Ovarian Carcinoma Xenografts Tumor Burden and Prolongs Survival of Mice Bearing Human A Synthetic Matrix Metalloproteinase Inhibitor Decreases

We have examined the effect of a synthetic low-molecular-weight ma trix metalloproteinase inhibitor, [4-(A'-hydroxyamino)-2/?-isobutyl-3S(thiopen-2-ylthiomethyl)-succinyl]-i.-phenylalanine-iV-methylamide (BB94), on human ovarian carcinoma xenografts growing in nude mice. The xenografts grew as thick intraperitoneal mucinous ascites containing freefloating tumor cell clumps. The ascites increased in volume, causing death approximately 3 weeks after introduction. Treatment with BB-94 caused resolution of ascitic disease. Tumor burden was dramatically reduced, and survival increased 5-6-fold. The increase in survival was dose dependent. The effects observed with BB-94 appeared to be due to its matrix metal loproteinase inhibiting effects, inasmuch as its inactive diastereoisomer had no effect on tumor biology. Following treatment with BB-94, freefloating clumps of tumor cells became surrounded by a capsule of host cells. These clumps of tumor cells typically formed one small (approxi mately 8 mm) avascular tumor of bright white appearance loosely at tached to fat in the peritoneum. Tumor cells within these capsules often appeared to be necrotic. Gel substrate analysis demonstrated that acti vated M, 92,000 type IV collagenase was present in the xenografts. We propose that inhibition of this enzyme causes the transition of ascites to solid tumors, concomitantly slowing tumor cell growth and allowing the development of tumor stroma. INTRODUCTION MMPs2 are a family of homologous enzymes capable of degrading components of the extracellular matrix. Different MMPs share highly homologous zinc-binding active sites, hemopexin-like domains, and cleavable NH2-terminal sequences the removal of which results in activation of the enzymes. Members of the MMP family are distin guished by their substrate specificities. Interstitial collagenase (MMP-1) degrades interstitial collagen and is the only MMP capable of doing so. Both M, 72,000 and M, 92,000 type IV collagenases (MMP-2 and MMP-9, respectively) digest types IV and V collagen and to a lesser extent proteoglycans and fibronectin (Refs. I and 2 and references therein), while stromelysin (MMP-3) preferentially de grades proteoglycan and fibronectin and has limited degradative ef fects on type IV collagen (3). MMPs are involved in tissue remodeling during wound healing (4) and embryogenesis (5). They also contribute to the damage which occurs in rheumatoid arthritis (6) and facilitate the passage of tumor cells across the basement membrane in metastasis (7-9). Given the involvement of MMPs in several disease states, consid erable interest has developed in the therapeutic potential of synthetic inhibitors. Inhibition of MMPs would be expected to limit the forma tion of metastasis by preventing basement membrane degradation and may have additional beneficial effects by inhibiting angiogenesis (10) and promoting the formation of stroma causing encapsulation of the Received 11/13/92; accepted 2/25/93. The costs of publication of this article were defrayed in pan by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ' To whom requests for reprints should be addressed, at Imperial Cancer Research Fund. P. O. Box 123. Lincoln's Inn Fields. London WC2A 3PX. England. 2 The abbreviations used are: MMP, matrix metalloproteinase; BB-94, [4-(N-kydroxyamino)-2/f-isobutyl-35-(thiopen-2-ylthiomethyl)-succinyl]-i.-phenylalanine-/V-methylamide; SDS, sodium dodecyl sulfate; PMN. polymorphonuclear neutrophils; TNF, tumor necrosis factor. tumor. BB-94 is a low-molecular-weight (MW = 478) synthetic com pound which inhibits a broad spectrum of MMPs. Here we report that this compound radically changes the composition of stroma and the biological behavior of ovarian carcinoma xenografts grown in nude mice, resulting in a dramatic reduction in tumor burden and a con comitant increase in survival. MATERIALS AND METHODS Ovarian Carcinoma Xenografts. The xenografts were established from human primary ovarian tumors. Both were maintained and passaged i.p. LA was established from a 72-year-old woman with a poorly differentiated muci nous cysadenocarcinoma. and HU was established from a 23-year-old woman with a moderately differentiated serous cystadenocarcinoma (11). BB-94. BB-94 was provided by British Biotechnology. Ltd. (Cowley, Ox ford, England). The structural formula of this compound is given in Fig. I. Essentially. BB-94 contains a peptide backbone which binds the molecule to matrix metalloproteinases and a hydroxamic acid group which binds to the catalytically active /.ine atom. The concentrations (nM) of BB-94 producing 50% inhibition against the following matrix metalloproteinases are: interstitial collagenase. 3; stromelysin. 20: M, 72,000 type IV collagenase, 4; M, 92.000 type IV collagenase. 1-10. BB-94 is a fine white powder and was sonicated into suspension at 2.5 mg/ml in phosphate-buffered saline (pH 7.2) plus Tween-20 (0.01%). This preparation was injected into nude mice i.p. An inactive diastereoisomer of BB-94. BB-1268 was prepared in an identical way. Gel Substrate Analysis. Gel substrate analysis was performed essentially as described by Brown et al. (12). Solid tumor was removed from BB-94treated HU-bearing mice and homogenized in SDS electrophoresis sample buffer (0.15 MTris, pH 8.8; 10% glycerol, v/v; 1% SDS w/v). Homogenized samples were applied directly without heating or reduction to a 5% stacking polyacrylamide gel laid over an 11% (w/v) polyacrylamide gel containing 1 mg/ml gelatin and 0.1% (w/v) SDS. Gels were run at room temperature at 180 V. After incubation of gels in 2.5% Triton X-100 for 30 min to remove SDS, the gels were incubated for 16 h in 50 m.MTris-HCl (pH 7.6) containing 0.2 M NaCl, 5 trivi CaCK, and 0.02% (w/v) Brij-35. Gels were stained for 3 h in 30% methanol/10% glacial acetic acid containing 0.5% (w/v) Coomassie brilliant blue G 250 and destained in the same solution in the absence of dye. Conditioned media from RPMI-7951 cells and HT-1080 stimulated with 10 ng/ml 12-O-tetradecanoylphorbol-l3-acetate for 36 h were used as type IV collagenase standards (12). Statistical Analysis. Student's r test was used in all statistical analysis.