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ααα-Trifluorothymidine (TFT), an anticancer nucleoside analog, is a potent thymidylate synthase inhibitor. TFT exerts its antitumor activity primarily by inducing DNA fragmentation after incorporation of the triphosphate form of TFT into the DNA. Although an oral combination of TFT and a thymidine phosphorylase inhibitor has been clinically developed, there is little information regarding TFT absorption. Therefore, we investigated TFT absorption in the rat small intestine. After oral administration of TFT in rats, more than 75% of the TFT was absorbed. To identify the uptake transport system, uptake studies were conducted by using everted sacs prepared from rat small intestines. TFT uptake was saturable, significantly reduced under Na(+)-free conditions, and strongly inhibited by the addition of an endogenous pyrimidine nucleoside. From these results, we suggested the involvement of concentrative nucleoside transporters (CNTs) in TFT absorption into rat small intestine. In rat small intestines, the mRNAs coding for rat CNT1 (rCNT1) and rCNT2, but not for rCNT3, were predominantly expressed. To investigate the roles of rCNT1 and rCNT2 in TFT uptake, we conducted uptake assays by using Xenopus laevis oocytes injected with rCNT1 complementary RNA (cRNA) and rCNT2 cRNA. TFT uptake by X. laevis oocytes injected with rCNT1 cRNA, and not rCNT2 cRNA, was significantly greater than that by water-injected oocytes. In addition, in situ single-pass perfusion experiments performed using rat jejunum regions showed that thymidine, a substrate for CNT1, strongly inhibited TFT uptake. In conclusion, TFT is absorbed via rCNT1 in the intestinal lumen in rats.

To clarify the cause of the canine individual variability in plasma concentration after oral administration of GTS-21, we evaluated in vitrothe metabolism to 4-OH-GTS-21 in liver microsomes of the same individuals from in vivopharmacokinetic study. First, we applied to the Michaelis-Menten kinetic parameters to a dispersion model, and compared hepatic availability (FH) and hepatic clearance (CLH) values from in vitrowith bioavailability (F), hepatic plasma flow (QPH), and plasma clearance (CLP) values from in vivo. The ratios of CLHto QPHwere ranged 0.74 to 0.94, suggesting that GTS-21 is a hepatic plasma flow-limiting drug. A significant correlation of FHand Fin the four dogs (r=0.995, p= 0.005) indicates that the variability is predominantly caused by GTS-21 O4-demethylase activity. Second, we specified the cytochrome P450 (CYP) enzymes that are involved with the metabolism by chemical inhibition. α-Naphthoflavone, furafylline, quinidine, quinine, and troleandomycin significantly inhibited GTS-21 O4-demethylase activity. Thus CYP1A, CYP2D15, and CYP3A12 were involved with O4-demethylation. The variability in control activity decreased on addition of α-naphthoflavone and furafylline. Third, we quantified the contents of CYP1A and CYP3A12 by enzyme-linked immunosorbent assay. The content of CYP1A was consistent with GTS-21 O4-demethylase activity. We concluded that canine liver CYP1A causes the individual variability in GTS-21 plasma concentration after oral administration.