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Background
There is evidence that P-gp has multiple substrate binding pockets and as such handles different substrates with different specificities. Furthermore, inhibition can occur via direct competition for efflux or by non-competitive mechanisms. Collectively, this means that the efficacy of an inhibitor will vary between substrates. Recent work has also shown that common genetic polymorphisms within the ABCB1 gene alter the extent of inhibition and this is an important consideration for inter-individual variability in inhibition. Finally, previous studies have also shown that ABCB1 gene expression is influenced by seizures as well as certain AEDs.
This work will utilise in-vitro and ex-vivo methodology to assess inhibition and induction of ABCB1 in a substrate dependent manner. Genetic diversity will then be superimposed in order to establish whether inhibition is influenced by common polymorphisms within the ABCB1 gene. In order to do this we will use a combination of assays some of which are established in Liverpool and some which will require optimisation. Assays have been developed for investigating substrate affinity for and inhibition of wild-type ABCB1 in Caco-2, MDCKII and Xenopus Laevis oocyte systems, including a validated ABCB1 RNAi with greater than 80% knockdown (AO). However, further work will be required to optimise an oocyte expression system to study genetic variants of ABCB1. For induction studies, methods are in place for assessing expression of ABCB1 mRNA and protein, but assays will need to be developed for RNAi knockdown of nuclear receptors.
WP03 will work in co-operation with WP01 and WP08 to ensure that substrate specificities of all ligands and inhibitors of transporters are accurately characterised and quantified, and the effects of genetic variation determined.
Task 1:
In-vitro characterisation of inhibition of transport by ABCB1 to identify compounds with the best inhibitory profile to be used in-vivo
Since different substrates interact with ABCB1 in different binding pockets and inhibition can be competitive or non-competitive, it is not possible to predict the degree of inhibition of ABCB1 mediated transport of (R)-[11C]-verapamil or [11C]laniquidar. Therefore, this task will identify the most potent inhibitor for each of these probes. In addition, we will determine which AEDs are substrates for ABCB1 and the most potent inhibitors for each of them. Although work focuses on AEDs as paradigms, these techniques will be applicable to other CNS drugs – these will be chosen based on work in WP01 and 04. The knowledge gained from this task will allow us to more clearly rationalise the in-vivo observations from other work packages and dissect which drugs are potentially influenced by the increased expression of P-gp observed in refractory disease. The work will also provide tools to screen for inhibitors should pharmaco-enhancement of AEDs and other CNS drugs by inhibition of P-gp be identified as a valid therapeutic strategy.
Task 2:
Comparison of inhibition of wild-type and variant forms of ABCB1 by model inhibitors
A recent study demonstrated that genetic polymorphisms in ABCB1 are linked to altered substrate affinity and altered drug/inhibitor interactions. To determine the functional effects of three common SNPs (C1236T, G2677T and C3435T) and their associated haplotypes on P-gp inhibition, we will use a Xenopus Laevis oocyte expression system. We will introduce individual point mutations and their combinations (haplotypes) into the vector containing ABCB1 using site-directed mutagenesis. Re-sequencing will then be performed to confirm nucleotide sequence identity of the inserts. P-gp localization will be measured by confocal fluorescence microscopy and we will compare inhibition (by multiple inhibitors) of P-gp transport in wild-type cells as well as cells expressing single- double- or triple- mutants.
Task 3:
Characterisation of mechanism of induction of ABCB1 by drugs
Previous studies have shown that ABCB1 expression can be induced by various drugs including rifampicin and phenobarbital. We recently showed that carbamazepine induced ABCB1 in peripheral blood mononuclear cells at the mRNA and protein level. We already have methodology in place for assessing induction of ABCB1 in Caco-2 cells (frequently used to study ABC transporters) and primary human hepatocytes. We will investigate the impact of AEDs on ABCB1 expression using both of these systems. In addition to assessing the degree of induction, we will investigate relationships with intracellular permeation of the compounds. A number of in-vitro and ex-vivo systems will be utilized to probe the mechanisms involved in regulation of ABCB1 expression via relevant transcription factors and response elements. Results will be compared to and further inform those from WP08
