WO1994009825A9 - Diagnostic et traitement d'osteoarthrite au moyen d'une gelatinase de 92 kd - Google Patents

Diagnostic et traitement d'osteoarthrite au moyen d'une gelatinase de 92 kd

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Publication number
WO1994009825A9
WO1994009825A9 PCT/US1993/010217 US9310217W WO9409825A9 WO 1994009825 A9 WO1994009825 A9 WO 1994009825A9 US 9310217 W US9310217 W US 9310217W WO 9409825 A9 WO9409825 A9 WO 9409825A9
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WO
WIPO (PCT)
Prior art keywords
gelatinase
subject
cartilage
detecting
chondrocytes
Prior art date
Application number
PCT/US1993/010217
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English (en)
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WO1994009825A1 (fr
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Publication date
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Priority to AU62804/94A priority Critical patent/AU6280494A/en
Publication of WO1994009825A1 publication Critical patent/WO1994009825A1/fr
Publication of WO1994009825A9 publication Critical patent/WO1994009825A9/fr

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Definitions

  • 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.
  • Osteoarthritis is a degenerative disease that results in the breakdown of articular cartilage. It is notoriously difficult to diagnose early in its course.
  • 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.
  • MRI magnetic resonance imaging
  • 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.
  • 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 Chem (1988)) 263 :6579-6587 ; cultured human alveolar macrophages (Hibbs, M.S. et al., J Clin Invest (1987)) £0:1644-1650; human neutrophils (Hibbs, M.S. et al., J Biol Chem (1985) 260:2493-2500: normal human macrophages (Wilhelm, S.M.
  • 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.
  • 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.
  • 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.
  • 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
  • Figure 4 is a photocopy of a nitrocellulose replica of conditioned media from chondrocytes or keratinocytes and developed with antibody to 92 kD _ j - 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
  • Figure 7 shows the relative expression of the 92 kD gelatinase in fibrillated and non-fibrillated
  • 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.
  • the invention features the critical importance of a 92 kD gelatinase in the etiology of degenerative diseases effecting chondrocytes, especially osteoarthritis.
  • 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.
  • 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.
  • the preproenzy e 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.
  • organomercurials such as p-aminophenyl mercuric acid (APMA)
  • the collagen-binding fibronectin-like domain also present in the 72 kD type IV collagenase;
  • the 92 kD type IV gelatinase/collagenase from the transformed lung fibroblast was completely glycosylated.
  • 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.
  • antibodies “immunospecific" 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.
  • 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.
  • the insights provided by the invention herein showing the unique association of the 92 kD gelatinase 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 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 identification of the condition as osteoarthritis or the alternative degenerative diseases can be effected by evaluating additional indicia associated with these particular diseases.
  • 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.
  • antibody preparations specific for this enzyme are useful.
  • antipodies prepared simply against the 92 kD enzyme may be crossreactive with the 72 kD type IV gelatinase.
  • antibody preparations obtained using standard immunization protocols with the 92 kD protein must be screened for this crossreactivity.
  • monoclonal antibodies are obtained which can be conveniently screened in this way.
  • 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.
  • 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.
  • a gelatin substrate can be used to bind the gelatinase in the biological sample which can then be detected with these antibodies.
  • PCT application W091/11714 Optimal conditions for this type of assay are disclosed in the cited application.
  • the expression of the gene encoding the 92 kD precursor can be conducted using mRNA encoding the gelatinase as the index of production.
  • 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.
  • 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.
  • 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.
  • antibodies which are immunoreactive with the 92 kD gelatinase are effective.
  • immunospecificity is not required as it is not particularly undesirable to inhibit the 72 kD form as well.
  • 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.
  • Suitable inhibitors include interleukin 1 receptor antagonist; IL-1 receptor protein, anti-IL-1 antibodies and IL-1 converting enzyme inhibitor.
  • . 5 - 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.
  • 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.
  • 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
  • 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.
  • 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 ⁇ g/ l of bovine pituitary extract and supplements (Clonetics Laboratories, San Diego, CA) .
  • the cells were treated with 50 ng/ l 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 ⁇ - 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 min. to allow proteins to renature. The gels r. were then incubated overnight in substrate buffer (50 mM Tris, pH 8.0, 10 mM CaCl 2 ) at 37°C.
  • substrate buffer 50 mM Tris, pH 8.0, 10 mM CaCl 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.
  • 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.
  • 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.
  • 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) 2.65:17238-17245).
  • 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 L 39 were 32 P 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 ethidium 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.
  • 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.
  • Cells were plated at 1.5 x 10 5 cell/cm 2 on poly-L-lysine- coated dishes and cultivated in serum free medium which consisted of DMEM/F12 (1:1) medium, 50 ⁇ g/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-l ⁇ (100 U/ml) in the serum free medium for 24 h.
  • IL-l ⁇ stimulation results are shown in Figure 6. Chondrocytes and cartilage were cultured without addition (lane 1, 3) or with IL-l ⁇ (lane 2, 4) for 2 days. Substrate gel analysis of the conditioned medium samples showed that IL-l ⁇ -induced expression of the 92 kD gelatinase (arrow) . Conditioned medium from sti ulated 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.
  • Figure 4 Expression patterns of the 92- and 72 kD type IV collagenase/gelatinase in response to IL-l ⁇ were also examined by Northern blot analysis (Fig. 5) .
  • IL-l ⁇ IL-l ⁇
  • 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.
  • PCR polymerase chain reaction
  • 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.
  • 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.

Abstract

Une gélatinase de 92 kD est présente de manière unique dans les chondrocytes ou le fluide synovial de patients souffrant d'affections dégénératives du cartilage. On pense que la gélatinase a une fonction active dans l'étiologie de la maladie. En conséquence, la détection de la présence ou de l'absence de la gélatinase de 92 kD peut être utilisée pour effectuer un diagnostic et appliquer un traitement au moyen de composés ou de protocoles inhibant sa fonction ou sa production.
PCT/US1993/010217 1992-10-23 1993-10-22 Diagnostic et traitement d'osteoarthrite au moyen d'une gelatinase de 92 kd WO1994009825A1 (fr)

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US96590892A 1992-10-23 1992-10-23
US07/965,908 1992-10-23

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AU5542794A (en) * 1992-10-29 1994-05-24 Miles Inc. Diagnostic assay for latent matrix metalloproteinase no. 9
US6140099A (en) * 1994-05-20 2000-10-31 The Trustees Of The University Of Pennsylvania Method of delaying fetal membrane rupture by inhibiting matrix metalloproteinase-9 activity
US5641636A (en) * 1994-05-20 1997-06-24 University Of Pennsylvania Method of predicting fetal membrane rupture based on matrix metalloproteinase-9 activity

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US4992537A (en) * 1989-05-15 1991-02-12 Washington University cDNA encoding 92-kDA type IV collagenase

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