Author: Attarat Pattanawongsa
Pattanawongsa, Attarat, 2018 The in vitro Characterisation of Drug-Drug Interactions Arising from Inhibition of Drug Metabolising Enzymes, Flinders University, College of Medicine and Public Health
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The overarching theme of the studies described in this thesis is the in vitro characterisation of drug-drug interactions (DDIs) arising from drug metabolising enzymes. Two studies characterised potential DDIs arising from the inhibition of UDP-glucuronosyltransferase (UGT) enzymes while the third investigated the molecular basis of the inhibition of cytochrome P450 (CYP), namely CYP2C8, by glucuronide conjugates.
The first major study (Chapter 3) primarily aimed to characterise the inhibition of UGT2B10 by 34 amine-containing antidepressant and antipsychotic drugs and identify potential perpetrators of DDIs. Initial experiments confirmed that cotinine is selective substrate of UGT2B10 while desloratadine is a selective inhibitor, and these compounds may be used as 'probes’ for reaction phenotyping. Amitriptyline, doxepin and mianserin were the most potent inhibitors of human liver microsomal UGT2B10, with Ki values < 1 µM. In vitro – in vivo extrapolation (IV-IVE) suggested that all three drugs may act as perpetrators of DDIs arising from inhibition of UGT2B10-catalysed drug glucuronidation. Molecular modelling demonstrated that moderate to potent inhibitors all contained a hydrophobic domain (comprising a tetra-, tri- or bi-cyclic ring structure or a single aromatic ring as the central ‘scaffold’) and an amine functional group that was located 3 C-C or C-N bond lengths from the central scaffold.
Studies described in Chapter 4 continued the theme of the in vitro characterisation of human UGT inhibition. Canagliflozin (CNF), dapagliflozin (DPF) and empagliflozin (EPF) are the first SGLT2 inhibitors introduced into clinical practice for the treatment of type 2 diabetes. While all three ‘flozins’ inhibited UGT enzyme activity to some extent, CNF inhibited recombinant UGT1A1, UGT1A9 and the extrahepatic UGT1A10 with IC50 values ranging from 6.9 to 9.5 µM. IV-IVE using mean Ki values for CNF inhibition of human liver microsomal UGT1A1 (7.2 µM) and UGT1A9 (3.0 µM) predicted that CNF may perpetrate DDIs with drugs glucuronidated by these enzymes. The results indicate that characterisation of the DDI potential of new SGLT2 inhibitors currently in clinical development is warranted.
In Chapter 5, the focus changed from inhibition of glucuronide formation to inhibition of CYP2C8 by glucuronide conjugates. Docking of the mechanism-based inhibitor gemfibrozil glucuronide and the diclofenac and estradiol glucuronides, both known to be hydroxylated by CYP2C8, in the CYP2C8 X-ray crystal structure confirmed that the side-chains of Asn99, Ser100, Ser103, Thr107, Ser114 and Gln214 were within hydrogen bonding distance to the polar groups of the glucuronide moiety. Based on these observations, 14 mutants (8 single, 2 double, 1 triple, 1 quadruple, and 2 quintuple) were generated by site-directed mutagenesis, and each was characterised for time-dependent inhibition (TDI) and non-TDI by the three glucuronides.
Although TDI studies with the multiple mutants was not possible due to their instability when pre-incubated with NADPH, the non-TDI (co-incubation) experiments with all mutants and the TDI experiments with the single mutants demonstrated that neither Asn99, Ser100, Ser103, Thr107, Ser114 nor Gln214 alone are ‘critical’ for glucuronide conjugate binding in the CYP2C8 active site. Rather, all six residues appear to contribute to the binding of the glucuronic acid moiety to a similar extent.
Keywords: Drug-drug interactions, inhibition of UGT2B10, inhibition of UGT enzymes, inhibition of CYP2C8, SGLT2 inhibitors, CYP2C8 inhibition by glucuronides, in vitro-in vivo extrapolation
Subject: Clinical Pharmacology thesis
Thesis type: Doctor of Philosophy
Completed: 2018
School: College of Medicine and Public Health
Supervisor: Professor John O. Miners