The Antiepileptic Drugs Phenobarbital and Carbamazepine Reduce Transport of Methotrexate in Rat Choroid Plexus by Down-Regulation of the Reduced Folate Carrier

Abstract

Intrathecal methotrexate (MTX) has been associated with severe neurotoxicity. Because carrier-associated removal of MTX from the cerebrospinal fluid (CSF) into blood remains undefined, we determined the expression and function of MTX transporters in rat choroid plexus (CP). MTX neurotoxicity usually manifests as seizures requiring therapy with antiepileptic drugs (AEDs) such as phenobarbital (PB). Because we have demonstrated that PB reduces activity of MTX influx carrier reduced folate carrier (Rfc1) in liver, we investigated the influence of the AEDs PB, carbamazepine (CBZ), or gabapentin on Rfc1-mediated MTX transport in CP. Reverse transcriptase-polymerase chain reaction and Western blot analysis showed similar expression of the MTX influx carrier Rfc1 and organic anion transporter 3 or efflux transporter multidrug resistance-associated protein 1 (Mrp1) and breast cancer resistance protein (Bcrp) in rat CP tissue and choroidal epithelial Z310 cells. Confocal microscopy revealed subcellular localization of Rfc1 and Bcrp at the apical and of Mrp1 at the basolateral CP membrane. Uptake, efflux, and inhibition studies indicated MTX transport activity of Rfc1, Mrp1, and Bcrp. PB and CBZ but not gabapentin significantly inhibited Rfc1-mediated uptake of MTX in CP cells. Studies on the regulatory mechanism showed that PB significantly inhibited Rfc1 translation but did not alter carrier gene expression. Altogether, removal of intrathecal MTX across the blood-CSF barrier may be achieved through Rfc1-mediated uptake from the CSF followed by MTX extrusion into blood, particularly via Mrp1. Antiepileptic treatment with PB or CBZ causes post-transcriptional down-regulation of Rfc1 activity in CP. This mechanism may result in enhanced MTX toxicity in patients with cancer who are receiving intrathecal MTX chemotherapy by reduced CSF clearance of the drug.