Narawa Tomoya, Tsuda Yasuyuki, Itoh Tomoo
School of Pharmacy, Kitasato University, Tokyo, Japan.
Drug Metab Pharmacokinet. 2007 Feb 25;22(1):33-40. doi: 10.2133/dmpk.22.33.
Stereoselectivity of the human reduced folate carrier (RFC1) in Caco-2 cells was examined using methotrexate (L-amethopterin, L-MTX) and its antipode (D-amethopterin, D-MTX) as model substrates. The initial uptake rate of L-MTX into Caco-2 cells followed Michaelis-Menten kinetics with a Km value of approximately 1 microM. The Eadie-Hofstee plot of the RFC1-mediated L-MTX uptake showed that it was mediated by a single transport system, RFC1. Dixon plots revealed that L-MTX uptake was inhibited competitively by folic acid (FA), L-MTX and D-MTX, with Ki values of approximately 0.8, 1.5 and 180 microM, respectively. The results showed that the affinities of FA and L-MTX to RFC1 were approximately 120-fold greater than that of D-MTX. The uptake of L- and D-MTX into Caco-2 cells was also measured using LC-MS/MS analysis, which revealed that the L-MTX uptake was at least 7-fold greater than that of D-MTX. The present study revealed significant stereoselectivity of RFC1 toward amethopterin enantiomers with the L-isomer being much more favored.
使用甲氨蝶呤（L-氨甲蝶呤，L-MTX）及其对映体（D-氨甲蝶呤，D-MTX）作为模型底物，研究了人还原型叶酸载体（RFC1）在Caco-2细胞中的立体选择性。L-MTX进入Caco-2细胞的初始摄取速率遵循米氏动力学，Km值约为1 microM。RFC1介导的L-MTX摄取的伊迪-霍夫斯泰曲线表明，它是由单一转运系统RFC1介导的。狄克逊图显示，叶酸（FA）、L-MTX和D-MTX竞争性抑制L-MTX摄取，Ki值分别约为0.8、1.5和180 microM。结果表明，FA和L-MTX对RFC1的亲和力比对D-MTX的亲和力大约高120倍。还使用LC-MS/MS分析测量了L-MTX和D-MTX进入Caco-2细胞的摄取情况，结果显示L-MTX的摄取至少比D-MTX高7倍。本研究揭示了RFC1对氨甲蝶呤对映体具有显著的立体选择性，L-异构体更受青睐。
