The transport of CNS-active cationic drugs across the blood-brain barrier

• Main Author: Sekhar, Gayathri Nair
• Other Authors: Thomas, Sarah Ann ; Dreiss, Cecile Ayako
• Published: King's College London (University of London) 2016
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.700768

The physical, transport, and metabolic barriers presented by the cerebral microvessel endothelium - the blood- brain barrier (BBB) - protect the brain from damage from toxic substances and ensure the delivery of nutrients required for the functioning of the brain thus creating a stable neuronal microenvironment. However, the BBB poses a challenge to many CNS-active drugs which must cross the BBB to reach their target. Of the drugs that target the brain, many are cationic at physiological pH making their transport into the brain even harder with the additional potential for drug-drug interactions. Therefore, this thesis has been an attempt to understand the transport characteristics of cationic therapeutic drugs across the BBB. One of the foci of this study has been the anti-human African trypanosomiasis (HAT) drugs pentamidine and e ornithine, both positively charged at physiological pH. Pentamidine is a stage 1 drug for HAT that was previously found in our laboratory to not cross the mouse BBB in vivo as it was a substrate for eux transporters, particularly P-glycoprotein. The present study observed that pentamidine is taken up by Organic Cation Transporter 1 (OCT1) in hCMEC/D3 (human) and bEnd.3 (mouse) cell lines where it accumulates and is euxed by ATP-dependent mechanisms out of the cell. In addition, the predominant localisation of OCT1 at the luminal membrane of the BBB may explain the low permeability of pentamidine into the brain. Considering pentamidine is a substrate for P-glycoprotein at the BBB, inhibitors of Pglycoprotein were used to increase pentamidine delivery into the brain. Pluronic® triblock polymers P85, P105, and F68 were thus chosen for their proven ability to inhibit P-glycoprotein and for their promising results from clinical trials. They were used at concentrations of 0.01%, 0.025%, 0.1%, and 0.5% along with pentamidine in an attempt to increase its delivery across the BBB, concomitantly reducing any side-e ects. In vitro assays carried out on MDCK-hMDR cell line suggested high concentrations of P85 and P105 were cytotoxic even though P85 was able to signi cantly increase pentamidine permeability at 0.5% and 0.1%. Greater speci city to a target could circumvent toxicity issues in the future. The second anti-HAT drug studied was e ornithine that treats stage 2 of HAT. To treat stage 2 of the disease it must cross the BBB, yet it was not found to cross the BBB in the in vivo study on mice carried out in our laboratory. Results obtained from assessing the accumulation of e ornithine in hCMEC/D3 and bEnd.3 cell lines, the in vitro models of the BBB, also suggested limited entry of e ornithine into the BBB. Nonetheless, the assays indicated that e ornithine could be a weak substrate for system y+ at the BBB. There was also evidence for its interaction with OCTs in the bEnd.3 cell line. Second part of the thesis focussed on the antipsychotic drugs haloperidol and amisulpride, both cationic drugs at physiological pH. Haloperidol, an extensively used drug both as an antipsychotic and for palliative care, has the potential to interact with other drugs at the BBB. Haloperidol was found to be a substrate for OCTs at the BBB in both hCMEC/D3 and bEnd.3 cell lines and was found to accumulate readily inside the BBB cells. Similarly, amisulpride is an antipsychotic that is also used to treat delusions and aggression in Alzheimer's disease (AD). Previous studies found increased central dopamine receptor occupancy in AD patients when amisulpride was administered at very low doses. Unlike haloperidol, amisulpride had very low accumulation inside the BBB cells. Results suggested amisulpride to be a substrate for the OCTs and the eux transporters MATE1 and PMAT at the BBB. Amisulpride entry into the brain was also tested in wildtype and transgenic AD mice to nd that amisulpride entry into the transgenic mice brains was signi cantly greater than wildtype mice brains and this was not due to a `leaky' BBB of the AD model. Changes to the expression levels of OCT1, 2, 3 and P-glycoprotein transporters in wildtype and AD model mice BBB were determined using Western blotting and no di erences were found. Further exploration of capillaries isolated from human brain samples from control and AD a ected patients was carried out and signi cant region-speci c changes to the expression levels of MATE1 was observed. There was also a tendency for region-speci c decrease in PMAT expression levels. MATE1 and PMAT are proton-dependent transporters that eux substrates out of the BBB. This decrease in eux transporter expression at the BBB of AD patients could explain the increased sensitivity in AD patients to amisulpride.