Stereoselective Recognition of Amethopterin Enantiomers by the Rat Proton-Coupled Folate Transporter

Abstract

Key words methotrexate; amethopterin; enantiomer; species difference; stereoselective transport; protoncoupled folate transporterFolates are essential nutrients for cell growth in various mammalian cells and are transported across cell membranes by the reduced folate carrier (RFC, SLC19A1) or the protoncoupled folate transporter (PCFT, SLC46A1).1-4) The cDNA of the RFC has been cloned in several different species, including mice, hamsters, and humans, and its transport characteristics have been fully elucidated.5-11) The RFC can only transport reduced folates, with maximal folate transport being observed at neutral pH. 2,4,12) Although the PCFT was originally identified as a heme carrier protein, subsequent research revealed that folates are good substrates of the PCFT.3)The PCFT is expressed in the epithelial cells of the small intestine and involved in the absorption of both reduced and oxidized folates, with a H + gradient providing the driving force for both of these adsorption processes. 12) Given that the microclimate pH at the surface of epithelial cells is acidic, the PCFT could also play a major role in the intestinal absorption of folates.Hereditary folate malabsorption (HFM) is a rare autosomal recessive disorder in humans. This disease is characterized by symptoms similar to those associated with folate deficiency and generally appears within 3-6 months of birth. Infants with HFM develop anemia, diarrhea, immune deficiency, peripheral neuropathies, and seizures.13) Several studies on patients with HFM have revealed a loss of PCFT function due to mutations in the gene encoding the PCFT.14,15) Loss of PCFT function leads to decreased intestinal absorption of folate and reduced folate transport into the central nervous system. 14)These findings demonstrate the importance of the role played by the PCFT in intestinal absorption and the distribution of folates to the brain.Methotrexate (L-amethopterin, L-MTX) is used clinically as an antineoplastic and antirheumatic drug. The molecular structure of L-MTX is similar to that of folic acid (FA), meaning that L-MTX is rapidly absorbed in the small intestine by the PCFT following its oral administration. D-Amethopterin (D-MTX) is the optical isomer of L-MTX, and the absorption of D-MTX from the human small intestine occurs at a significantly slower rate than that of L-MTX. It was reported that the area under the plasma concentration-time curve (AUC) following the oral administration of L-MTX is approximately 40-fold greater than that of a dose of D-MTX. 16)Our previous studies revealed that the human PCFT (hPCFT)-mediated transport of the different enantiomers of MTX occurred in a stereoselective manner.17) That particular study used hPCFT-expressing human embryonic kidney (HEK) 293 cells to show that both L-and D-MTX were substrates of the hPCFT and that the K m value of L-MTX (4.98 µM) was significantly lower than that of D-MTX (211 µM). The results of that study also revealed that the observed stereoselective transport of MTX occurred as a consequence of sig...