Effect of glucuronides on metabolic enzymes and active hepatic uptake: in vitro assessment and prediction of drug-drug interaction risk

  • Rebecca Sullivan

Student thesis: Phd


The potential contribution of drug metabolites to drug-drug interactions (DDIs) is increasingly recognised. The latest FDA guidance recommends investigation of the effect of metabolites on CYP450 enzymes if present at greater than or equal to25% of parent systemic exposure. In addition, the clinical relevance of transporter mediated DDIs is highlighted in the latest EMA and FDA guidelines. Glucuronidation is a major conjugation pathway and increasing numbers of glucuronides have been reported to inhibit metabolic enzymes and/or uptake transporters e.g., gemfibrozil glucuronide inhibits both CYP2C8 and OATP1B1. However, their inhibitory effects on a range of metabolic enzymes and transporters has not been characterised in a systematic manner and their potency in relation to parent compounds is unknown.The aim of this Thesis was to comprehensively investigate the inhibitory potential of 18 compounds in total including 10 glucuronides, parent compounds and reference inhibitors of interest against CYP2C8, CYP3A4 and UGT1A1 in human liver microsomes, using repaglinide as a probe substrate. Similar studies were performed on OATP1B1 in HEK293 cells, using estradiol 17β glucuronide (E17βG) as an initial probe substrate. For a subset of glucuronides and parent drugs, additional studies were conducted using pitavastatin as a clinically relevant OATP1B1 probe. A pre-incubation with inhibitor was included in both transporter and metabolism experiments to assess potential for time-dependent inhibition. In addition, the clinical exposure of glucuronides was assessed in relation to FDA guidance exposure limits. The in vitro data generated and inhibitor clinical exposure data collated were used to predict glucuronide DDI risk initially using basic models (1 + I/Ki). Subsequently, the extent of DDI was predicted using static mechanistic models incorporating the fraction of victim drug metabolised (fmCYP2C8) or transported (ftOATP1B1) by the enzyme or transporter of interest using repaglinide and pitavastatin as relevant victim drugs.CYP2C8 IC50 values were characterised for 5/10 glucuronides (IC50 8.6 - 54.1 μM for telmisartan and diclofenac glucuronides, respectively) which demonstrated similar or greater inhibitory potency to their parent compounds except for mefenamic acid which was 3-fold more potent than its glucuronide. The choice of enzyme co-factor conditions influenced the CYP2C8 inhibitory potential of mefenamic acid and clopidogrel glucuronides, no changes were seen for other glucuronides. Time-dependent increase in potency was observed for clopidogrel and gemfibrozil glucuronides resulting in 3 to 10-fold more potent inhibition than parent compounds. Minimal inhibition of CYP3A4 and UGT1A1 by glucuronides was observed. OATP1B1 IC50 values with E17βG as a probe substrate were obtained for 8/10 glucuronides (IC50 1.2 - 55.1 μM for telmisartan and raloxifene glucuronides, respectively) and all 5 parent compounds investigated (IC50 0.73 - 47.8 μM). For repaglinide, diclofenac and gemfibrozil glucuronides, OATP1B1 IC50 values were comparable to parents. Ezetimibe glucuronide was 4-fold more potent than its parent but the opposite was observed for telmisartan. The OATP1B1 inhibitory potency of parents and glucuronides was in the same rank order; however, differential pre-incubation effects were seen between glucuronide-parent pairs. Using pitavastatin as a probe, IC50 values were within 2-fold of those obtained using E17βG except for gemfibrozil, diclofenac and ezetimibe where up to 12-fold more potent OATP1B1 inhibition was observed using E17βG. A mean increase in OATP1B1 inhibitory potency of 1.7 and 2.3-fold was observed following pre-incubation with inhibitor using E17βG and pitavastatin as probe substrates, respectively.In conclusion, glucuronides were found to inhibit both CYP2C8 and OATP1B1 in vitro causing comparable or more potent inhibition than their parent drugs in the majority of cases. Inclusion of a pre-incubation step wit
Date of Award1 Aug 2016
Original languageEnglish
Awarding Institution
  • The University of Manchester
SupervisorAleksandra Galetin (Supervisor)


  • Drug-drug interaction
  • Glucuronide

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