Abstracts

Rationale: Changes in expression of drug transporters at the blood-brain barrier (BBB) may impact on brain penetration of antiepileptic drugs (AEDs) and contribute to the phenomenon of drug resistant epilepsy. Monocarboxylate transporters (MCTs) are members of the solute carrier (SLC) transporter family. Three isoforms, MCT1, MCT4 and MCT8, are expressed in cerebrovascular endothelial cells and are believed to contribute to the functionality of the BBB. This study employed an immortalised human cerebral microvascular endothelial cell line (hCMEC/D3) and a panel of compounds proposed to inhibit MCT transporters and aimed to determine whether these MCTs are involved in the active transport of commonly used AEDs. Methods: Transport of [³H]-phenytoin (25µM), [³H]-carbamazepine (20µM), [³H]-sodium valproate (300µM), [³H]-lamotrigine (10µM), [³H]-gabapentin (20µM), [³H]-topiramate (10µM), and [³H]-levetiracetam (6µM) was assessed in cultures of hCMEC/D3 cells, with [¹⁴C]-lactic acid (2µM) used as a positive control. Cells were exposed to AED plus inhibitor or vehicle (1% DMSO) for 60 minutes at 37⁰C. MCT inhibitors comprised salicylic acid (1mM), 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS; 1mM), imatinib (100µM), simvastatin (10µM), 4-acetamido-4′-isothiocyanato-2,2′-stilbenedisulfonic acid (SITS; 1mM), 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB; 100µM), and 4-(hydroxymercuri)benzoic acid (4-MBA; 100µM). Transport was determined by scintillation counting of cellular radioactive content in comparison to standards of known concentration, with experiments performed in triplicate on at least three occasions (n≥9). Results were expressed as mean percentage of control transport (±SEM), with statistical analysis performed by ANOVA with Dunnett correction for multiple comparisons. Results: Transport of gabapentin was decreased to 3.0% (±0.3%; p<0.001) of control in the presence of 4-MBA (MCT1/MCT4 inhibitor) and increased to 255% (±14.5%; p<0.001) and 159% (±13.8%; p<0.005) of control with NPPB (MCT4 inhibitor) and DIDS (non-specific MCT inhibitor), respectively. Transport of phenytoin was decreased to 57% (±5.2%; p<0.005) of control in the presence of simvastatin (MCT4 inhibitor), whereas transport of lamotrigine was decreased to 33% (±2.5%; p<0.05) of control with imatinib (MCT8 inhibitor). Changes in relative transport of AEDs for all other drug and inhibitor combinations failed to reach statistical significance. Conclusions: This study suggests that the contribution of MCTs to the brain penetration of AEDs is modest, at best. Gabapentin appears to undergo active transport in the hCMEC/D3 cell line, possibly mediated by MCT4, but a lack of inhibitor selectivity and contradictory findings prevented identification of the transporter involved. Phenytoin and lamotrigine were similarly subject to transport, potentially by MCT4 and MCT8 respectively, but again the identity of the MCT isoform could not be confirmed. Further investigation is required to clarify the contribution of individual MCT transporters to the brain penetration of these AEDs.