Drug transport and drug-drug interactions at the blood-brain barrier

Membrane transporters are increasingly recognised as being important in determining drug pharmacokinetics at whole body, organ, and cellular levels. At the blood-­‐brain barrier (BBB), membrane transporters determine the passage of drugs into and out of the brain. About 30 % of all patients are classed as non-­‐responders for both epilepsy and schizophrenia. Drug transporters from the adenosine 5'-­‐ triphosphate (ATP)-­‐binding cassette (ABC) transporter family or from the solute carrier (SLC) superfamily may contribute to these drug resistant phenotypes but most have received limited attention. Treatment response to carbamazepine (CBZ) has been associated with genetic polymorphisms in ABCC2, particularly -­‐24C>T, c.1249G>A, and c.3972C>T. However, the results have been conflicting and inconclusive amongst the different studies. A functional and clinical analysis was undertaken to investigate the impact of ABCC2 on CBZ treatment response. In vitro, no ABCC2-­‐mediated CBZ transport could be observed in efflux assays with an ABCC2-­‐transfected human fibrosarcoma cell line (Rht14-­‐10) and a dog kidney cell line (MDCKII). In addition, uptake into inside-­‐out vesicles derived from the Rht14-­‐10 cell line was negative. Clinical analysis of patients from the SANAD (Standard and New Antiepileptic Drugs) trial (assessing the clinical end-­‐points time to first seizure (n = 229) and time to 12-­‐month remission (n = 134)) did not show any significant associations between the three ABCC2 gene polymorphisms, -­‐24C>T, c.1249G>A, c.3972C>T, and clinical outcomes. In an attempt to identify currently unrecognised human drug transporters with potential relevance to epilepsy and schizophrenia, screening of transport of CBZ, lamotrigine (LTG), topiramate (TPM), levetiracetam, valproate, phenytoin, and clozapine (CLP) was undertaken using an immortalised human brain endothelial cell line (hCMEC/D3) as an in vitro model of the BBB. Accumulation of TPM was significantly enhanced by 44-­‐53 % in the presence of the typical ABCC efflux transporter inhibitors MK571 and montelukast. Furthermore, CLP uptake was significantly reduced by 94 % and 83 % in the presence of the typical organic cation transporter inhibitors prazosin and verapamil, respectively. CLP uptake into the hCMEC/D3 cell line followed classical Michaelis-­‐Menten kinetics with Vmax of 3288 (pmol/million cells)/min and Km of 35.93 μM. To identify the exact underlying transporters involved in TPM efflux and CLP uptake, both functional siRNA screening was undertaken and transport was investigated in transfected cell lines. None of the known functional ABCC transporters were shown to transport TPM. In addition, none of the expressed and functionally characterised organic cation transporters from the SLC22A family, as well as transporters from the SLC6A, SLC28A, and SLC29A families, had an effect on CLP accumulation. LTG has recently been identified as a substrate for SLC22A1 (OCT1). Interaction with the human immunodeficiency virus protease inhibitors lopinavir/ritonavir and the antipsychotic CLP was therefore investigated. At clinically relevant concentrations, lopinavir was found to significantly reduce SLC22A1-­‐mediated uptake of LTG by 39 %. In addition, CLP was a potent inhibitor of SLC22A1-­‐mediated LTG uptake yielding an IC50 of 1.8 μM. Similarly low IC50 values were obtained with primary human hepatocytes from two patients (IC50 = 7.9 μM and IC50 = 3.9 μM, respectively) and the hCMEC/D3 cell line (IC50 = 2.0 μM). The clinical consequences of these observations will require further in vivo pharmacokinetic and epidemiological research. In conclusion, the results presented in this thesis demonstrate that membrane transporters can be involved in the passage of AEDs and antipsychotics across the BBB and other membrane barriers. However, currently available in vitro methods proved to be insufficient to identify and characterise the underlying transporters involved and to further evaluate the impact on treatment efficacy. The development of large-­‐scale functional screening methodologies will be crucial for a more systematic and comprehensive understanding of drug transport processes involved in determining access of drugs to the central nervous system. This will help in improving drug efficacy and drug safety, allow prediction of drug-­‐drug interactions, and eventually allowed a more personalised approach to prescribing in diseases such as epilepsy and schizophrenia.