Synergistic activity of troxacitabine (Troxatyl) and gemcitabine in pancreatic cancer.

BACKGROUND: Gemcitabine, a deoxycytidine nucleoside analog, is the current standard chemotherapy used as first-line treatment for patients with locally advanced or metastatic cancer of the pancreas, and extends life survival by 5.7 months. Advanced pancreatic cancer thus remains a highly unmet medical need and new therapeutic agents are required for this patient population. Troxacitabine (Troxatyl) is the first unnatural L-nucleoside analog to show potent preclinical antitumor activity and is currently under clinical investigation. Troxacitabine was recently evaluated as a first-line therapy in 54 patients with advanced adenocarcinoma of the pancreas and gave comparable overall results to those reported with gemcitabine in recently published randomized trials. METHODS: The human pancreatic adenocarcinoma cell lines, AsPC-1, Capan-2, MIA PaCa-2 and Panc-1, were exposed to troxacitabine or gemcitabine alone or in combination, for 72 h, and the effects on cell growth were determined by electronic particle counting. Synergistic efficacy was determined by the isobologram and combination-index methods of Chou and Talalay. Mechanistic studies addressed incorporation of troxacitabine into DNA and intracellular levels of troxacitabine and gemcitabine metabolites. For in vivo studies, we evaluated the effect of both drugs, alone and in combination, on the growth of established human pancreatic (AsPC-1) tumors implanted subcutaneously in nude mice. Statistical analysis was calculated by a one-way ANOVA with Dunnett as a post-test and the two-tailed unpaired t test using GraphPad prism software. RESULTS: Synergy, evaluated using the CalcuSyn Software, was observed in all four cell-lines at multiple drug concentrations resulting in combination indices under 0.7 at Fa of 0.5 (50% reduction of cell growth). The effects of drug exposures on troxacitabine and gemcitabine nucleotide pools were analyzed, and although gemcitabine reduced phosphorylation of troxacitabine when cells were exposed at equal drug concentrations, there was no effect on phosphorylated pools at drug combinations that were synergistic. The amount of troxacitabine incorporated into DNA was also not affected by the presence of gemcitabine. In vivo testing against a human pancreatic (AsPC-1) xenograft mouse tumor model indicated that both drugs were more than additive at well-tolerated doses and schedule. The biological basis for this synergy is unclear as we did not observe changes in apoptosis, DNA repair, troxacitabine incorporation into DNA or troxacitabine metabolism in the presence of gemcitabine. CONCLUSION: These data, together with phase I clinical data showing tolerability of both agents when combined, suggest combination therapy with troxacitabine and gemcitabine warrants further evaluation in advanced pancreatic cancer patients.

BCH-1868 [(-)-2-R-dihydroxyphosphinoyl-5-(S)-(guanin-9'-yl-methyl) tetrahydrofuran]: a cyclic nucleoside phosphonate with antitumor activity.

Nucleoside phosphonates are widely used therapeutic agents with a broad spectrum of antiviral activity. However, only a few of them are reported to have antitumor activity. In this study, we show that a tetrahydrofuran phosphonate analogue of guanosine, (-)-2-R-dihydroxyphosphinoyl-5-(S)-(guanin-9'-ylmethyl) tetrahydrofuran (BCH-1868), previously reported as having antiviral activity, also displays antitumor activity. In vitro, BCH-1868 inhibited the proliferation of several murine and human cancer cell lines with IC50s in the microM range independently of the tissue type or the presence of multidrug resistance protein MRP/gp190. In vivo, BCH-1868 was active against a variety of human tumor xenograft models (Caki-1, HT-29, DU 145, COLO 205, and CCRF-CEM). In all tumors tested, a significant tumor growth inhibition was noted at 40-50 mg/kg (daily x 5), but no tumor regression was observed in the settings used. To better understand these results, we partially characterized, at the cellular level, the mechanism of action of this new cyclic nucleoside phosphonate and investigated its pharmacokinetic characteristics in mice. We showed that BCH-1868 exerts its antitumor activity by an inhibitory mechanism at the level of DNA polymerase a, resulting in arrest of DNA synthesis and a block of cell division at the S phase of the cell cycle. Low-circulating plasma concentration (Cmax = 87 microM; area under the curve = 1138 micromol x min/liters; after a bolus i.v. injection of 10 mg/kg) and rapid clearance of the drug (terminal half-life, t1/2 = 16 min) may contribute to the modest antitumor efficacy observed in vivo.

Antitumor activity of troxacitabine (Troxatyl) against anthracycline-resistant human xenografts.

PURPOSE: We have recently identified a deoxycytidine nucleoside analogue, troxacitabine (beta- L-dioxolane cytidine, Troxatyl; Shire BioChem), which has potent antitumor activity against both leukemia and solid tumors. In contrast to the cytidine nucleoside analogues currently in clinical use (cytarabine and gemcitabine), troxacitabine is a poor substrate of nucleoside transporters and enters cells primarily by passive diffusion. This unusual property led us to evaluate the efficacy of troxacitabine in multidrug resistant (MDR) and multidrug resistance-associated protein (MRP) tumors. METHODS: The in vitro antiproliferative activity of troxacitabine was investigated in the human nasopharyngeal epidermoid carcinoma cell line, KB, and its vincristine-resistant derivative (KBV), as well as in human leukemia cell lines of myeloid and lymphoblastoid origin, HL60 and CCRF-CEM, respectively, and their MDR (HL60/R10 and CCRF-CEM/VLB) and MRP (HL60/ADR) derivatives, using the thymidine incorporation assay. For in vivo studies, we compared the antitumor efficacy of troxacitabine with that of doxorubicin and vinblastine in xenograft models of these solid and hematological human anthracycline-resistant tumor xenografts. RESULTS: Troxacitabine demonstrated potent antiproliferative activity against both P-glycoprotein-positive (KBV, HL60/R10, CCRF-CEM/VLB) and P-glycoprotein-negative (HL60/ADR) multidrug-resistant cell lines with IC(50) values ranging from 7 to 171 n M. Tumor regression was observed in the KBV xenograft following a 5-day treatment with 20, 50 and 100 mg/kg of troxacitabine, with percent total growth inhibition (TGI) of 81, 96 and 97, respectively, and some cures at the two highest dose levels. In the HL60, HL60/R10, HL60/ADR and CCRF-CEM/VLB xenografts, the effect of troxacitabine was evaluated on survival time. In the HL60 promyelocytic human xenograft models, troxacitabine treatment (25, 50 and 100 mg/kg per day for 5 days) was initiated 10 days after tumor cell inoculation, once animals had developed disseminated tumors. In all three promyelocytic leukemia xenografts, troxacitabine was quite potent, producing T/C values of 162% to 315% as well as complete cures at the higher dose levels. In the CCRF-CEM/VLB T-lymphoblastoid leukemia xenograft, troxacitabine treatment (10, 30 or 250 mg/kg total doses using different schedules) was initiated 20 days after tumor cell inoculation. Troxacitabine was not as potent in this model but did result in significant antileukemic activity (T/C of 131%) when administered at 10 mg/kg on days 20, 27 and 34. CONCLUSIONS: These results indicate that troxacitabine has a potent in vivo antitumor activity associated with tumor regressions and complete cures in animals with tumors refractory to current chemotherapeutic agents.

Complementary antineoplastic activity of the cytosine nucleoside analogues troxacitabine (Troxatyl) and cytarabine in human leukemia cells.

PURPOSE: Troxacitabine (BCH-4556, l-(-)-OddC, Troxatyl) is a novel beta- l-nucleoside analogue with potent antineoplastic activity both in vitro and in several tumor models in vivo, and is presently in phase II clinical trials. The combination of the cytosine analogues troxacitabine and araC (1-beta- d-arabinofuranosylcytosine, cytarabine) has shown promising activity in patients with acute myelogenous leukemia. To further examine the interactions between these two analogues, we investigated the in vitro and in vivo effects of their combination against a human leukemia cell line, CCRF-CEM. METHODS: . The in vitro cytotoxic effect of the combination of troxacitabine and araC on the survival of CCRF-CEM cells was measured using a standard MTT assay and combination indices were generated with the CalcuSyn software. For in vivo studies, we evaluated the effect of both drugs, alone and in combination, on survival of CCRF-CEM tumor-bearing animals. Mechanistic studies addressed recovery of DNA synthesis, intracellular levels of araC metabolites, feedback inhibition by triphosphate species and pharmacokinetics of both drugs. RESULTS: The combination of troxacitabine and araC in vitro was synergistic with combination indices between 0.1 and 0.7. This appeared to be related to the impact of the combination on DNA synthesis recovery, which was significantly delayed following exposure to the combination of troxacitabine and araC compared to either agent alone. Analysis of the effect of troxacitabine on the intracellular metabolites of araC revealed that troxacitabine did not inhibit araC deamination and caused a slight decrease in the overall intracellular accumulation of araCTP. The lower accumulation of araCTP could not be attributed to feedback inhibition caused by troxacitabine triphosphate on dCK. Furthermore, our in vivo experiments demonstrated that the combination of araC and troxacitabine was better at slowing down the progression of leukemia in SCID mice than either agent used alone without additive toxicities. Injections of 10 mg/kg troxacitabine i.p. daily for 5 days in combination with araC at 10 mg/kg led to an increase in median survival time of 58 days compared to 49.5 and 53.5 days for araC and troxacitabine, respectively, given as single agents. This represents an increase in life span of 17%, respectively when compared to araC alone. A pharmacokinetic study revealed that troxacitabine did not influence the disposition of araC when coadministered. CONCLUSIONS: Overall, our results show that the antileukemic activity of troxacitabine and araC is complementary when the two nucleoside analogues are combined in vivo. These effects appear to be related to their interaction at the level of DNA repair rather than to pharmacokinetic interactions. These results encourage the use of troxacitabine and araC in combination in patients with acute leukemia.