Chiral Recognition of Amethopterin Enantiomers by the Reduced Folate Carrier in Caco-2 Cells

Summary: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 μM. 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 μM, 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.