[CANCER RESEARCH 58, 4947-4956, November I. 1998] Antitumor Activity of SCH 66336, an Orally Bioavailable Tricyclic Inhibitor of Farnesyl Protein Transferase, in Human Tumor Xenograft Models and Wap-ras Transgenic Mice Ming Liu,1 Matthew S. Bryant, Jianping Chen, Suining Lee, Bohdan Yaremko, Phil Lipari, Michael Malkowski, Eric Ferrari, Loretta Nielsen, Nicholas Prioli, Janet Dell, Dineshwar Sinha, .lamed Syed, Walter A. Korfmacher, Amili A. Nomeir, C-C. Lin, Lynn Wang, Arthur G. Taveras, Ronald J. Doll, F. George Njoroge, Alan K. Mallams, Stacy Remiszewski, Joseph J. Catino, Viyyoor M. Girijavallabhan, Paul Kirschmeier, and W. Robert Bishop Departments of Biological Research-Oncology ¡M.L. J. C.. 5. L. B. Y.. P. L. M. M.. E. F., L. N., N. P.. J. D., L. W.. J. J. C.. P. K.. W. R. B.¡, Chemical Research ¡A.G. T.. R. J. D.. F. G. N., A. K. M.. S. R.. V. M. G.I. Drug Metabolism and Pharmacokinetics [M. S. B.. W. A. K.. A. A. N.. C-C. L.¡.and Safety Evaluation Center [D. S.. J. S.¡.Schering- Plough Research Institute. Kenilworth, New Jersey 07033 ABSTRACT We have been developing a series of nonpeptidic, small molecule lai - nesyl protein transferase inhibitors that share a common tricyclic nucleus and compete with peptide/protein substrates for binding to farnesyl pro tein transferase. Here, we report on pharmacological and in vivo studies with SCH 66336, a lead compound in this structural class. SCH 66336 potently inhibits Ha-Ras processing in whole cells and blocks the trans formed growth properties of fibroblasts and human tumor cell lines expressing activated Ki-Ras proteins. The anchorage-independent growth of many human tumor lines that lack an activated ras oncogene is also blocked by treatment with SCH 66336. In mouse, rat, and monkey sys tems, SCH 66336 has excellent oral bioavailability and pharmacokinetic properties. In the nude mouse, SCH 66336 demonstrated potent oral activity in a wide array of human tumor xenograft models including tumors of colon, lung, pancreas, prostate, and urinary bladder origin. Enhanced in vivo efficacy was observed when SCH 66336 was combined with various cytotoxic agents (cyclophosphamide, 5-fluorouracil, and vin- cristine). In a Ha-Ras transgenic mouse model, prophylactic treatment with SCH 66336 delayed tumor onset, reduced the average number of tumors/mouse, and reduced the average tumor weight/animal. In a ther apeutic mode in which gavage treatment was initiated after the transgenic mice had developed palpable tumors, significant tumor regression was induced by SCH 66336 in a dose-dependent fashion. This was associated with increased apoptosis and decreased DNA synthesis in tumors of animals treated with SCH 66336. Enhanced efficacy was also observed in this model when SCH 66336 was combined with cyclophosphamide. SCH 66336 is presently being evaluated in Phase I clinical trials. INTRODUCTION In normal cells. Ras switches between an inactive guanosine 5'- diphosphate-bound and an active GTP2-bound state, which can initiate several intracellular signaling pathways (1, 2). Ras signaling is ter minated by hydrolysis of GTP to guanosine 5'-diphosphate in a reaction that is stimulated by guanosine triphosphatase-activating proteins. As a consequence of specific mutational events in the Ras sequence, oncogenic Ras proteins have greatly reduced capacity to hydrolyze GTP. This leads to constitutive activation of downstream signaling pathways resulting in altered regulation of cellular prolifer ation (1, 3). Received 6/10/98; accepted 8/28/98. The costs of publication of this article were defrayed in part 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. 1To whom requests for reprints should be addressed, at Schering-Plough Research Institute. 2015 Galloping Hill Road, K-15-4-4945, Kenilworth. NJ 07033. Phone: (908) 740-7136: Fax: (908) 740-7115. 2 The abbreviations used are: GTP. guanosine 5'-triphosphate; FPT. farnesyl protein transferase; GGPT-1, geranylgeranyl protein transferase-1 ; mpk. mg/kg body weight; HP/3CD. hydroxypropyl-ß-cyclodexlrin; q.i.d.. four times/day; Cytoxan. cyclophospha mide; 5-FU. 5-fluorouracil: TÚNEL, terminal deoxynucleolidyl transferase-mediated dUTP-biotin nick end labeling: b.i.d.. twice/day; BrdUrd. 5-bromo-2'-deoxy-uridine: MC. methyl cellulose; Cmax. peak serum concentration. Four isoforms of the Ras protein exist: Ha-Ras, N-Ras, Ki-Ras 4A, and K-Ras 4B (3). They are products of three genes, with Ki-Ras4A and Ki-Ras4B being splice variants of the same gene. Oncogenic mutations of the different isoforms predominate in different tumors (4). Ha-ras mutations are found in carcinomas of the bladder, kidney, and thyroid; N-ras mutations are found in myeloid and lymphoid cancers, liver carcinoma, and melanoma; whereas Ki-ras mutations predominate in colon, lung, and pancreatic carcinoma. The functional differences of the four isoforms remain unknown. Many lines of evidence suggest that antitumor activity can be achieved by interfering with the function of oncogenic Ras proteins (5-7). Signal transduction by Ras is dependent on its plasma mem brane localization. This localization is supported by a series of post- translational modifications. The first modification is farnesylation of a cysteine residue near the COOH-terminus of Ras catalyzed by FPT. Ras prenylation is thought to be the critical modification for proper membrane localization and function (8-10). Therefore, FPT inhibi tion is a potential mechanism to interfere with Ras-driven tumor growth. Recent studies suggest that prenylation of Ras proteins is complex. In vitro, both Ki- and N-Ras proteins are substrates for a related protein prenyl transferase, GGPT-1 (11, 12). Although this reaction occurs with a lower catalytic efficiency than the farnesylation of these proteins, geranylgeranylation of Ki- and N-Ras proteins has been observed in cells treated with FPT inhibitors (13, 14). In contrast, the Ha-Ras protein is not a substrate for GGPT-1 in vitro or in cells. Because geranylgeranylated forms of Ras can support cellular trans formation when overexpressed (9), this reaction represents a potential resistance mechanism for Ras-transformed cells to the effects of FPT inhibitors. Despite this alternative prenylation, inhibitors of FPT dem onstrate in vitro and in vivo antitumor activity in tumor models expressing a mutationally activated Ki-ras gene (15-19). Thus, in some cases, blocking farnesylation of proteins other than Ras may contribute to the observed antitumor properties. We have previously reported on a series of nonpeptidic, small molecule FPT inhibitors (15, 20-24).3 These compounds share a common tricyclic nucleus, compete with peptide/protein substrates for binding to FPT, and inhibit growth of Ras-transformed fibroblasts and human cancer cells in vitro and in vivo. Here, we report on in vivo studies with SCH 66336, an improved compound in this structural class. The data demonstrate that SCH 66336 possesses potent antitu- mor activity against a wide variety of tumors after oral administration in mice. This compound is presently undergoing Phase I human clinical trials as an anticancer chemotherapeutic agent. ' R. J. Doll, el al. SCH 66336 chemical synthesis, manuscript in preparation. 4947