Determinants of transcriptional regulation of transport and oxidative processes in human model systems

Williamson, Beth Determinants of transcriptional regulation of transport and oxidative processes in human model systems. Doctor of Philosophy thesis, University of Liverpool.

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Abstract

Initial predictions of drug response and drug-drug interactions (DDIs) are made following high-throughput in vitro screening. Such assays are indispensible in the pharmaceutical industry to determine the metabolism, transport and pharmacokinetics of new chemical entities. However, they often fail when extrapolated to in vivo response due to unsuitable pharmacokinetic or pharmacodynamic prediction. The primary aim of this thesis was to investigate and understand the differences in the expression profiles of drug disposition genes, between transformed hepatic cell lines and primary human hepatocytes. Primary human hepatocytes were also analysed to determine uptake contribution, induction and genotype of key drug disposition-relevant genes. The loss of hepatic phenotype in HepG2 and Huh7 cells is partly due to the altered expression of transcriptional regulators including; chaperones, co-chaperones, co-activators and co-repressors. Indeed, Chapter 2 of this thesis shows lower levels of the Gadd45β and PGC1α gene expression in HepG2 cells corresponds to a deficient expression and activity of cytochrome P450 3A4 (CYP3A4), with the levels reducing further as cell passage increases, in comparison to primary human hepatocytes. HepG2 cells were transfected with a novel complex transfection of Gadd45β and PGC1α with the aim to improve CYP3A4 activity in Chapter 3. CYP3A4 activity was improved by 54% and induction response was enhanced in comparison to control cells with no off-target effects. Over the last decade it has become apparent that transporters can play a significant role in the disposition of many drugs. Organic anion transporting polypeptide (OATP) transporters have received considerable recent attention since they mediate sodium-independent uptake of a broad array of xenobiotics. A method to determine the specific contribution of OATP1B1 in the hepatic uptake was successfully optimised and applied for 5 therapeutic drugs in Chapter 4. Future application of this strategy is likely to have broad importance in determining relative contribution that individual transporters play in drug disposition. To prevent accumulation and toxicity of xenobiotics, biotransformation and transport of foreign compounds occurs. However, these processes can be altered by induction or inhibition mechanisms. Rifampicin is a first line drug in tuberculosis (TB) treatment but it is a potent inducer of CYPs and transporters. DDIs during TB treatment are common but the induction potential of different rifamycins has not been comprehensively ranked. Chapter 5 investigated the induction potential of rifampicin, rifapentine and rifabutin. Rifampicin significantly induced CYP3A4, ABCB1, OATP1B1 and ABCC2 in primary human hepatocytes. Induction by rifabutin was observed for CYP3A4, OATP1B3 whilst rifapentine only significantly induced OATP1B1. This work serves as a basis for further study into the extent to which rifamycins induce key metabolism and transporter genes. Nuclear receptors (NR) regulate the expression of CYPs and drug transporters influencing pharmacokinetics. PXR and VDR have been found to synergistically increase CYP3A4 expression and activity in intestinal cell lines. This effect has been observed in vivo with seasonal variations apparent for CYP3A4 substrates. In Chapter 6, novel associations between vitamin D receptor polymorphisms and expression of it and its target genes involved in drug disposition were shown in D2 intestinal biopsies. This thesis reports generation of model systems and their application to enable many questions to be answered relating to pharmacokinetics and DDIs. The thesis forms a solid platform from which to further investigate these issues in future studies.

Item Type:	Thesis (Doctor of Philosophy)
Additional Information:	Date: 2013-09 (completed)
Uncontrolled Keywords:	Transporters, Cytochrome P450, OATP1B1, Induction, Rifampicin, Nuclear receptors, Nuclear receptor co-regulators
Subjects:	?? QP ??
Divisions:	Faculty of Health and Life Sciences > Institute of Systems, Molecular and Integrative Biology
Depositing User:	Symplectic Admin
Date Deposited:	08 Aug 2014 09:46
Last Modified:	16 Dec 2022 04:41
DOI:	10.17638/00015053
Supervisors:	Owen, Andrew
URI:	https://livrepository.liverpool.ac.uk/id/eprint/15053