Transport of Pregabalin Via L-Type Amino Acid Transporter 1 (SLC7A5) in Human Brain Capillary Endothelial Cell Line

Abstract

PurposeThe anti-epileptic drug pregabalin crosses the blood-brain barrier (BBB) in spite of its low lipophilicity. This study was performed to determine whether L-type amino acid transporters (LAT1/SLC7A5 and LAT2/SLC7A8) contribute to the uptake of pregabalin.MethodsPregabalin uptake by LATs-transfected HEK293 cells or hCMEC/D3 cells, an in vitro human BBB model, was measured by LC-MS/MS analysis. Expression of LAT1 mRNA in hCMEC/D3 cells was determined by quantitative RT-PCR analysis.ResultsOverexpression of LAT1, but not LAT2, in HEK293 cells significantly increased the cellular uptake of pregabalin, and the LAT1-mediated uptake was saturable with a Km of 0.288 mM. LAT1-mediated amino acid uptake was inhibited specifically and almost completely in the presence of 1 mM pregabalin. The uptake of pregabalin by hCMEC/D3 cells was sodium-independent, saturable (Km = 0.854 mM), and strongly inhibited by large amino acids at 1 mM, 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid, a specific system L inhibitor, at 1 mM and by JPH203, a LAT1-selective inhibitor, at 10 μM. Pregabalin uptake in hCMEC/D3 cells was also decreased by 75% by the silencing of LAT1 gene using LAT1 siRNA.ConclusionsOur results indicate that LAT1, but not LAT2, recognizes pregabalin as a substrate. It is suggested that LAT1 mediates pregabalin transport at the BBB.Electronic supplementary materialThe online version of this article (10.1007/s11095-018-2532-0) contains supplementary material, which is available to authorized users.

Establishment of an in Vitro Human Blood-Brain Barrier Model Derived from Induced Pluripotent Stem Cells and Comparison to a Porcine Cell-Based System

“…In addition, the vectorial transport was three-fold higher in the AB direction than in the BA direction ( Figure 6A) and reduced by about 70% in the presence of JPH203, a selective LAT1 inhibitor. This is consistent with the observed 75% inhibition in the uptake of the LAT1 specific substrate pregabalin, after LAT-1 mRNA silencing in the human endothelial cell line hCMEC/D3 [37]. Phenylalanine, another LAT-1 substrate, had a permeability value two-fold higher than in PBEC [2] similar to what was observed for leucine.…”

Putative roles of SLC7A5 (LAT1) transporter in pain

“…GBP and PGB are transported into the neuronal cytoplasm via a L-type amino acid transporter ( Su et al, 1995 ). The influx of gabapentinoid drugs was demonstrated to be mediated by LAT1 in brain endothelial cells and in HEK-293 cells stably expressing LAT1 ( Dickens et al, 2013 , Takahashi et al, 2018 ) ( Fig. 2 ).…”

“…In line with this evidence, gabapentin can resemble a L-form of large neutral amino acids ( Su et al, 1995 ), giving it strong structural similarity to endogenous substrates of LAT1 ( Singh and Ecker, 2018 ). On the other hand, short interfering RNA (siRNA) mediated suppression of LAT1 ( Dickens et al, 2013 , Takahashi et al, 2018 ) provided evidence that GBP is transported specifically by this transporter since its uptake was significantly reduced compared to LAT2 targeted siRNA ( Dickens et al, 2013 ). Importantly, BCH inhibits gabapentinoid transport ( Su et al, 2005 , Akanuma et al, 2018 ) and prevents the effect of GBP on HVA Ca 2+ channels ( Hendrich et al, 2008 , Biggs et al, 2014 ).…”

“…LAT1 is strongly expressed in malignant tumors presumably to support their continuous growth and proliferation ( Yanagida et al, 2001 ). In addition to transporting L-leucine, L-phenylalanine, L-histidine, and L-tryptophan, LAT1 also recognizes thyroid hormones ( Friesema et al, 2001 ) and some pharmaceutical compounds such as L-DOPA ( Kageyama et al, 2000 ), 2-(1-(aminomethyl)-cyclohexyl)acetic acid (gabapentin; GBP) ( Dickens et al, 2013 ), 3-(aminomethyl)-5‑methyl-hexanoic acid (pregabalin; PGB) ( Takahashi et al, 2018 ), among others. LAT1 only serves as a transporter for a handful of drugs, which makes it relatively unique to gabapentinoid pain therapeutics and their mechanism of action.…”

“…Global LAT1 knockout mice were found to be embryonically lethal ( Poncet et al, 2014 ), which could be due to its essential activity in cells of importing large neutral amino acids. In the sensory system, the best known role of LAT1 is to mediate the influx of GBP and PGB ( Dickens et al, 2013 , Takahashi et al, 2018 ), which are two front-line medications for management of neuropathic pain (see gabapentinoid transport section). Therefore, in this review we will address the expression, regulation, function and relevant interactions of this transporter that link it to the onset, development and/or maintenance of pain.…”