Preclinical and Clinical Pharmacodynamic Assessment of L- 778,123, a Dual Inhibitor of Farnesyl:Protein Transferase and Geranylgeranyl:Protein Transferase Type-I Robert B. Lobell, 1 Dongming Liu, 2 Carolyn A. Buser, Joseph P. Davide, Elizabeth DePuy, Kelly Hamilton, Kenneth S. Koblan, Yih Lee, Scott Mosser, Sherri L. Motzel, James L. Abbruzzese, Charles S. Fuchs, Eric K. Rowinsky, Eric H. Rubin, Sunil Sharma, Paul J. Deutsch, Kathryn E. Mazina, Briggs W. Morrison, Lynne Wildonger, Siu-Long Yao, and Nancy E. Kohl Departments of Cancer Research [R. B. L., D. L., C. A. B., J. P. D., K. H., K. S. K., S. M., N. E. K.], Drug Metabolism [E. D.,Y. L.], Laboratory Animal Resources [S. L. M.], and Clinical Pharmacology [P. J. D., K. E. M., B. W. M., L. W., S-L. Y.], Merck Research Laboratories, West Point, Pennsylvania 19486 and Rahway, New Jersey 07065; University of Texas M.D. Anderson Cancer Center, Houston, Texas 77030 [J. L. A.]; Dana-Farber Cancer Institute, Boston, Massachusetts 02115 [C. S. F.]; Institute for Drug Development, Cancer Therapy and Research Center, San Antonio, Texas 78245 [E. K. R.]; University of Medicine and Dentistry New Jersey-Cancer Institute, New Brunswick, New Jersey 08901 [E. H. R.]; and Memorial Sloan-Kettering Cancer Center, New York, New York 10021 [S. S.] Abstract Farnesyl:protein transferase (FPTase) inhibitors were developed as anti-Ras drugs, but they fail to inhibit Ki-Ras activity because Ki-Ras can be modified by geranylgeranyl:protein transferase type-I (GGPTase-I). L-778,123, an inhibitor of FPTase and GGPTase-I, was developed in part because it can completely inhibit Ki-Ras prenylation. To support the clinical development of L-778,123, we developed pharmacodynamic assays using peripheral blood mononuclear cells (PBMCs) to measure the inhibition of prenylation of HDJ2 and Rap1A, proteins that are FPTase- and GGPTase-I substrates, respectively. We validated these assays in animal models and show that inhibition of HDJ2 prenylation in mouse PBMCs correlates with the concentration of FPTase inhibitors in blood. In dogs, continuous infusion of L-778,123 inhibited both HDJ2 and Rap1A prenylation in PBMCs, but we did not detect inhibition of Ki-Ras prenylation. We reported previously results from the first L-778,123 Phase I trial that showed a dose-dependent inhibition of HDJ2 farnesylation in PBMCs. In this report, we present additional analysis of patient samples from this trial and a second Phase I trial of L-778,123, and demonstrate the inhibition of both HDJ2 and Rap1A prenylation in PBMC samples. This study represents the first demonstration of GGPTase-I inhibition in humans. However, no inhibition of Ki-Ras prenylation by L-778,123 was detected in patient samples. These results confirm the pharmacologic profile of L-778,123 in humans as a dual inhibitor of FPTase and GGPTase- I, but indicate that the intended target of the drug, Ki-Ras, was not inhibited. Introduction The Ras proteins are M r 21,000 GTP binding proteins that affect cell proliferation and survival. The Ras isoforms include the splicing variants of the Ki-ras gene, Ki4A-Ras and Ki4B- Ras, and the Ha-Ras and N-Ras isoforms. Ras functions at the plasma membrane, where it transduces signals from extracellular growth factor receptors to downstream effec- tors including the Raf and phosphatidylinositol 3' kinases. Ras is normally activated by GTP binding and deactivated by GTP hydrolysis, biochemical events that are facilitated by auxiliary protein factors. The Ras pathway is dysregulated in 30% of all human cancers through mutations that inacti- vate the GTPase activity of Ras, giving rise to oncogenic proteins that constitutively send proliferation and survival signals (1, 2). Ras is post-translationally modified by an isoprenoid lipid that serves to anchor the protein at the plasma membrane, and which is required for its biological and/or transforming functions (3– 6). FPTase 3 catalyzes this lipid modification by conjugating the 15-carbon farnesyl group to a cysteine res- idue four amino acids from the COOH terminus of Ras (7). Farnesylation occurs on the cysteine that is part of the CA 1 A 2 X motif, where C is cysteine, A is typically an aliphatic amino acid, and X is typically serine or methionine for sub- strates of FPTase. In the past decade, many FTIs have been identified, and several of these are currently under clinical evaluation as a treatment for cancer (8, 9). Cancer cells transformed with oncogenic Ha-ras are growth inhibited by FTIs and are typically more responsive than cells harboring wild-type ras or oncogenic forms of Ki-ras or N-ras (10 –12). Similarly, mammary tumors formed in transgenic mice because of expression of oncogenic Ha- ras undergo dramatic regression when animals are treated with an FTI (13, 14). In contrast, mammary tumor growth in transgenic mice expressing oncogenic Ki- ras or wild-type Received 3/11/02; revised 5/7/02; accepted 5/13/02. 1 To whom requests for reprints should be addressed, at Department of Cancer Research, Merck Research Laboratories, WP26 – 462, West Point, PA 19486. Phone: (215) 652-8813; Fax (215) 993-3398; E-mail: rob_lobell@ merck.com. 2 Present address: Exelixis Pharmaceuticals Inc., South San Francisco, CA 94080. 3 The abbreviations used are: FPTase, farnesyl:protein transferase; FTI, farnesyl:protein transferase inhibitor; GGPTase-I, geranylgeranyl:protein transferase type-I; GGTI, geranylgeranyl:protein transferase type-I inhib- itor; PBMC, peripheral blood mononuclear cell; PD, pharmacodynamic; DPI, dual prenyltransferase inhibitor; MTD, maximum tolerated dose; MDCK, Madin-Darby canine kidney cell. 747 Vol. 1, 747–758, July 2002 Molecular Cancer Therapeutics on December 23, 2021. © 2002 American Association for Cancer Research. mct.aacrjournals.org Downloaded from