Stereoselective Transport of Amethopterin Enantiomers by the Proton-coupled Folate Transporter

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  Stereoselective transport of methotrexate (L-amethopterin, L-MTX) and its antipode (D-amethopterin, D-MTX) by the proton-coupled folate transporter (PCFT) was examined using PCFT-expressing HEK293 cells (PCFT-HEK293 cells). Uptake of both L-MTX and D-MTX was pH-dependent and decreased with an increase in the extracellular pH from 5.0 to 7.4. The initial uptake rate of L-MTX into PCFT-HEK293 cells followed Michaelis-Menten kinetics with a K_m value of approximately 5.0 μM. Dixon plots revealed that L-MTX uptake was inhibited competitively by unlabeled L-MTX, D-MTX, and folic acid (FA), with K_i values of approximately 3.6, 180, and 2.1 μM, respectively. The initial uptake rate of D-MTX into PCFT-HEK293 cells also followed Michaelis-Menten kinetics with a K_m value of 211 μM. The V_max value of D-MTX was similar to that of L-MTX. The present study revealed that the transport of MTX enantiomers by PCFT is highly stereoselective with the uptake clearance of L-MTX being approximately 40-fold greater than that of D-MTX. It was also revealed that this high stereoselectivity results from the difference in K_m values, and not V_max values, between the enantiomers. The observed stereoselectivity was consistent with the differences in the intestinal absorption of MTX enantiomers in humans.