Han Y H, Kato Y, Watanabe Y, Terao K, Asoh Y, Sugiyama Y
Graduate School of Pharmaceutical Sciences, University of Tokyo, Bunkyo-ku, Tokyo, Japan.
Drug Metab Dispos. 2001 Apr;29(4 Pt 1):394-400.
The hepatic uptake and biliary excretion of a novel methotrexate derivative, N-[4-[(2,4-diamminopteridine-6-yl)methyl]-3,4-dihydro-2H-1,4-benzothiazin-7-yl]carbonyl-L-homoglutamic acid (MX-68), were examined in rats in vitro using isolated hepatocytes and bile canalicular membrane vesicles (CMVs), respectively. The uptake of MX-68 by isolated rat hepatocytes showed a saturable component (Km = 2.15 microM and Vmax = 2.34 pmol/min/mg of protein) and was inhibited by ATP-depletors and anionic compounds such as taurocholate and probenecid. [3H]MX-68 uptake was also inhibited by folate analogs such as methotrexate and 5CH3-tetrahydrofolate, but the effect of these compounds was slightly less than that of unlabeled MX-68. On replacing Na+ with choline, MX-68 uptake remained unchanged, whereas the methotrexate uptake was reduced. Uptake of MX-68 increased as the extracellular pH fell from 7.5 to 5.5. These results suggest that MX-68 is taken up via active transport systems. The uptake of MX-68 by CMVs prepared from normal rats exhibited clear ATP dependence, whereas ATP had only a minimal effect on the uptake by CMVs from Eisai-hyperbilirubinemic rats with a hereditary deficiency in canalicular multispecific organic anion transporter (cMOAT). The initial uptake rate of ATP-dependent MX-68 transport showed saturation with kinetic parameters similar to those of methotrexate. MX-68 inhibited the ATP-dependent transport of 2,4-dinitrophenyl-S-glutathione, a typical substrate for cMOAT, the inhibition constant (162 microM) being comparable with the Km of ATP-dependent MX-68 transport. These results suggest that the biliary excretion of MX-68 via the bile canalicular membrane is mediated mainly by cMOAT. In conclusion, active transport systems are involved in membrane penetration of MX-68 both at sinusoidal and canalicular sides in the liver, the latter being mainly involved with methotrexate (MTX) whereas the former differs partially from that for MTX.
分别使用分离的大鼠肝细胞和胆小管膜囊泡（CMV）在体外研究了一种新型甲氨蝶呤衍生物N-[4-[(2,4-二氨基蝶啶-6-基)甲基]-3,4-二氢-2H-1,4-苯并噻嗪-7-基]羰基-L-高谷氨酸（MX-68）的肝脏摄取和胆汁排泄。分离的大鼠肝细胞对MX-68的摄取呈现出饱和成分（Km = 2.15 microM，Vmax = 2.34 pmol/分钟/毫克蛋白质），并且受到ATP消耗剂以及阴离子化合物如牛磺胆酸盐和丙磺舒的抑制。[3H]MX-68的摄取也受到叶酸类似物如甲氨蝶呤和5CH3-四氢叶酸的抑制，但这些化合物的作用略小于未标记的MX-68。用胆碱替代Na+时，MX-68的摄取保持不变，而甲氨蝶呤的摄取减少。随着细胞外pH从7.5降至5.5，MX-68的摄取增加。这些结果表明MX-68是通过主动转运系统摄取的。从正常大鼠制备的CMV对MX-68的摄取表现出明显的ATP依赖性，而ATP对来自具有胆小管多特异性有机阴离子转运体（cMOAT）遗传性缺陷的艾塞那-高胆红素血症大鼠的CMV摄取只有最小的影响。ATP依赖性MX-68转运的初始摄取速率显示出饱和，其动力学参数与甲氨蝶呤相似。MX-68抑制了2,4-二硝基苯基-S-谷胱甘肽（一种cMOAT的典型底物）的ATP依赖性转运，抑制常数（162 microM）与ATP依赖性MX-68转运的Km相当。这些结果表明MX-68通过胆小管膜的胆汁排泄主要由cMOAT介导。总之，主动转运系统参与了MX-68在肝脏窦状隙和胆小管侧的膜穿透，后者主要与甲氨蝶呤（MTX）有关，而前者与MTX部分不同。
