David Dickens

Transport of gabapentin by LAT1 (SLC7A5).

Gabapentin is used in the treatment of epilepsy and neuropathic pain. Gabapentin has high and saturable permeability across the BBB, but no mechanistic studies underpinning this process have been reported. The aim of the current study was to investigate the transport of gabapentin in a model of the BBB, identify the important drug transporter(s) and to use mathematical modelling to quantify the processes involved. A human brain endothelial cell line (hCMEC/D3) was utilised as an in-vitro model of the BBB. Uptake of radiolabeled gabapentin into cells in the presence of chemical inhibitors, siRNA or overexpressed drug transporters of interest was investigated. Gabapentin was demonstrated to be a LAT1 substrate in brain endothelial cells (LAT1-process; Km=530μM and Vmax=7039pmoles/million cells/min versus other-processes; Km=923μM and Vmax=3656pmoles/million cells/min) and in transfected HEK 293 LAT1 cells (LAT1-process; Km=217μM and Vmax=5192pmoles/million cells/min versus otherprocesses; Km=1546μM and Vmax=3375pmoles/million cells/min). At physiological concentrations of gabapentin, LAT1 mediated transport was 3 or ~10-fold higher than the other transport processes in the two systems, respectively, demonstrating clear selectivity for gabapentin. In-silico structural homology modelling confirmed that LAT1 could have the LeuT conserved fold and functions by the alternative access mechanism. Mathematical modelling of this mechanism revealed revised significance of Vmax and Km so that a low Km may not necessarily imply a high affinity transport process. Gabapentin was negative for OCT like transport and LAT2 activity in the hCMEC/D3 and OCT1 transfected cells. Our data shows that gabapentin is a substrate for the influx transporter LAT1 at therapeutic concentrations.

Lamotrigine is a substrate for OCT1 in brain endothelial cells

The mechanisms that underpin the passage of lamotrigine at the blood-brain barrier to its site of action in the brain is poorly understood. Lamotrigine has been postulated to be delivered to its site of action in the brain favourably despite its physicochemical properties. The aim of this study was to investigate the transport of lamotrigine in an in-vitro model of the BBB. In this study, lamotrigine was found to have a distribution coefficient of 0 at pH 7.4 indicating that it was not highly lipophilic. Human brain endothelial cells (hCMEC/D3) were used to probe the interaction of lamotrigine with drug transporters. The uptake of lamotrigine into hCMEC/D3 cells was found to be an active process (K(m) = 62 ± 14 μM; V(max) = 385 ± 30 pmol/min/million cells). Furthermore, use of a panel of transporter inhibitors indicated that this active uptake was mediated by organic cation transporter 1 (OCT1). OCT1 mRNA and protein were shown to be expressed in hCMEC/D3 cells. KCL22 cells overexpressing OCT1 were then used to validate these findings. Lamotrigine was confirmed to be a substrate and inhibitor in OCT1-transfected KCL22 cells. A putative pharmacokinetic drug-drug interaction (DDI) between quetiapine and lamotrigine was recently reported in patients and we show here that quetiapine is a potent inhibitor of the OCT1-mediated transport of lamotrigine. This is the first time that a specific influx transporter has been shown to transport lamotrigine. The clinical implications of these findings with respect to the efficacy of lamotrigine and its potential for DDI require further investigation.

MAP4K3 modulates cell death via the post-transcriptional regulation of BH3-only proteins

Intracellular signal transduction networks involving protein kinases are important modulators of cell survival and cell death in multicellular organisms. Functional compromise of these networks has been linked to aberrant apoptosis in diseases such as cancer. To identify novel kinase regulators of cell death, we conducted an RNAi-based screen to identify modulators of the intrinsic apoptosis pathway. Using this approach, we identified MAP4K3 as a novel apoptosis inducer. Here, we present evidence that this pro-apoptotic kinase orchestrates activation of BAX via the concerted posttranscriptional modulation of PUMA, BAD, and BIM. Additionally, we found decreased levels of this kinase in pancreatic cancer samples, suggesting a tumor suppressor role for MAP4K3 in pancreatic tumorigenesis.

A multi-system approach assessing the interaction of anticonvulsants with P-gp.

30% of epilepsy patients receiving antiepileptic drugs (AEDs) are not fully controlled by therapy. The drug transporter hypothesis for refractory epilepsy proposes that P-gp is over expressed at the epileptic focus with a role of P-gp in extruding AEDs from the brain. However, there is controversy regarding whether all AEDs are substrates for this transporter. Our aim was to investigate transport of phenytoin, lamotrigine and carbamazepine by using seven in-vitro transport models. Uptake assays in CEM/VBL cell lines, oocytes expressing human P-gp and an immortalised human brain endothelial cell line (hCMEC/D3) were carried out. Concentration equilibrium transport assays were performed in Caco-2, MDCKII ±P-gp and LLC-PK1±P-gp in the absence or presence of tariquidar, an inhibitor of P-gp. Finally, primary porcine brain endothelial cells were used to determine the apparent permeability (Papp) of the three AEDs in the absence or presence of P-gp inhibitors. We detected weak transport of phenytoin in two of the transport systems (MDCK and LLC-PK1 cells transfected with human P-gp) but not in the remaining five. No P-gp interaction was observed for lamotrigine or carbamazepine in any of the seven validated in-vitro transport models. Neither lamotrigine nor carbamazepine was a substrate for P-gp in any of the model systems tested. Our data suggest that P-gp is unlikely to contribute to the pathogenesis of refractory epilepsy through transport of carbamazepine or lamotrigine.

The B-cell lymphoma 2 (BCL2)-inhibitors, ABT-737 and ABT-263, are substrates for P-glycoprotein

Inhibition of BCL2 proteins is one of the most promising new approaches to targeted cancer therapy resulting in the induction of apoptosis. Amongst the most specific BCL2-inhibitors identified are ABT-737 and ABT-263. However, targeted therapy is often only effective for a limited amount of time because of the occurrence of drug resistance. In this study, the interaction of BCL2-inhibitors with the drug efflux transporter P-glycoprotein was investigated. Using (3)H labelled ABT-263, we found that cells with high P-glycoprotein activity accumulated less drug. In addition, cells with increased P-glycoprotein expression were more resistant to apoptosis induced by either ABT-737 or ABT-263. Addition of tariquidar or verapamil sensitized the cells to BCL2-inhibitor treatment, resulting in higher apoptosis. Our data suggest that the BCL2-inhibitors ABT-737 and ABT-263 are substrates for P-glycoprotein. Over-expression of P-glycoprotein may be, at least partly, responsible for resistance to these BCL2-inhibitors.

ABCB1 single nucleotide polymorphisms (1236C > T, 2677G > T, and 3435C > T) do not affect transport activity of human P-glycoprotein

Background P-glycoprotein (P-gp) is a multidrug efflux transporter that has a defined role in the absorption and disposition of drugs. Many studies have investigated the potential influence of ABCB1 polymorphisms on the disposition of its substrates. However, there remains significant controversy regarding the role of these polymorphisms. Our aim was to generate a P-gp expression system for single nucleotide polymorphisms (SNPs) and the reference sequence to assess the functional significance of these variants on transport. Materials and methods P-gp reference, a P-gp ATPase deficient mutant (G534D) and a triple SNP variant of P-gp (1236C>T, 2677G>T, and 3435C>T) were expressed in Xenopus laevis oocytes and used to assess the influence of these SNPs on transport of digoxin and imatinib. The inhibition of P-gp-mediated transport in Caco-2 cells and oocytes was also assessed. Results No effect of the triple SNP variant of P-gp on molecular transport of digoxin or imatinib was observed. The rank order of inhibition of P-gp in Caco-2 cells and ABCB1-injected oocytes was tariquidar>elacridar>PSC-833>laniquidar>cyclosporine>verapamil>dipyridamole. Conclusion These data suggest there is no functional consequence of these SNPs for molecular transport of model substrates or inhibition by model inhibitors for P-gp. Transporter-injected oocytes may be a useful tool for probing the mechanism for unexplained drug–drug interactions or to characterize therapeutic transport inhibitors.

Misoprostol-induced fever and genetic polymorphisms in drug transporters SLCO1B1 and ABCC4 in women of Latin American and European ancestry

AIM Misoprostol, a prostaglandin analogue used for the treatment of postpartum hemorrhage and termination of pregnancy, can cause high fevers. Genetic susceptibility may play a role in misoprostol-induced fever. SUBJECTS & METHODS Body temperature of women treated with misoprostol for termination of pregnancy in the UK (n = 107) and for postpartum hemorrhage in Ecuador (n = 50) was measured. Genotyping for 33 single nucleotide polymorphisms in 15 candidate genes was performed. Additionally, we investigated the transport of radiolabeled misoprostol acid across biological membranes in vitro. RESULTS The ABCC4 single nucleotide polymorphism rs11568658 was associated with misoprostol-induced fever. Misoprostol acid was transported across a blood-brain barrier model by MRP4 and SLCO1B1. CONCLUSION Genetic variability in ABCC4 may contribute to misoprostol-induced fever in pregnant women. Original submitted 21 January 2015; Revision submitted 24 April 2015.

Modulation of LAT1 (SLC7A5) transporter activity and stability by membrane cholesterol

LAT1 (SLC7A5) is a transporter for both the uptake of large neutral amino acids and a number of pharmaceutical drugs. It is expressed in numerous cell types including T-cells, cancer cells and brain endothelial cells. However, mechanistic knowledge of how it functions and its interactions with lipids are unknown or limited due to inability of obtaining stable purified protein in sufficient quantities. Our data show that depleting cellular cholesterol reduced the Vmax but not the Km of the LAT1 mediated uptake of a model substrate into cells (L-DOPA). A soluble cholesterol analogue was required for the stable purification of the LAT1 with its chaperon CD98 (4F2hc,SLC3A2) and that this stabilised complex retained the ability to interact with a substrate. We propose cholesterol interacts with the conserved regions in the LAT1 transporter that have been shown to bind to cholesterol/CHS in Drosophila melanogaster dopamine transporter. In conclusion, LAT1 is modulated by cholesterol impacting on its stability and transporter activity. This novel finding has implications for other SLC7 family members and additional eukaryotic transporters that contain the LeuT fold.

Development of Fluorine-18 Labeled Metabolically Activated Tracers for Imaging of Drug Efflux Transporters with Positron Emission Tomography

Increased activity of efflux transporters, e.g., P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP), at the blood-brain barrier is a pathological hallmark of many neurological diseases, and the resulting multiple drug resistance represents a major clinical challenge. Noninvasive imaging of transporter activity can help to clarify the underlying mechanisms of drug resistance and facilitate diagnosis, patient stratification, and treatment monitoring. We have developed a metabolically activated radiotracer for functional imaging of P-gp/BCRP activity with positron emission tomography (PET). In preclinical studies, the tracer showed excellent initial brain uptake and clean conversion to the desired metabolite, although at a sluggish rate. Blocking with P-gp/BCRP modulators led to increased levels of brain radioactivity; however, dynamic PET did not show differential clearance rates between treatment and control groups. Our results provide proof-of-concept for development of prodrug tracers for imaging of P-gp/BCRP function in vivo but also highlight some challenges associated with this strategy.

A comprehensive functional and clinical analysis of ABCC2 and its impact on treatment response to carbamazepine

At the blood–brain barrier, overexpression of the drug efflux transporter ABCC2 (also known as MRP2) has been proposed as a mechanism for impaired carbamazepine (CBZ) treatment response in epilepsy. However, investigation of the impact of ABCC2 polymorphisms on CBZ treatment efficacy has produced conflicting and inconclusive results. A series of in vitro cell efflux and plasma membrane vesicle uptake assays were undertaken to investigate whether CBZ was an ABCC2 substrate. In addition, the effect of three common ABCC2 polymorphisms, −24C>T, c.1249G>A and c.3972C>T, on the efficacy of CBZ in epilepsy (assessed using the clinical end points time to first seizure and time to 12-month remission from the SANAD (Standard and New Antiepileptic Drugs) trial) was determined. CBZ was found not to be a substrate for human ABCC2 in vitro. Clinically, no significant association was observed for the ABCC2 genetic variants and CBZ treatment outcomes. This comprehensive analysis does not support a role for ABCC2 in CBZ treatment efficacy.