Narawa Tomoya, Shimizu Rikako, Takano Shuhei, Tsuda Yasuyuki, Ono Kiyoko, Yamada Hideo, Itoh Tomoo
School of Pharmaceutical Sciences, Kitasato University, Tokyo, Japan.
Chirality. 2005 Oct;17(8):444-9. doi: 10.1002/chir.20184.
Stereoselectivity of the human reduced folate carrier (RFC1) was examined in Caco-2 cells using methotrexate (l-amethopterin or l-MTX) and its antipode (d-amethopterin or d-MTX) as model substrates. The initial uptake rate of folic acid (FA) was concentration dependent, with a K(m) value of approximately 0.6 microM. The Eadie-Hofstee plot of the RFC1-mediated FA uptake revealed a single component for FA uptake into Caco-2 cells, demonstrating that only RFC1 is involved in FA uptake. l-MTX inhibited FA uptake in a competitive manner with a K(i) value of approximately 2 microM, similar to the K(m) value of l-MTX. d-MTX also competitively inhibited FA uptake with a K(i) value being approximately 120 microM, indicating that the affinity of d-MTX is ca. 60-fold less than that of l-MTX. The stereoselectivity of human RFC1 observed in the present study was consistent not only with the stereoselectivity of rabbit RFC1 observed in rabbit intestinal brush border membrane vesicles but also with the reported differences in oral absorption of amethopterin enantiomers in humans.
使用甲氨蝶呤（l-氨甲蝶呤或l-MTX）及其对映体（d-氨甲蝶呤或d-MTX）作为模型底物，在Caco-2细胞中检测了人还原型叶酸载体（RFC1）的立体选择性。叶酸（FA）的初始摄取速率呈浓度依赖性，K(m)值约为0.6 microM。RFC1介导的FA摄取的伊迪-霍夫斯泰 plot图显示，FA摄取到Caco-2细胞中只有一个成分，表明只有RFC1参与FA摄取。l-MTX以竞争性方式抑制FA摄取，K(i)值约为2 microM，与l-MTX的K(m)值相似。d-MTX也竞争性抑制FA摄取，K(i)值约为120 microM，表明d-MTX的亲和力约比l-MTX低60倍。本研究中观察到的人RFC1的立体选择性不仅与在兔肠刷状缘膜囊泡中观察到的兔RFC1的立体选择性一致，而且与报道的氨甲蝶呤对映体在人体中的口服吸收差异一致。
