SYSTEMIC THERAPY OF PROSTATE CANCER

Our overall goal is to develop more effective systemic therapy for patients with prostate cancer. Antimicro-tubule drugs are used as chemotherapeutic agents due to their effects on essential cellular functions such as mitosis, organelle transport, and maintenance of cell shape. We are continuing a series of clinical trials based on the hypothesis that inhibition of microtubule function is achieved with greatest therapeutic index by drugs that interact with the microtubule at distinct sites. This approach has yielded several estramustine-based antimicrotubule drug combinations that are effective against hormone refractory prostate cancer (HRPC). Our current clinical trials are intended to further improve upon the safety and efficacy of chemotherapy for all patients with prostate cancer. In the laboratory, we are continuing studies to elucidate the causes of resistance to estramustine, paclitaxel and other antimicrotubule agents. We are also investigating several novel agents in Phase I trials. We have begun pharmacodynamic studies to correlate changes in p21ras with the antitumor and toxic effects of new drugs directed against this pivotal signaling molecule.

The combination of weekly paclitaxel and oral estramustine phosphate (EMP) has been evaluated in a Phase I trial conducted by the Fox Chase Network and Indiana University. In clinical trial, we defined a safe dose of paclitaxel given as a short infusion for six of eight weeks, in combination with two doses of oral EMP. The higher dose of EMP proved to be too toxic, resulting in severe gastrointestinal and cardiovascular toxicity. In contrast, EMP taken three days per week at a reduced dosage of 280 mg twice daily (b.i.d.) was well-tolerated. In patients completing at least four weeks of treatment, we observed reduction of tumor volume by at least 50% in 6 of 11 patients (55%) with measurable soft-tissue metastases. Elevated serum levels of prostate specific antigen (PSA) were reduced by at least 50% in 10 of 16 patients (62.5%), and by at least 80% in 7 of 16 (43.8%). The median duration of these responses was eight months. This treatment program has been opened as a larger, Phase II trial within the Eastern Cooperative Oncology Group (ECOG) to determine the effects of weekly paclitaxel plus oral estramustine on pain, quality of life, PSA and measurable tumors.

We are also completing a Phase I trial of intravenous (IV) EMP in patients with HRPC and other solid tumors. Compared to the oral formulation, IV EMP appears to cause less nausea and cardiovascular toxicity, yet retains antitumor activity as determined by decreased serum PSA levels. The pharmacokinetics of IV EMP demonstrate higher peak plasma levels for the active metabolites estra- and estromustine, which may prove advantageous for combination therapy utilizing IV EMP with paclitaxel and other microtubule inhibitors. As predicted from preclinical studies, the dose-limiting toxicity of IV EMP appears to be hepatotoxicity. Based on these results, a larger, multicenter Phase II trial has commenced to determine more precisely the effectiveness of IV EMP in HRPC. A Phase II trial of IV EMP in combination with paclitaxel, both given weekly, is also planned.

Although recent clinical trials have demonstrated that cytotoxic therapy has an important role in the palliative treatment of patients with HRPC, such therapy is not curative and prolonged survival has not been definitely established. As such, there is an urgent need to identify novel approaches. Ras proteins (p21ras) play key roles in cell growth and survival, and activating mutations of p21ras are common in human cancer. Post-translational addition of a 15-carbon lipid moiety by farnesyl protein transferase 1 (FPT-1) onto proteins possessing a CAAX amino acid motif at the carboxyl terminus is required for localization and function of p21ras and several other cellular proteins. R115777 is a potent non-peptidomimetic inhibitor of FPT-1 in vitro and in vivo, inhibiting growth of cells with H-, N-, and K-ras mutations at median inhibitory concentrations (IC50s) ranging from 7 to 50 nM. We are completing a Phase I trial of R115777 in patients with refractory solid tumors to determine the dose-limiting toxicity, optimal dose, and pharmacokinetic properties of R115777 given orally, b.i.d. for 21 days. A novel Bayesian dose escalation schema, Escalation With Overdose Control (EWOC), is being utilized to estimate the maximum tolerable dose (MTD; dose producing 33% chance of dose-limiting toxicity) with maximal efficiency and safety.

Preliminary pharmacokinetic studies have shown that peak plasma concentrations (C_max) of R115777 were reached at 0.8 to 3 hours after oral dosing (n=9). Day 1 values of R115777 Cmax and AUC_0-12h (12-hour area under the plasma concentration versus time curve) increased linearly. Cmax increased from 320 to 2,266 ng/ml and AUC from 1,024 to 11,276 ng-hour/ml over the R115777 dose range of 60 to 420 mg/m² b.i.d. Plasma elimination of R115777 was biphasic, with an initial half-life of approximately five hours. Steady state was reached within two to three days, and was maintained throughout the 21-day dosing period. In parallel laboratory studies, we have observed increased cytosolic, or non-farnesylated, p21ras in peripheral blood mononuclear cells obtained from patients after 7 and 14 days of R115777 therapy. This finding indicates that we are achieving inhibition of FPT-1 in human tissues.

Neutropenia and thrombocytopenia, evident by day 15, have been the principal dose-limiting toxicities of R115777. Fatigue, skin rash and hepatic toxicity have also been observed in patients treated in the range of 200-250 mg/m² b.i.d. Estimated MTD based on current data is 240 mg/m². One patient with parotid carcinoma and a second patient with HRPC had stabilization of their tumors for over six months on R115777. Following completion of the Phase I trial, we plan to conduct a Phase II trial of R115777 in patients with prostate cancer. Other studies of R115777 are planned in patients with superficial bladder carcinoma, and in colon and pancreatic carcinomas, tumors that are associated with a high prevalence of ras mutations.

P21ras, a guanosine triphosphate (GTP) binding protein that regulates cell proliferation, requires farnesylation for its membrane association and transforming activity. To investigate the effects of p21ras inhibition on the cytotoxicity of paclitaxel, human prostate (DU145) and breast (MCF7) carcinoma cells were treated with paclitaxel and R115777. Exposure of DU145 and MCF-7 cells to 5 and 10 nM R115777 for 48 hours resulted in the complete inhibition of p21ras and an approximate 50% inhibition of lamin B farnesylation in both cell lines as determined by Western Blot analysis. Treatment of cells with 5 and 10 nM R115777 (<=IC 10) in combination with paclitaxel resulted in synergistic activity of these agents as determined by cytotoxicity assays. Similarly, cell cycle analysis of drug-treated DU145 cells (72 hours) resulted in enhanced G₂/M block in the 50 nM R115777 plus 1 nM paclitaxel group (38% of cells in G₂/M phase) when compared to cells treated with 50 nM R115777 alone (31.1%) or paclitaxel alone (23.8%). In contrast, cell cycle analysis of MCF-7 cells indicated enhanced G₁ block as a result of combination treatment. Five and 10 nM doses of R115777 that resulted in complete inhibition of p21ras farnesylation, and also in synergistic cytotoxicity with paclitaxel, failed to have any effect on the cell cycle distribution of these cells. These data suggest that additional factors are involved in the mechanism of synergy for these two agents. In our current experiments, we are examining the effects of R115777 on cytoskeletal protein morphology and function. The enhanced cytotoxicity of paclitaxel as a result of its combination with farnesyltransferase inhibitor may be of therapeutic importance.

Previous studies from our laboratory have shown that treatment of cells with the antimicrotubule agents, estramustine and paclitaxel, results in elevation of b_III tubulin isotype; DU145 cells that were made resistant to estramustine and paclitaxel had elevated b_III and b_IVa tubulin isotypes. These observations prompted us to clone and sequence the human bIII tubulin cDNA. To investigate further the function of b-tubulin isotypes in antimicrotubule drug response, DU-145 cells were transfected with b_III cDNA, which had been subcloned into the ZEOSV vector; control cells were transfected with vector alone. To distinguish between endogenous and transfected b_III, an 18 basepair hemagglutinin (HA) epitope tag was fused to the b_III cDNA prior to subcloning into the vector. Transfected cells were selected in the presence of Zeocin and cloned. Western Blot analysis using antibodies against the HA epitope and b_III demonstrated a 3- to 4-fold increase in the b_III protein when compared to control cells. Reverse transcription (RT)-PCR analysis corroborated this increase, showing a similar result with b_III mRNA, and also indicated a lack of coordinate increase of other b-tubulin isotype mRNAs. Immunofluorescent staining of the transfectant cells with antibodies against HA epitope demonstrated incorporation of HA tagged b_III into functional microtubules. To determine the function of b_III tubulin isotype in antimicrotubule drug response, cytotoxicity assays were performed with estramustine, paclitaxel, vinblastine and colchicine. Cytotoxicity profiles for all of these antimicrotubule agents were similar for b_III transfectant and vector transfectant cell lines. Currently, we are establishing double transfectants of b_III and b_IVa to determine if the combination of isotypes is necessary for antimicrotubule drug resistance.

BERLIN, J.D., PROPERT, K.J. TRUMP, D., WILDING, G., HUDES, G., GLICK, J., BURCH, P., KELLER, A., LOEHRER, P. 5-Fluorouracil and leucovorin therapy in patients with hormone refractory prostate cancer: An Eastern Cooperative Oncology Group Study (E1889). Am. J. Clin. Oncol. 21(2):171-176, 1998.

HUDES, S.R., LIPSITZ, S., GREM, J., MORRISEY, M., WEINER, L., FUGLER, J.W., BENSON, A. A Phase II study of 5fluorouracil, leucovorin, and Interferon-a in the treatment of patients with metastatic or recurrent gastric carcinoma. An Eastern Cooperative Oncology Group Study (E5292). Cancer 85:290-294, 1999.

HUDES. G. Signaling inhibitors in the clinic: New agents and new challenges. J. Clin. Oncol. 17:1093-1094, 1999.

SOKOL, D.B., HUDES, G.R. Cisplatin-based chemotherapy for testicular cancer in a patient with spinal muscular atrophy: A case report. Am. J. Clin. Oncol. (CCT) 21(4):420-421, 1998.

HUDES, G.R., NATHAN, F., KHATER, C., HAAS, N., CORNFIELD, M., GIANTONIO, B., GREENBERG, R., GOMELLA, L., LITWIN, S., ROSS, E., ROETHKE, S. AND MCALEER, C. Phase II trial of 96-hour paclitaxel plus oral estramustine phosphate in metastatic hormone-refractory prostate cancer. J. Clin. Oncol. 15(9):3156-3163, 1997.

RANGANATHAN, S., DEXTER, D.W., BENETATOS, C. A., HUDES, G.R. Cloning and sequencing of human b _III-tubulin cDNA: Induction of b_III isotype in human prostate carcinoma cells by acute exposure to antimicrotubule agents. Biochem. Biophys. Acta 1395:237-245, 1998.

RANGANATHAN, S., BENETATOS, C.A., COLARUSSO, P.J., DEXTER, D.W., HUDES, G.R. Altered b-tubulin isotype expression in paclitaxel-resistant human prostate carcinoma cells. Br. J. Cancer 77(4):562-566, 1998.

^§   Fox Chase researcher

a   C. Garay: Present address-Rhône-Poulenc Rorer, Collegeville, PA 19426

b   B. Hartley-Asp, M. Kopreski: Pharmacia-Upjohn, Kalamazoo, MI 49001

c   B. Roth: Indiana University Medical Center, University Hospital, Indianapolis IN 46202

d   I. Horak: Janssen Research, Titusville, NJ 08560

Illustrations or unpublished data in these reports should not be used without permission of the author.

Fox Chase Cancer Center

Scientific Report 1998