Organic cation transporter 1 (OCT1) is involved in pentamidine transport at the human and mouse blood-brain barrier (BBB)

Abstract

Pentamidine is an effective trypanocidal drug used against stage 1 Human African Trypanosomiasis (HAT). At the blood-brain barrier (BBB), it accumulates inside the endothelial cells but has limited entry into the brain. This study examined transporters involved in pentamidine transport at the human and mouse BBB using hCMEC/D3 and bEnd.3 cell lines, respectively. Results revealed that both cell lines expressed the organic cation transporters (OCT1, OCT2 and OCT3), however, P-gp was only expressed in hCMEC/D3 cells. Polarised expression of OCT1 was also observed. Functional assays found that ATP depletion significantly increased [3H]pentamidine accumulation in hCMEC/D3 cells (***p<0.001) but not in bEnd.3 cells. Incubation with unlabelled pentamidine significantly decreased accumulation in hCMEC/D3 and bEnd.3 cells after 120 minutes (***p<0.001). Treating both cell lines with haloperidol and amantadine also decreased [3H]pentamidine accumulation significantly (***p<0.001 and **p<0.01 respectively). However, prazosin treatment decreased [3H]pentamidine accumulation only in hCMEC/D3 cells (*p<0.05), and not bEnd.3 cells. Furthermore, the presence of OCTN, MATE, PMAT, ENT or CNT inhibitors/substrates had no significant effect on the accumulation of [3H]pentamidine in both cell lines. From the data, we conclude that pentamidine interacts with multiple transporters, is taken into brain endothelial cells by OCT1 transporter and is extruded into the blood by ATP-dependent mechanisms. These interactions along with the predominant presence of OCT1 in the luminal membrane of the BBB contribute to the limited entry of pentamidine into the brain. This information is of key importance to the development of pentamidine based combination therapies which could be used to treat CNS stage HAT by improving CNS delivery, efficacy against trypanosomes and safety profile of pentamidine.

Region-specific blood-brain barrier transporter changes leads to increased sensitivity to amisulpride in Alzheimer’s disease

“…lysozyme) (Schwake et al 2013). OCT1, OCT2 and 3 have been shown to be expressed in hCMEC/D3 and b.End3 cells in an earlier study by our group(Sekhar et al 2017) and OCTN1 and OCTN2 were shown to be expressed in both cell lines in this present study.…”

“…They are not listed on the misidentified cell line register (version 9 released 14 th October 2018). Both cell lines require different cell mediums to grow to confluence and their BBB phenotype has been confirmed using Western blots, confocal and transmission electron microscopy (Sekhar et al 2017)(section 4.10). Functional expression of several transporters has also been demonstrated by our group(Sekhar et al 2017)(Watson et al 2016)(Watson et al 2012).…”

“…The differences in the effect of inhibitors on the two cell lines may be explained by amisulpride being a substrate for multiple transporters and variations in the function/expression of OCT1, MATE and PMAT transporters possibly related to species differences (Shimomura et al 2013)(Wu et al 2015). The absence of any effect of prazosin on [ 3 H]amisulpride uptake in bEnd.3 cells can be explained by prazosin-associated toxicity in this cell line, which causes protein values to decrease over the course of the experiment, resulting in no net effect on V D values(Sekhar et al 2017). It is unlikely that this efflux transporter is an ABC transporter, as neither ATP-depletion or substrates for P-gp, BCRP, or the MRP family had an effect on [ 3 H]amisulpride accumulation in either cell line.…”

“…The hCMEC/D3 (human) and bEnd.3 (mouse) are well-established cell lines that model the BBB in vivo (Weksler et al 2013)(Brown et al 2007)(Sekhar et al 2017)(Park et al 2014). They are not listed on the misidentified cell line register (version 9 released 14 th October 2018).…”

“…hCMEC/D3 cells (passages 27-35) were provided under a MTA and maintained in Clonetics® endothelial cell growth medium-2 MV Bullet Kit (Cat# CC-3162 Lonza, UK) containing the endothelial basal medium, the SingleQuotsTM growth factor kit, foetal bovine serum (FBS), penicillin-streptomycin and HEPES (Sigma-Aldrich, UK) (Poller et al 2008)(Watson et al 2012)(Sekhar et al 2017).…”