6 is a photocopied photograph of an electrophoresis gel performed on conditioned media of chondrocytes cultured with and without the addition of IL-1.

Figure 7 shows the relative expression of the 92 kD gelatinase in fibrillated and non-fibrillated osteoarthritic cartilage chondrocytes.

Figure 8 is a photocopied photograph of a gel showing the expression of 92 kD gelatinase mRNA compared to beta-actin mRNA expression. The range of signal varies depending on the state of the cartilage in which the 92 kD gelatinase is detected. 92 kD gelatinase was detected in all osteoarthritic samples.

Modes of Carrying Out the Invention

The invention features the critical importance of a 92 kD gelatinase in the etiology of degenerative diseases effecting chondrocytes, especially osteoarthritis. By "92 kD gelatinase" is meant the 92 kD type IV collagenase/gelatinase described and claimed by Goldberg, G.I. et al. in U.S. Patent 4,992,537, the contents of which are incorporated herein by reference. This differs from the earlier described 72 kD type IV collagenase described by the same group in British Application 2,205,526A and U.S. Patent 4,923,818.

As described in the '537 patent, the 92 kD type IV collagenase was purified from SV40-transformed fetal lung fibroblasts. A cDNA clone representing the full- length protein was also obtained. The protein was also found in U-937 monocytic leukemia cells.

As described in the ^,537 patent, the preproenzyme is synthesized as a polypeptide of a predicted molecular weight of 78,426 containing a 19 amino acid signal peptide and is secreted as a single 92 kD glycosylated proenzyme. The 92 kD proenzyme can be activated by organomercurials such as p-aminophenyl mercuric acid (APMA) , which results in removal of 73 amino acids from the amino terminus, yielding an active form of about 84 kD; the proenzyme can also be activated by proteolysis. The in vitro substrate specificity seems identical to that of the 72 kD type IV collagenase of the prior art.

The complete cDNA encoding the preproenzyme was obtained and the sequence presented in the above patent. Analysis of the structure showed it to have five domains: 1 and 2. The amino terminal and zinc-binding domains shared by all members of the secreted etalloprotease gene family;

3. The collagen-binding fibronectin-like domain also present in the 72 kD type IV collagenase;

4. A carboxy terminal hemohexin-like domain shared by all enzymes of this family except PUMP-1;

5. A 54 amino acid long proline-rich domain homologous to the α2 chain of type V collagen.

In its secreted form, the 92 kD type IV gelatinase/collagenase from the transformed lung fibroblast was completely glycosylated.

Accordingly, as defined herein, the "92 kD gelatinase" refers to the protein encoded by the cDNA set forth in the above-referenced '537 patent and by its allelic variants, as well as substantially identical proteins. The definition also includes any activated form of the gelatinase, in particular the protein of 82- 84 kD obtained by activation with organomercurials or proteases.

By antibodies "i munospecific" with the 92 kD gelatinase is meant antibodies which are capable of distinguishing the 92 kD collagenase from the 72 kD collagenase of the prior art. While affinities for various moieties are relative, there should be a sufficient difference in affinity to identify the presence of absence of the 92 kD protein in fluids also containing the 72 kD protein using conditions and methods well known to those of skill in the art.

By antibodies "immunoreactive" with the 92 kD gelatinase is meant antibodies which are capable of inactivating this enzyme. These antibodies are useful in therapy, and it is immaterial whether or not these antibodies also immunoreact with the 72 kD gelatinase. These antibodies may be specifically immunoreactive with the zinc binding portion of the protein or with the collagen binding portion thereof or otherwise with sites in the enzyme necessary for activity. The antibodies may also be effective in preventing the activation of the 92 kD enzyme.

Diagnostic Protocols

The insights provided by the invention herein showing the unique association of the 92 kD gelatinase

10 with degenerative conditions of the cartilage provides methods for diagnosis and treatment of these conditions. Included among these conditions are osteoarthritis, degenerative joint disease, juvenile arthritis, psoriatic arthritis, rheumatoid arthritis, post-traumatic

15 arthritis, septic arthritis, ankylosing spondylitis, Reiter's syndrome, inflammatory arthritis, arthritis secondary to avascular necrosis, hemophiliac arthropathy, arthritis secondary to hemochromatosis, gouty arthropathy, and systemic lupus erythematosus. The

20 identification of the condition as osteoarthritis or the alternative degenerative diseases can be effected by evaluating additional indicia associated with these particular diseases.

With respect to diagnosis, the presence or

_ absence of these conditions may be ascertained by detecting either the 92 kD gelatinase protein or mRNA encoding it in chondrocyte-conditioned media or extracts or in the synovial fluid of a subject.

For the detection of the 92 kD proenzyme (or

_ its activated form) antibody preparations specific for this enzyme are useful. As there are regions of ho ology between the 92 kD glycosylated enzyme and the 72 kD enzyme of the prior art, as outlined in the '537 patent referenced above, antibodies prepared simply against the

_ 92 kD enzyme may be crossreactive with the 72 kD type IV gelatinase. Thus, antibody preparations obtained using standard immunization protocols with the 92 kD protein must be screened for this crossreactivity. Preferably, monoclonal antibodies are obtained which can be conveniently screened in this way.

Alternatively, antibodies may be prepared against the region of the 92 kD protein not shared with the 72 kD form. These, too, however, should be screened to verify the immunospecificity.

Preparation of the antibodies useful in the diagnostic assay of the invention is conducted by conventional immunization protocols using suitable mammalian subjects such as rats, rabbits, mice, sheep and the like. The peptide or protein is administered in amounts and on a schedule which are optimized for production of antibodies. The progress of immunization can be monitored by titrating the serum against the immunizing moiety. If peptide fragments of the 92 kD protein are used as immunogens, it may be necessary to couple them with immunogenic carriers to enhance their immunogenicity, as is well known in the art.

Because immunospecificity for the 92 kD protein or its activated form is required, monoclonal antibodies are preferred. These antibodies are prepared using known techniques by immortalizing immunoglobulin-producing cells from the peripheral blood lymphocytes or spleens of the immunized animals and screening the immortalized lymphocytes for secretion of antibodies of the proper specificity. Such screening is conducted by standard immunoassay methods such as fluorescent, enzyme or radioactive labeled complex formation.

The antibodies shown to be immunospecific for the 92 kD protein can then be used in immunoassays in a variety of protocols. A large number of such protocols including both competitive and direct assays is known in the art. In one assay particularly useful for this type of protein, a gelatin substrate can be used to bind the gelatinase in the biological sample which can then be detected with these antibodies. A detailed description of this approach is set forth in PCT application W091/11714. Optimal conditions for this type of assay are disclosed in the cited application.

Alternatively, the expression of the gene encoding the 92 kD precursor can be conducted using mRNA encoding the gelatinase as the index of production. To conduct this approach, the mRNA of chondrocytes is first obtained and purified and optionally separated according to size by gel electrophoresis. The mRNA, optionally size separated, is then subjected to Northern blot using conventional techniques and using a detection sequence which is specific for hybridization to the mRNA encoding the 92 kD gelatinase under the required stringency conditions. For example, the cDNA encoding the 92 kD gelatinase described in the '537 patent above can be used as a probe. Enhanced levels of mRNA encoding the 92 kD gelatinase provide an index for the presence of conditions characterized by cartilage degradation.

Therapeutic Protocols

The discovery that the 92 kD gelatinase is uniquely involved in the etiology of cartilage degenerative diseases provides essential information for the design of therapeutic protocols. Medicaments which are designed to inhibit the activity of the activated form of the 92 kD gelatinase are effective. Among these inhibitors are antibodies which are immunoreactive with the 92 kD gelatinase. In the context of therapeutic protocols, immunospecificity is not required as it is not particularly undesirable to inhibit the 72 kD form as well. Thus, antibodies prepared with respect to either the 72 kD or 92 kD form may be used provided they are immunoreactive with the activated form of the 92 kD protein or with its precursor. Alternative inhibitors in the form of peptides and binding proteins have been disclosed by Stetler-Stevenson et al. in WO90/1127 and WO90/10228. These inhibitors either prevent the activation of the precursor form of the protein or mimic or bind to essential domains. The disclosure of these two applications is incorporated herein by reference. Finally, as it has been shown herein that the 92 kD gelatinase is induced by interleukin-1, inhibitors of interleukin-1 may be administered to lower the levels of the gelatinase in the chondrocytes. Suitable inhibitors include interleukin 1 receptor antagonist; IL-1 receptor protein, anti-IL-1 antibodies and IL-1 converting enzyme inhibitor.

The administration of the medicaments of the invention is conducted using standard formulations and protocols such as those found in Remington's Pharmaceutical Sciences. Mack Publishing Co. , Easton, PA, latest edition. Depending on the nature of the medicament, the formulation may be designed for injection, such as intravenous, intramuscular, intraperitoneal or subcutaneous injection or for transmucosal or transdermal administration (appropriate for many peptide medicaments) or in some cases may be designed for oral use. Those of ordinary skill routinely optimize such administration regimes.

The following examples are intended to illustrate but not to limit the invention.

Example 1 Detection of 92 kD Gelatinase in Arthritic Cartilage Osteoarthritis cartilage samples were obtained from arthritic patients undergoing total joint arthroplasty. In each case, full-thickness cartilage slices (40-50 mg wet weight) were made, and cultured in DMEM/F12 in 24 cluster microwell culture plates (Costar, Cambridge, MA, U.S.A.) for 3 days. The medium was renewed on the second day of culture and the renewed culture medium was harvested on the fourth day of culture, and frozen at -20°C until use.

Normal human articular cartilage was obtained at autopsy within 24 h of death and processed immediately. Tissue from four adults (with ages ranging from 36 to 60 years) were studied. All cartilage used in this study had a normal macroscopic appearance. None showed overt degeneration with characteristic tissue loss. None of the patients surveyed had any clinical history of chemotherapy or radiotherapy. Full-thickness cartilage slices 940-50 mg wet weight) , similar size of the OA cartilage slices, were distributed in 24 cluster microwell culture plates and cultured for 3 days. The medium was renewed on the second day of culture and some of the normal cartilage slices were challenged with IL-lα (100 U/ml, Genzyme, Boston, MA, U.S.A.) for 2 days.

Human keratinocytes of neonatal foreskins were generously gifted by Dr. Toshiro Iwasaki (Department of Dermatology, Stanford University) . The cells were cultured in serum-free MCDB 153 medium with 0.1 mM calcium, 30 μg/ml of bovine pituitary extract and supplements (Clonetics Laboratories, San Diego, CA) . The cells were treated with 50 ng/ml of TPA for 24 h. to stimulate 92 kD gelatinase production.

Electrophoresis of samples of the conditioned medium from the above cultures on gelatin substrate gels demonstrated that all four arthritic cartilage cultures expressed a 92 kD gelatinase, whereas none of the normal cartilage cultures showed this enzyme.

Electrophoresis was conducted on 10% SDS- polyacrylamide gels impregnated with 1 mg/ml type I gelatin from porcine skin (Sigma Chemical Corp. , St. Louis, MO) as described by Chin, J.R. et al., J Biol Chem (1985) 260:12367-12376. Aliquots of medium were mixed with Laemmli sample buffer containing 2.5% SDS without β- mercaptoethanol and electrophoresed without boiling under nonreducing conditions at 4°C, constant current 20 mA. After electrophoresis, gels were washed in 2.5% TRITON X- 100 for 30 in. to allow proteins to renature. The gels were then incubated overnight in substrate buffer (50 mM Tris, pH 8.0, 10 mM CaCl₂) at 37°C.

After incubation, gels were stained with Coomassie blue R250 to reveal zones of lysis within the gelatin matrix. Zones of clearing represent gelatinolytic activity. Human keratinocytes stimulated by TPA was used as a positive control. As shown in Figure 1, four normal cartilage cultures (N; lanes 1-4) did not show the 92 kD gelatinase observed in the TPA- stimulated human keratinocytes (lane 9) , and in the OA samples (lanes 5-8) , 72 kD gelatinase, which appeared frequently as a doublet of gelatinolytic activity at 66 and 62 kD in non-reduced condition on SDS-PAGE (arrowheads) , was expressed by all cartilage studies (Fig. 1, arrowhead) . One OA sample showed an additional 82 kD gelatinolytic band, which might represent an active form of the 92 kD gelatinase (lane 8) . Molecular weight markers are in the far left lane.

These observations have now been extended to six other patients. Other experiments showed expression of the 92 kD gelatinase was prominent in samples of moderate to severe disease, and higher activity was observed in the center of erosion than in remote sites.

To characterize the gelatinase, 1,10 phenanthroline (2 mM) , PMSF (1 mM) , or pepstatin (1 mM) was added to the substrate buffer to identify which classes of proteinases were responsible for lysis of the substrate in the gels (Barrett, A.J., "Proteinases in Mammalian Cells and Tissues", A.J. Barrett, ed. , North- Holland Publishing Co. , Amsterdam, pp. 15-18) . As shown in Figure 2, after electrophoresis of conditioned medium samples from normal (lane 1) and OA cartilage (lane 2) , gelatin substrate gels were incubated in the presence of 2 mM 1,10 phenanthroline (1,10-Phe), 1 mM PMSF, 1 mM pepstatin A(Pep A) . All gelatinases, including the 92 kD (arrow) and the 72 kD gelatinase (arrowheads) were inhibited by 1,10 phenanthroline but resistant to inhibition by other proteinase inhibitors.

Some of the conditioned medium samples were incubated with 0.5 mM APMA for 3 h at 37°C to activate latent protease before loading to the substrate gels (Ishibashi, M. et al., Bioche J (1987) 241:527-5341. These results are shown in Figure 3. Before loading, conditioned media were incubated without (lane 1, 3) or with 0.5 mM APMA for 3 h at 37°C to activate latent protease (lane 2, 4). APMA transformed the 92 kD gelatinase (arrow) into a 82 kD species (asterisk) . On the other hand, the 72 kD gelatinase (arrowhead) became a triplet of gelatinolytic activity at 66, 64, and 62 kD by APMA treatment (asterisks) .

PMSF, a serine protease inhibitor, and pepstatin A, an acid proteinase inhibitor, had no effect on the 92 kD gelatinase activity. 72 kD gelatinase, which appeared frequently as a doublet of gelatinolytic activity at 66 and 62 kD in non-reduced condition on SDS- PAGE, was expressed by all cartilage studied (Fig. 1, arrowheads). It was also inhibited by 1,10 phenanthroline (Fig.2).

Example 2 Western Blot Analysis

Western blot analysis was carried out using a polyclonal antibody against 92 kD type IV collagenase/gelatinase.

For Western blot of chondrocyte proteins, supernatants from organ cultures were concentrated using a microconcentrator CENTRICON-30 (Amicon, Amicon Division, W.R. Grace & Co., Conn.). Protein concentration was measured using a protein assay kit (Bio-Rad Laboratories, Richmond, CA) . 20 μg of protein was resuspended in loading dye (125 mM Tris-HCl (pH 6.8), 4% (w/v) sodium dodecyl sulfate, 0.01% bromphenol blue, 20% glycerol) , reduced (10% jS-mercaptoethanol) , heated for 2 min at 95°C, loaded, and electrophoresed on a 10% polyacrylamide gel. After electrophoresis, the proteins were transferred in electroblotting buffer (20 mM Tris (pH 8.0), 150 mM glycine, 20% MeOH) overnight at 250 mA. Western blots were blocked in a milk solution (5% nonfat dry milk, 150 mM NaCl, 50 mM Tris, 0.05% Tween) at room temperature until solidified. The blots were exposed to a primary antibody against 92 kD type IV collagenase/gelatinase (Corcoran, M.L. et al., J Biol Chem (1992) 267:515-519) and subsequently developed by using goat anti-rabbit IgG horseradish peroxidase in conjunction with an enhanced chemiluminescence Western blotting detection system (Amersham Corp., U.K.). As shown in Figure 4, the 92 kD band in arthritic cartilage culture medium was recognized by the antibody whereas no such band was present in normal cartilage culture medium.

Example 3 Presence of Gelatinase mRNA

Northern blot analysis using cDNA probe for the 92 kD gelatinase and the 72 kD gelatinase was performed on total cytoplasmic RNA extracted from normal and arthritic cartilage chondrocytes and from keratinocytes.

Total RNA was extracted from human chondrocytes and TPA-treated keratinocytes using a solution containing 4M guanidine thiocyanate, 25 mM sodium citrate, 0.2% (w/v) n-lauroylsarcosine, and 0.7% (v/v) 3-mercapto- ethanol, followed by centrifugation at 35,000 rpm for 20 hr over a 5.6 M cesium chloride pat. The RNA pellet was solubilized in water, sequentially extracted with 4:1 (v/v) chloroform:n-butanol, 1:1 (v/v) phenol:chloroform and chloroform. The aqueous phase containing RNA was adjusted to 0.3 M sodium acetate. RNA was precipitated overnight with 2.5* volume of ethanol. The concentration of RNA was determined by spectrophotometric absorption at 260 nm. RNA was separated on formamide/formaldehyde agarose gel and transferred to nitrocellulose membrane (Schleicher & Schuell, Keene, NH) by capillary blotting overnight. The membranes were air-dried, then baked at 80°C for 2 h.

A cDNA for 72 kD type IV collagenase/ gelatinase was isolated from the cDNA library of primary human synovial fibroblasts as described before (MacNaul, K.L. et al., J Biol Chem (1990) 265:17238-17245) . A 0.9 bp Pst I fragment of the 72 kD type

IV/gelatinase, a 2.3 kb Xba I fragment of the 92 kD type IV collagenase/ gelatinase (Wilhelm, S.M. et al., J Biol Chem (1989) 264:17213-17221) . and a fragment of the human ferritin L³⁹ were ³²P labeled by random priming (Amersham Corp. UK) . Ferritin L mRNA is constitutively expressed and can be used as a standard for nonspecific mRNA induction or variations in RNA quantities loaded onto gels. Comparisons of ethidiu bromide-stained gels with corresponding Ferritin L mRNA levels confirmed that Ferritin L mRNA levels reflected total RNA levels.

Blots were probed sequentially with a 92 kD type IV collagenase/gelatinase cDNA, a 72 kD type IV collagenase/gelatinase cDNA, and human ferritin L cDNA. Blots were hybridized at 42°C overnight and washed at 60°C in 0.5 x SSC + 0.1% SDS for 1 h. The membranes were exposed to Kodak X-OMAT films with a DuPont lightening intensifying screen at -80°C. To strip blots, nylon membranes were put into boiled 0.1 x SSC + 0.1% SDS until the solution became cooled down. Consistent with zymographic and immunoblot results, mRNA for the 92 kD gelatinase was strongly expressed in arthritic cartilage chondrocytes but not normal chondrocytes (Fig. 5) . Normal cartilage (lane 1) , OA cartilage (lane 2), normal chondrocytes cultured in the serum free medium without (lane 3) or with (lane 4) IL-1 for 24 h, and keratinocytes stimulated with TPA. RNAs were resolved on a formamide/formaldehyde gel. The 72 kD gelatinase was expressed in both chondrocytes at the mRNA level, consistent with protein results, with expression levels as similar as the arthritic cartilage chondrocytes.

Example 4

Regulation of the 92 kD Gelatinase in Normal Cartilage and Chondrocytes by IL-1

To see the effects of IL-1 on the production of the gelatinases by normal cartilage and normal chondrocytes, zymographies of these culture media were performed. Normal human articular chondrocytes were isolated from the samples described above by sequential digestion at 37°C, with 1 mg/ l of trypsin (1:250, Grand Island Biological Company, Grand Island, NY) for 1 hr and 2 mg/ml of each collagenase (CLS-II and CLS-IV; Worthington Chemical company. Freehold, NJ) for 12-16 h. Cells were plated at 1.5 x 10⁵ cell/cm² on poly-L-lysine- coated dishes and cultivated in serum free medium which consisted of DMEM/F12 (1:1) medium, 50 μg/ml of Gentamicin, 2 x 10^~8 M of selenium, and a lipid supplement (Jones, D.G. et al., J Orthop Res (1990) 8.:227-233). After 3 days, the medium was replaced, followed by culture in the presence or absence of IL-lα (100 U/ml) in the serum free medium for 24 h.

The results of IL-lα stimulation are shown in Figure 6. Chondrocytes and cartilage were cultured without addition (lane 1, 3) or with IL-lα (lane 2, 4) for 2 days. Substrate gel analysis of the conditioned medium samples showed that IL-lα-induced expression of the 92 kD gelatinase (arrow) . Conditioned medium from stimulated cultures of keratinocyte exhibited the 92 kD type IV collagenase/gelatinase.

IL-1 induced the production of the 92 kD gelatinase by both normal cartilage and chondrocytes in 48 h. Western blot analysis of the IL-1 stimulated cartilage culture medium revealed that the newly produced 92 kD gelatinase by normal cartilage was recognized by the polyclonal antibody against 92 kD type IV collagenase/gelatinase. These results are shown in Figure 4. Expression patterns of the 92- and 72 kD type IV collagenase/gelatinase in response to IL-lα were also examined by Northern blot analysis (Fig. 5) . mRNA for the 92 kD type IV collagenase/gelatinase was inducible by IL-lα, consistent with zymographic and Western blot results. On the other hand, IL-lα, had no effect on expression of the 72 kD type IV collagenase/gelatinase.

Example 5 PCR METHOD FOR DETECTING 92 KD GELATINASE RNA

Cartilage samples were obtained from 15 osteoarthritic (OA) patients at the time of total joint arthroplasty. When possible, osteoarthritic samples from each joint were separated into fibrillated and non- fibrillated segments based on gross appearance. Normal cartilage was obtained at autopsy within 24 hours of death and processed immediately to serve as controls. Chondrocytes were isolated from OA and normal articular cartilage samples by cutting the tissue into small pieces followed by treatment with 2mg/ml of type II and type IV collagenase for 14-18 hours.

RNA was extracted from collagenase-digested cartilage chondrocytes by lysing the cells in a solution containing 4 M guanidinium isothiocyanate and centrifuging for 14-18 hours on 5.7 M cesium-chloride cushion. 160 ng of total RNA was converted to single- stranded cDNA using random hexamer and M-MLV reverse transcriptase. A target sequence in reverse-transcripted cDNA was amplified using polymerase chain reaction (PCR) with sequence specific oligonucleotide primers designed for amplification of the proline-rich domain (exon 9) of human 92 kD gelatinase. Radiolabelled nucleotide was added in the reaction.

The design of the sequence specific primer for 92kd collagenase/gelatinase was as follows:

5'primer (#2092)

5'-ATGTACCCTATGTACCGCTTCACTG-3' 25mer GC=12 Tm=74°C

Corresponds to the sense of nucleotide sequence 1274-

1298.

3'primer (antisense, #2091)

5'-CGAAGATGTTCACGTTGCAGGCATC-3' 25mer GC=13 Tm=76°C

Corresponds to the antisense nucleotide sequence 1583-

1559.

Expected length of amplified products:310 bp

Amplification program:

95°C, 2 min.

95-60-72(l'+l'+l') X 30 cycles

72°C, 3 min.

For quantitation, expression of 92 kD gelatinase mRNA was compared to both 18S rRNA and beta- actin mRNA expression. Both 18S rRNA and beta-actin mRNA were amplified at same time in the same reaction to monitor any difference in amplification conditions. Amplified products were separated on 5% polyacrylamide gel. Gels were directly subjected to autoradiography using Kodak X-OMAT autoradiography film and exposed at -70°C at illustrated in Figure 8.

The target sequence for this study proved to be unique to the 92 kD gelatinase by homology search in Genebank database; the amplified products showed a single product coinciding with the exact molecular size predicted from the primer sequences. Using this RT-PCR ethod combined with densitometric analysis, all of OA samples (N=15) exhibited the 92 kD gelatinase while chondrocytes from normal controls did not. In 8 of 10 samples, divided into nonfibrillated and fibrillated segments, there was an average of 4-fold greater signal for 92 kD gelatinase in the fibrillated site. Only two pairs showed weaker expression of the 92 kD gelatinase in the fibrillated segments. This data is summarized in Figure 7. The data for expression of the 92 kD gelatinase mRNA in each cartilage sample is shown relative to beta-actin mRNA expression. Similar results were obtained when the data was normalized to 18S rRNA.

The RT-PCR method showed a positive expression for the 92 kD gelatinase mRNA in human osteoarthritic cartilage but not in normal cartilage. The majority of samples demonstrated a positive correlation between expression of the 92 kD gelatinase and the degree of fibrillation. This is consistent with Example 3 above using the northern blot technique. However, two sample pairs showed weaker expression of the 92 kD gelatinase in fibrillated sites. Extensively damaged cartilage either contains fewer viable cells or is so damaged that the cells which are present can no longer produce proteolytic enzymes. These data confirmed that sites of fibrillation coincide with elevated gene expression for 92 kD gelatinase.

Claims

ClaimsWe claim:

1. A method to assess the presence or absence of cartilage degeneration in a subject, which method comprises detecting the presence or absence of a 92 kD gelatinase precursor as determined by SDS-PAGE under non- reducing conditions or its activated products in the chondrocytes or synovial fluid of said subject; wherein the presence of said gelatinase or its activated forms indicates the presence of a degenerative condition of the cartilage in the subject.

2. The method of claim 1 wherein said detecting is conducted by

(a) treating said chondrocytes or culture medium in which said chondrocytes have been cultured or said synovial fluid with an antibody or an antibody fragment which specifically binds said gelatinase or its activated form under conditions wherein when said gelatinase is present, a complex is formed between said antibody or said antibody fragment and said gelatinase or activated form; and

(b) detecting the presence or absence of said complex.

3. The method of claim 1 wherein said detecting is conducted by

(a) separating a 92 kD fraction as determined by SDS-PAGE under non-reducing conditions and other fractions according to size from an extract of said chondrocytes or said synovial fluid;

(b) subjecting the 92 kD fraction to activation; and

(c) detecting the presence or absence of gelatinase activity in the activated 92 kD fraction.

4. The method of claim 1 wherein said detecting is conducted by

(a) subjecting an extract of said chondrocytes or said synovial fluid to the activity of a protease which cleaves the 92 kD gelatinase as determined by SDS- PAGE under non-reducing conditions to obtain the 82 kD mature form thereof as determined by SDS-PAGE under non- reducing conditions;

(b) separating said 82 kD fraction and other fractions contained therein according to size;

(c) recovering the 82 kD fraction; and

(d) detecting the presence or absence of gelatinase activity in said 82 kD fraction.

5. The method of claim 1 wherein said degenerative condition is osteoarthritis.

6. A method to assess the presence or absence of cartilage degeneration in a subject, which method comprises detecting the presence or absence of mRNA encoding 92 kD gelatinase as determined by SDS-PAGE under non-reducing conditions in the chondrocytes of said subject wherein the presence of said mRNA indicates the presence of a degenerative condition of cartilage in the subject.

7. The method of claim 6 wherein said detecting is conducted by Northern blot analysis.

8. The method of claim 6 wherein said detecting is conducted by polymerase chain reaction amplification methodology.

9. A method to treat a subject diagnosed as having a degenerative condition of the cartilage so as to alleviate said condition, which method comprises administering to said subject an effective amount of an inhibitor of 92 kD gelatinase.

10. A method to treat a subject diagnosed as having a degenerative condition of the cartilage so as to alleviate said condition, which method comprises administering to said subject an effective amount of a moiety capable of specifically binding to a 92 kD gelatinase.

11. The method of claim 10 wherein said moiety is an antibody immunoreactive with 92 kD gelatinase or an immunologically reactive fragment thereof.

12. A method to treat a subject diagnosed as having a degenerative condition of the cartilage so as to alleviate said condition, or to treat a subject with a propensity for such a condition so as to prevent its occurrence, which method comprises administering to said subject an effective amount of an inhibitor of IL-1.