Methotrexate is excreted into the bile by canalicular multispecific organic anion transporter in rats

Abstract

Methotrexate [(+) amethopterin, L-MTX] has two carboxyl groups in its structure and is eliminated mainly by excretion into urine and bile. To investigate the biliary excretion mechanism of L-MTX, we performed in vivo and in vitro studies using mutant rats, Eisai hyperbilirubinemic rats (EHBRs), whose canalicular multispecific organic anion transporter (cMOAT) is defective as a consequence of heredity. After i.v. administration of L-MTX to EHBRs, its plasma disappearance and biliary excretion was slower than in normal Sprague Dawley rat (SDR). ATP-dependence and overshoot phenomena were observed in the uptake of [3H]L-MTX by canalicular membrane vesicles (CMV) prepared from SDR, whereas no ATP-dependence was observed in CMV from EHBRs. The ATP-dependent uptake of L-MTX by SDR CMV exhibited saturable kinetics with a K(m) of 295 μM. L-MTX competitively inhibited the ATP-dependent uptake of [3H]2,4-dinitrophenyl-S-glutathione, a typical substrate for cMOAT, and the inhibition constant (K(i)) of L-MTX was comparable with its own K(m). These results suggest that L-MTX is excreted into bite by cMOAT. The inhibitory effects of L-MTX and its optical isomer, (-) amethopterin (D- MTX), on the uptake of [3H]L-MTX differed with K(i)s of 326 and 93 ~M, respectively, indicating that the biologically inactive D form has a higher affinity for cMOAT than L-MTX.