Introduction

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Irinotecan, 7-ethyl-10-[4-(1-piperidino)-1-piperidino]-carbonyloxy camptothecin (CPT-11)¹ is a potent anticancer drug with a broad spectrum of antitumor activity (Slichenmyer et al., 1993). As a prodrug, CPT-11 is converted in vivo by carboxylesterase to its active metabolite, SN-38 (Kojima et al., 1993). The -hydroxy--lactone ring in CPT-11 and its metabolites are unstable, and conversion to the carboxylate form at physiological pH is easy (Fassberg and Stella, 1992). Biliary excretion is a major elimination pathway for CPT-11 and its metabolites (Kaneda and Yokokura, 1990). Our previous investigation using isolated rat canalicular membrane vesicles (CMVs) showed that two saturable transport components are involved in ATP-dependent uptake of the carboxylate form of CPT-11: canalicular multispecific organic anion transporter (cMOAT) is responsible as the low-affinity component (K_m = 236 µM, V_max = 1992 pmol/min/mg protein), whereas its high-affinity component (K_m = 3.39 µM, V_max = 115 pmol/min/mg protein) is governed by a transporter other than cMOAT, which is also expressed in Eisai hyperbilirubinemic rats with a genetic cMOAT deficiency (Chu et al., 1997b). Gupta et al. (1996) found that cyclosporin A (CsA) inhibits the biliary excretion of CPT-11 and its metabolites in rats. This suggests that one of the possible candidates for the high-affinity component of CPT-11 transport is P-glycoprotein (P-gp). However, CsA as well as other substrates and/or modulators of P-gp are potent inhibitors of not only P-gp, but also cMOAT and canalicular bile acid transporter. Böhme et al. (1993) reported that the inhibition constant (K_i) of CsA and (3'-oxo-4-butenyl-4-methyl-threoninel)-(Val²)-cyclosporin (PSC-833), the nonimmunosuppressive analog of CsA, for P-gp was one-half and 100 times smaller than that for cMOAT, respectively. Accordingly, in the present study, we investigated the effect of these compounds on the ATP-dependent uptake of the carboxylate form of CPT-11 by varying their concentrations. To assess the specificity of such an inhibitory effect, we also carried out control experiments to determine the effect of these compounds on the uptake of S-(2,4-dinitrophenyl)-glutathione (DNP-SG), a substrate of cMOAT.

	Experimental Procedures

Top Abstract Introduction Experimental Procedures Results and Discussion References

Materials. CPT-11 was obtained from Daiichi Pharmaceutical Co. Ltd. (Tokyo, Japan) and Yakult Honsha Co. Ltd. (Tokyo, Japan). The lactone and carboxylate forms of CPT-11 were obtained by dissolving them in 50 mM phosphate buffer at pH 3.0 or 9.0 and leaving them overnight (Chu et al., 1997b). CsA, verapamil, taurocholic acid (TCA), ATP, AMP, creatine phosphate, and creatine phosphokinase were purchased from Sigma (St. Louis, MO). PSC-833 was kindly provided by Novartis (Basel, Switzerland). Unlabeled and ³H-labeled DNP-SG (50.0 µCi/nmol) were synthesized as described previously (Chu et al., 1997a). All other chemicals were commercial products and of analytical grade. Male Sprague-Dawley rats weighing 250 to 300 g were purchased from Charles River Japan Inc. (Kanagawa, Japan).

Uptake Study Using CMVs. CMVs were prepared from male SD rats as described previously (Chu et al., 1997a). Uptake of the carboxylate or lactone form of CPT-11 was allowed to take place for 2 min at 37°C (Chu et al., 1997a). The total volume of incubation mixture was 20 µl, and the final protein and ATP concentration was 1 mg protein/ml and 5 mM, respectively. CsA or PSC-833 was dissolved in ethanol and diluted to a final concentration with transport buffer (250 mM sucrose, 10 mM Tris/HCl, 10 mM MgCl₂, pH7.4, 1000-fold). In the control study, the same concentration of ethanol also was added to the transport medium. The concentration of the carboxylate and lactone forms of CPT-11 in filters and medium was determined by HPLC as described previously (Chu et al., 1997a). To evaluate the effect of several compounds on the uptake of DNP-SG, 1.0 µM [³H]DNP-SG was added to the transport medium. Radioactivity retained on the filter was determined using a liquid scintillation counter (Chu et al., 1997a). The ATP-independent uptake of the substrate was determined in the presence of 5 mM AMP- and ATP-regenerating system. The ATP-dependent uptake was obtained by subtracting the uptake in the presence of AMP from that in the presence of ATP.

	Results and Discussion

Top Abstract Introduction Experimental Procedures Results and Discussion References

ATP-dependent uptake of the carboxylate form of CPT-11 at 5 µM, at which the high-affinity component is mainly attributed to the uptake (Chu et al., 1997b), was inhibited significantly by PSC-833, verapamil, and CsA (Fig. 1A), whereas no significant inhibitory effect of PSC-833 and verapamil was observed at 250 µM CPT-11 (Fig. 1B). There was also no inhibitory effect of verapamil on DNP-SG uptake, and only the highest concentration of PSC-833 examined (10 µM) exhibited clear inhibition (Fig. 1C). Thus, verapamil and PSC-833 specifically inhibited the high-affinity component of the carboxylate form of CPT-11, suggesting that the transporter responsible for the high-affinity component may be P-gp. However, possible involvement of mrp family transporters other than cMOAT cannot be ruled out. Of these compounds, verapamil seems to be the only inhibitor that is uniquely associated with P-gp. The present finding also is supported by our recent observation that the carboxylate form of CPT-11 (5 µM) exhibited significant ATP-dependent uptake in membrane vesicles isolated from the KB-C2 cell line, a human epidermoid KB carcinoma-derived cell line that overexpresses P-gp (Chu et al., 1999). Unlike verapamil and PSC-833, CsA inhibited the ATP-dependent uptake of CPT-11 at 250 µM (Fig. 1B). This might result from its similar affinity for P-gp and cMOAT (Böhme et al., 1993). The K_i of PSC-833 for P-gp is much lower than that for cMOAT (0.3 and 29 µM, respectively) (Böhme et al., 1993). Thus, it might be reasonable to assume that its effect on CPT-11 transport at 5 µM could be differentiated from that on CPT-11 transport at 250 µM (Fig. 1). TCA exhibited a minimal inhibitory effect (Fig. 1), suggesting that canalicular bile acid transporter is not responsible for the CPT-11 transport. It is believed that P-gp mainly accepts amphipathic cationic or neutral compounds as substrates (Alexander et al., 1989). Interestingly, our present finding suggests that the carboxylate form of CPT-11, a monovalent anion, is a substrate of both cMOAT and P-gp. It should be noted that topotecan (Sikic et al., 1997), with a structure similar to CPT-11, and estradiol 17-(-D-glucuronide) (Vore et al., 1996), a well known endogenous substrate of cMOAT, are also substrates of P-gp. Thus, P-gp may accept certain types of anionic compounds and act in coordination with cMOAT to detoxify endogenous compounds and exogenous xenobiotics.

View larger version (16K): [in this window] [in a new window]   Fig. 1.   The effect of several compounds on ATP-dependent uptake of the carboxylate form of CPT-11 at substrate concentrations of 5 (A) and 250 µM (B) and of [3H]DNP-SG (C) by rat CMVs. CMVs were incubated with 5 µM (A) and 250 µM (B) of the carboxylate form of CPT-11 or 1 µM [3H]DNP-SG (C) in the presence of various compounds for 2 min. ATP-dependent uptake was obtained by subtracting the uptake in the presence of 5 mM AMP from that in the presence of 5 mM ATP. Data are the mean ± S.E. of three CMV preparations. *P .05 and **P .01, significantly different from controls.