Species differences in troxacitabine pharmacokinetics and pharmacodynamics: implications for clinical development.

PURPOSE: Troxacitabine is the first unnatural L-nucleoside analog to show potent preclinical antitumor activity and is currently under clinical investigation. Significant differences in troxacitabine toxicity between mice, rats, monkeys, and humans were observed during preclinical and clinical evaluations. To better understand the different toxicity and efficacy results observed between the human xenograft mouse tumor models used for preclinical assessment and the clinical study results, the pharmacodynamics and pharmacokinetics of troxacitabine were reassessed in murine and human models. EXPERIMENTAL DESIGN: Clonal and thymidine incorporation assays were used to investigate the in vitro antiproliferative activity of troxacitabine on a selected panel of mouse and human tumor cell lines and normal hemapoietic cells. Analysis of the intracellular metabolites of [14C]troxacitabine was determined in mouse and human T-lymphocytes obtained from peripheral blood. The antitumor efficacy of troxacitabine administered either as single or repeated high-dose bolus administrations or as low-dose continuous infusions was evaluated in the human colon HT-29 xenograft model. We also determined plasma concentrations of troxacitabine using the different administration schedules. RESULTS: Five to nine hundred-fold lower concentrations of troxacitabine were required to inhibit cell growth in human compared with murine tumor and normal hemapoietic cell lines. Furthermore, the sensitivity of cells of both species to troxacitabine was strongly time dependent, requiring >24 hours exposure for maximum activity. Analysis of the intracellular metabolites of [14C]troxacitabine in T-lymphocytes obtained from peripheral blood revealed subsequently higher levels of mono-, di-, and triphosphates in human compared with mouse. Antitumor efficacy studies revealed that prolonged exposure schedules (up to 6 days) showed equivalent efficacy to repeated high-dose bolus administrations. Five-day continuous infusion of 20 mg/mL troxacitabine via subcutaneous implanted mini-osmotic pump maintained systemic concentrations of 262 ng/mL (1.2 micromol/L) for the duration of administration, which are clinically achievable plasma concentrations, and led to significant antitumor activity [treated versus control (T/C) of 27% and tumor regression during treatment]. CONCLUSIONS: These studies support the hypothesis that troxacitabine infusions might be the administration regimen with the greatest likelihood of fully exploiting clinically the potent preclinical antitumor activity of troxacitabine.

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.

Novel nucleotide phosphonate analogues with potent antitumor activity.

We have identified several nucleotide phosphonates demonstrating in vitro antiproliferative activity in several human cancer cell lines with IC(50) values in the microM range. The synthesis as well as structure-activity relationship are described.