Jeff Torri

PURPOSE: This phase I study was performed to evaluate the safety and pharmacokinetics of escalating doses of Marimastat (British Biotech, Inc, Oxford, United Kingdom) in patients with advanced malignancies and to determine the phase II recommended dose to be used in subsequent studies. PATIENTS AND METHODS: A standard phase I design was used in this study, in which consecutive groups of three patients were treated with escalating doses of the study drug. Marimastat was administered orally at 25, 50, or 100 mg twice daily to consecutive groups of patients with advanced lung cancer. An additional three patients were added at the highest dose studied (100 mg orally twice daily) to assess whether the inflammatory polyarthitis observed at that dose level can be prevented by a concurrent administration of nonsteroidal antiinflammatory drugs (NSAIDS) and/or low-dose corticosteroids. Blood was drawn for safety monitoring, pharmacokinetic analysis, and plasma levels of metalloproteinase (MMP)-2 and MMP-9 (determined by zymography). A total of 12 patients were studied. RESULTS: The most significant toxicity at the highest dose studied (100 mg orally twice daily) was a symptomatic inflammatory polyarthritis that persisted for up to 8 weeks after discontinuation of the study drug and was dose-limiting. The estimated plasma elimination half-life of Marimastat was 4 to 5 hours. The mean maximum concentration (Cmax) at a reasonably well-tolerated dose (50 mg orally twice daily) was 196 ng/mL and was reached within 1 to 2 hours (Tmax) after administration. Areas under the curve (AUC) tended to correlate with the dose of Marimastat. Zymographic analysis of peripheral-blood ratios of activated proenzymatic forms of MMP-2 and -9 did not show any consistent patterns of change in MMP levels or in a degree of their activation during the course of treatment. CONCLUSION: Marimastat was well absorbed from the gastrointestinal tract, with high levels of the study drug detected in plasma within hours after drug administration. Plasma concentrations of Marimastat achieved at dose levels 2 and 3 (50 mg and 100 mg orally twice daily) were substantially higher than those required for MMP inhibition in vitro. The dose-limiting toxicity (DLT) was severe inflammatory polyarthritis, which seemed to be a cumulative toxicity.

A novel matrix-degrading enzyme was identified from human breast cancer cells. This enzyme appears as major gelatinase in hormone-dependent breast cancer cell lines and has as an apparent molecular mass of 80 kDa on gelatin zymography. The 80-kDa enzyme has a unique metal ion specificity. In addition to calcium ions, the gelatinolytic activity can be supported by manganese and/or magnesium. Unlike 92- and 72-kDa gelatinases and other known members of the metalloproteinase family, the 80-kDa protease is not activated by p-aminophenylmercuric acetate and its gelatinolytic activity is not inhibited by tissue inhibitor of metalloproteinase 2. It is active over the pH range 7.5-9.5 with an optimum at pH 8.5. The enzyme degrades gelatin and type IV collagen. The proteolytic activity of the enzyme is inhibited by EDTA and leupeptin. These unique features clearly distinguish the 80-kDa protease from the known 92-and 72-kDa gelatinases. The expression of 80-kDa enzyme can be detected in hormone-dependent human breast cancer cell lines in vitro and in tumors grown from these cells in athymic nude mice.

A major, apparently novel extracellular matrix-degrading protease was previously identified and partially isolated from hormone-dependent but not from hormone-independent human breast cancer cells (Shi, Y. E., Torri, J., Yieh, L., Wellstein, A., Lippman, M. E., and Dickson, R. B. (1993) Cancer Res. 53, 1409-1415). Although initially the 80-kDa protease was identified from breast cancer cell-conditioned medium, immunofluorescence staining of breast cancer cells with anti-80-kDa protease monoclonal antibody 21-9 showed that in addition to its detection in intracellular compartments, the protease was uniformly localized around periphery of the cells with more intensive staining on the pseudopodia and membrane ruffles. A surface biotinylation technique confirmed the plasma membrane localization of the protease. In addition, the 80-kDa protease could not be washed from the membrane fraction of homogenized breast cancer cells with high concentrations of salts or with EDTA.The 80-kDa protease may noncovalently associate with other protein(s) to form complexes, the 95- and 110-kDa proteases. Both complexes showed gelatinolytic activity and bore the epitopes recognized by monoclonal antibody 21-9. Furthermore, both complexes could be converted to 80-kDa forms by boiling in SDS in the absence of reducing agents. Expression of this novel, integral membrane gelatinase could allow breast cancer cells an alternative to other previously described matrix-degrading enzymes for degradation of the extracellular matrix in close proximity to their surfaces. A major, apparently novel extracellular matrix-degrading protease was previously identified and partially isolated from hormone-dependent but not from hormone-independent human breast cancer cells (Shi, Y. E., Torri, J., Yieh, L., Wellstein, A., Lippman, M. E., and Dickson, R. B. (1993) Cancer Res. 53, 1409-1415). Although initially the 80-kDa protease was identified from breast cancer cell-conditioned medium, immunofluorescence staining of breast cancer cells with anti-80-kDa protease monoclonal antibody 21-9 showed that in addition to its detection in intracellular compartments, the protease was uniformly localized around periphery of the cells with more intensive staining on the pseudopodia and membrane ruffles. A surface biotinylation technique confirmed the plasma membrane localization of the protease. In addition, the 80-kDa protease could not be washed from the membrane fraction of homogenized breast cancer cells with high concentrations of salts or with EDTA. The 80-kDa protease may noncovalently associate with other protein(s) to form complexes, the 95- and 110-kDa proteases. Both complexes showed gelatinolytic activity and bore the epitopes recognized by monoclonal antibody 21-9. Furthermore, both complexes could be converted to 80-kDa forms by boiling in SDS in the absence of reducing agents. Expression of this novel, integral membrane gelatinase could allow breast cancer cells an alternative to other previously described matrix-degrading enzymes for degradation of the extracellular matrix in close proximity to their surfaces.