Exposure-dependent incorporation of trifluridine into DNA of tumors and white blood cells in tumor-bearing mouse.

PURPOSE: Trifluridine (TFT) is an antitumor component of a novel nucleoside antitumor agent, TAS-102, which consists of TFT and tipiracil hydrochloride (thymidine phosphorylase inhibitor). Incorporation of TFT into DNA is a probable mechanism of antitumor activity and hematological toxicity. The objective of this study was to examine the TFT incorporation into tumor- and white blood cell-DNA, and to elucidate the mechanism of TFT-related effect and toxicity. TFT effect on the colony formation of mouse bone marrow cells was also investigated. METHODS: Pharmacokinetics of TFT was determined in nude mice after single oral administration of TAS-102, while the antitumor activity and body weight change were evaluated in the tumor-bearing nude mice after multiple oral administrations for 2 weeks. TFT concentrations in the blood- and tumor-DNA were determined by LC/MS/MS. The colony formation was evaluated by CFU-GM assay. RESULTS: TFT systemic exposure in plasma increased dose-dependently. The tumor growth rate and body weight gain decreased dose-dependently, but TFT concentrations in the DNA of tumor tissues and white blood cells increased dose-dependently. TFT inhibited colony formation of bone marrow cells in a concentration-dependent manner. CONCLUSIONS: A significant relationship between systemic exposure of TFT and pharmacological effects including the antitumor activity and body weight change was well explained by the TFT incorporation into DNA. TFT inhibited proliferations of mouse bone marrow cells and human colorectal carcinoma cells implanted to nude mice dose-dependently. The highest tolerable TFT exposure provides the highest antitumor activity, and the hematological toxicity may serve as a potential surrogate indicator of TAS-102 efficacy.

The N-oxide metabolite contributes to bladder selectivity resulting from oral propiverine: muscarinic receptor binding and pharmacokinetics.

We characterized contribution of N-oxide metabolites [1-methyl-4-piperidyl diphenylpropoxyacetate N-oxide (M-1) and 1-methyl-4-piperidyl benzilate N-oxide (M-2)] to the binding of muscarinic receptors in relation to the pharmacokinetics of propiverine in rats. The in vitro muscarinic receptor binding activity of M-2 was equipotent to that of propiverine, whereas M-1 was much less active. After the oral administration of propiverine (24.8-248 micromol/kg), there was relatively selective and longer-lasting binding of muscarinic receptors in the rat bladder compared with the submaxillary gland as shown by a significant increase in the apparent dissociation constant (K(d)) for specific binding of [N-methyl-(3)H]scopolamine ([(3)H]NMS). In addition, the intravesical instillation of M-2 produced a significant increase in K(d) for specific [(3)H]NMS binding in the rat bladder. Extremely high concentrations of M-1 and M-2 were detected in plasma after the oral administration of propiverine. The concentration of unbound M-2 was much higher than that of M-1 and propiverine in the rat plasma. The sum of maximal plasma unbound propiverine equivalents (C(max)) after the oral administration of propiverine at doses of 24.8, 74.3, and 248 micromol/kg was 66.0, 303, and 509 nM, respectively. The sum of corresponding area under the time-concentration curve from 0 to 12 h was 194, 2123, and 4645 nM . h, respectively. In fact, the unbound concentration of M-2 comprised more than 90% of sum of unbound propiverine equivalents in the plasma. After oral treatment with propiverine, the bladder showed the highest concentration of M-2, indicating specific distribution of this metabolite into the target organ. Thus, M-2 may contribute greatly to the relatively selective and long-lasting occupation of bladder muscarinic receptors after oral administration of propiverine.

Validation of a simple liquid chromatography-tandem mass spectrometric method for the determination of propiverine hydrochloride and its N-oxide metabolite in human plasma.

A simple high-performance liquid chromatography (HPLC)-tandem mass spectrometric method has been developed for determination of propiverine hydrochloride and its metabolite, propiverine N-oxide (M-1) in human plasma using stable isotopes, propiverine hydrochloride-d10 and M-1-d10, as internal standards. The analytes were extracted with dichloromethane from 0.2 ml of plasma in neutral condition (pH 7.0) and separated by HPLC on a C18 reversed-phase column using methanol-1% acetic acid (50:50) as a mobile phase, and detected using positive electrospray ionization in selected reaction monitoring (SRM) mode. The method was validated over a concentration range of 2-500 ng/ml for propiverine hydrochloride and 4-1000 ng/ml for M-1 using 0.2 ml of human plasma per assay. The method developed was successfully applied to analysis of propiverine hydrochloride and M-1 in clinical studies.