Enoxaparin and methods of its use

This present invention discloses inhibitors of matrix metalloproteinases, and novel methods of their use. The inhibitors may be useful for treating conditions that involve enhanced activity of at least one of the matrix metalloproteinases neutrophil collagenase (MMP-8), aggrecanase, hADAMTS1, and gelatinase A (MMP-2). Such disorders may include, but are not limited to, degenerative joint disorders (e.g., osteoarthroses), spondyloses, chondrolysis associated with joint trauma or prolonged joint immobilization (often occurring after meniscus or patellar injuries or ligament tears), connective tissue disorders (e.g., collagenoses), wound healing disturbances, periodontal disorders, chronic disorders of the locomotor system (e.g., inflammatory, immunologically, or metabolism-related acute and chronic arthritides), arthropathies, myalgias, or disturbances of bone metabolism.

EXAMPLES 
 Example 1 
 Effect of enoxaparin on the aggrecanase of porcine chondrocytes To generate aggrecanase activity, porcine chondrocytes were stimulated with 10 ng/ml IL-1&agr; for 4 days (Hughes, C. E., Little, C. B., Buettner, F. H., Bartnik, E., Caterson, B., “Differential expression of aggrecanase and matrix metallo-proteinase activity in chondrocytes isolated from bovine and porcine articular cartilage,” J. Biol. Chem., 273: 30576-30582 (1998)). In a 96-well cell culture plate, 200 &mgr;l of the chondrocyte supernatant containing aggrecanase activity were mixed with 100 &mgr;l of (cell culture medium/buffer) DMEM per well. 5 &mgr;l of enoxaparin, dissolved in an appropriate concentration in H 2 O, were added as an inhibitor one hour before addition of 5 &mgr;g of rAgg1mut substrate (Buttner et al., Biochem. J., 333: 159-165 (1998)). The mixture was incubated at 37° C. for 17 h and then transferred into an ELISA plate in order to detect the neoepitopes generated by the aggrecanase activity (Büttner et al., Trans. Orthop. Res. Soc., 23: 916 (1998)) with the antibody BC-3 (Hughes et al., Biochem. J., 305, 799-804 (1995)). The enzymatic activity of a representative experiment, expressed as the average BC-3 signal (extinction), is provided in Table 1. 1 TABLE 1 Digestion of rAgg1mut by porcine chondrocyte aggrecanase: Inhibition by enoxaparin Enoxaparin Average BC-3 Standard (&mgr;g/ml) signal n &equals; 2 deviation 0 1.15 0.031 0.0167 1.24 0.020 0.167 1.13 0.031 1.67 1.05 0.027 16.7 0.70 0.049 167 0.33 0.041 1670 0.44 0.027 16700 0.27 0.063 no aggrecanase and no enoxaparin 0.06 0.002 no aggrecanase and 16700 &mgr;g/ml enoxaparin 0.07 0.019 The IC 50 was determined to be about 80 &mgr;g/ml enoxaparin using this experimental approach. As the results indicate, enoxaparin inhibits the aggrecanase activity of porcine chondrocytes in the digestion of the substrate rAgg1mut. 
 Example 2 
 Effect of enoxaparin on the aggrecanase of human de-differentiated chondrocytes To generate aggrecanase activity, 50,000 de-differentiated human chondrocytes were stimulated in a 96-well cell culture plate with 0.01 ng/ml IL-1&agr; and 3 U of TNF&agr; in 200 &mgr;l of DMEM/F12 medium (1:1) per well for 47 h. In a 96-well cell culture plate, 200 &mgr;l of the chondrocyte supernatant containing aggrecanase activity were mixed with 5 &mgr;l of enoxaparin (Clexane), dissolved in an appropriate concentration in H 2 O, as an inhibitor an hour before addition of substrate (2.5 &mgr;g of rAgg1mut). The mixture was incubated at 37° C. for 4 h and then transferred into an ELISA plate in order to detect the neoepitopes generated by aggrecanase activity with the antibody BC-3. The enzymatic activity from a representative experiment, expressed as the BC-3 signal (extinction), is provided in Table 2. 2 TABLE 2 Standard Standard Enoxaparin Clexane 40 Clexane 20 deviation deviation &mgr;g/ml n &equals; 2 n &equals; 2 (Clex 40) (Clex 20) 0 1.20 0.043 0.0167 1.13 1.05 0.091 0.017 0.167 1.0 0.97 0.025 0.044 1.67 0.99 0.92 0.064 0.009 16.7 0.95 0.87 0.037 0.048 167 0.72 0.72 0.043 0.002 1670 0.29 0.42 0.007 0.084 no aggrecanase and 0.41 0.016 no enoxaparin no aggrecanase and 0.19 0.22 1670 &mgr;g/ml enoxaparin The IC 50 was determined to be about 200 &mgr;g/ml enoxaparin in this experiment. As the results demonstrate, enoxaparin inhibits the aggrecanase activity of de-differentiated human chondrocytes in the digestion of the substrate rAgg1mut. 
 Example 3 
 Effect of enoxaparin on the aggrecanase activity of recombinant human ADAMTS1 protein To generate the aggrecanase activity of human ADAMTS1, 293 cells were transfected with an ADAMTS1 expression plasmid by the calcium phosphate method. An expression plasmid was constructed that harbors the coding sequence of the human ADAMTS1 gene, followed by an inserted C-terminal FLAG tag. The gene was placed under the control of the CMV promoter. The transfection supernatant was passed through an M2 (anti-FLAG) antibody agarose column. The hADAMTS1 was bound to the M2 antibody via its FLAG tag. The recombinant hADAMTS1 was then eluted from the M2 antibody column with free FLAG peptide. The human ADAMTS1 that was partially purified in this manner was employed in the aggrecanase assay described below. In a 96-well cell culture plate, 10 &mgr;l of eluate containing recombinant human ADAMTS1 and 300 &mgr;l of DMEM with 5 &mgr;l of enoxaparin (Clexane) inhibitor (dissolved in an appropriate concentration in H 2 O) were mixed one hour before the addition of substrate (1 &mgr;g of rAgg1mut). The mixture was incubated at 37° C. for 4 h and then transferred into an ELISA plate in order to detect the neoepitopes generated by the aggrecanase activity with the antibody BC-3. The enzymatic activity of the representative experiment, expressed as the average BC-3 signal (extinction), is shown in Table 3. 3 TABLE 3 Enoxaparin Average BC-3 &mgr;g/ml signal n &equals; 2 0 1.36 0.0167 1.35 0.167 1.34 1.67 1.32 16.7 0.87 167 0.27 1670 0.23 no hADAMTS1/no enoxaparin 0.11 The IC 50 was determined to be about 25 &mgr;g/ml enoxaparin in this assay. As the results indicate, enoxaparin inhibits the aggrecanase activity of recombinant human ADAMTS1 in the digestion of the substrate rAgg1 mut. 
 Example 4 
 Enoxaparin does not inhibit the catalytic domain of MMP-3 in digesting recombinant substrate rAgg1mut In a 96-well cell culture plate, 31.3 &mgr;l of recombinant human MMP-3 (catalytic domain, G98-P273, prepared by the method of Ye et al., Biochemistry, 31:11231-11235 (1992)) in MMP-3 digestion buffer (0.1 M MES; 0.1 M NaCl; 0.01 M CaCl 2 ; 0.5% Brij; pH 6.0) was mixed with 5 &mgr;l of enoxaparin (Clexane) inhibitor, dissolved in an appropriate concentration in H 2 O, were mixed one hour before the addition of substrate (5 &mgr;g of rAgg1mut). The mixture was incubated at 37° C. for 8 h and then transferred to an ELISA plate in order to detect the neoepitopes generated by MMP-3 activity (cleavage at amino acids N341-F342) with the antibody BC-14 (Hughes et al., Biochem. J., 305:799-804 (1995)). Table 4 shows the results of a representative experiment. 4 TABLE 4 Enoxaparin Average BG-14 Standard &mgr;g/ml signal n &equals; 2 deviation 0 0.97 0.044 0.0167 1.02 0.004 1.67 1.03 0.075 167 0.96 0.019 16700 0.94 0.042 no MMP-3 and no enoxaparin 0.14 0.057 no MMP-3 and 16700 &mgr;g/ml enoxaparin 0.12 0.050 As the results indicate, enoxaparin showed no effect on the catalytic domain of MMP-3 on digestion of the recombinant substrate rAgg1 mut. 
 Example 5 
 Preparation and determination of the enzymatic activity of gelatinase A (MMP-2) and neutrophil collagenase (MMP-8) Gelatinase A (MMP-2) and neutrophil collagenase (MMP-8) were purchased from Roche and Biocon, respectively. To measure the enzymatic activity or the enzyme-inhibitory effect, 10 &mgr;l of enzyme-containing buffer solution were incubated with 10 &mgr;l of H 2 O which contains the enzyme inhibitor for 15 minutes. MMP-2 (20 mUnit) or MMP-8 (20 ng) were incubated by the method described by Knight (Knight et al., FEBS Letters, 296:263 (1992)) with 10 &mgr;l of a 10% strength (v/v) aqueous dimethyl sulfoxide solution containing 0.1 mmol/l of a fluorogenic substrate (7-methoxycoumarin-4-yl)acetyl-Pro-Leu-Gly-Leu-3-(2′,4′-dinitrophenyl)-L-2,3-diaminopropionyl-Ala-Arg-NH 2 (Bachem, Heidelberg, Germany). The progress of the enzymatic reaction was followed by fluorescence spectroscopy (328 nm (ex)/393 nm (em)). The fluorescence was measured for 15 minutes. The initial rate of the enzymatic reaction was measured without addition of inhibitor, and the resulting values were defined as 100% activity. The compounds tested included unfractionated heparin (UF) with a molecular weight of about 15,000 daltons, a 3,000 dalton (LMW) heparin fraction (both obtainable from Sigma), and enoxaparin. The MMP activities of MMP-2 and MMP-8 were assayed with each test compound. The inhibitiors listed in Table 5 were each measured at a concentration of 1 &mgr;g/ml and were compared with a control without inhibitor (100%). 5 TABLE 5 MMP-2 MMP-8 Compound inhibition (%) inhibition (%) UF heparin 41 1 LMW heparin 27 8 Enoxaparin 70 28