title: 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.
authors.affiliation: King's College London, Institute of Pharmaceutical Science, Waterloo, London United Kingdom.
authors.forename: Gayathri N
authors.identifier: http://orcid.org/0000-0002-9779-9327
authors.initials: GN
authors.lastname: Sekhar
authors.affiliation: King's College London, Institute of Pharmaceutical Science, Waterloo, London United Kingdom.
authors.forename: Ana R
authors.initials: AR
authors.lastname: Georgian
authors.affiliation: King's College London, Institute of Pharmaceutical Science, Waterloo, London United Kingdom.
authors.forename: Lisa
authors.initials: L
authors.lastname: Sanderson
authors.affiliation: King's College London, Centre for Ultrastructural Imaging, King's College London, London Bridge United Kingdom.
authors.forename: Gema
authors.initials: G
authors.lastname: Vizcay-Barrena
authors.affiliation: King's College London, Institute of Pharmaceutical Science, Waterloo, London United Kingdom.
authors.forename: Rachel C
authors.initials: RC
authors.lastname: Brown
authors.affiliation: King's College London, Institute of Pharmaceutical Science, Waterloo, London United Kingdom.
authors.forename: Paula
authors.initials: P
authors.lastname: Muresan
authors.affiliation: King's College London, Centre for Ultrastructural Imaging, King's College London, London Bridge United Kingdom.
authors.forename: Roland A
authors.initials: RA
authors.lastname: Fleck
authors.affiliation: King's College London, Institute of Pharmaceutical Science, Waterloo, London United Kingdom.
authors.forename: Sarah A
authors.initials: SA
authors.lastname: Thomas
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001692:Biological Transport / Q000235:genetics / Q000502:physiology; D001812:Blood-Brain Barrier / Q000378:metabolism*; D015153:Blotting, Western; D001921:Brain / Q000378:metabolism; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D051379:Mice; D018613:Microscopy, Confocal; D046529:Microscopy, Electron, Transmission; D050814:Octamer Transcription Factor-3 / Q000235:genetics / Q000378:metabolism; D027701:Organic Cation Transport Proteins / Q000235:genetics / Q000378:metabolism; D027702:Organic Cation Transporter 1 / Q000235:genetics / Q000378:metabolism*; D000075102:Organic Cation Transporter 2; D010419:Pentamidine / Q000378:metabolism*
chemical_list: D050814:Octamer Transcription Factor-3; D027701:Organic Cation Transport Proteins; D027702:Organic Cation Transporter 1; D000075102:Organic Cation Transporter 2; C494174:Pou5f1 protein, mouse; C491012:Slc22a2 protein, mouse; D010419:Pentamidine; D000255:Adenosine Triphosphate