The glucuronidation kinetics of the prototypic substrates 4-methylumbelliferone (4MU) and 1-naphthol (1NP) by human UDP-glucuronosyltransferases (UGT) 1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B7, 2B15, and 2B17 were investigated. Where activity was demonstrated, inhibitory effects of diclofenac, probenecid, and the solvents acetone, acetonitrile, dimethyl sulfoxide, ethanol, and methanol were characterized. All isoforms except UGT1A4 glucuronidated 4MU, whereas all but UGT 1A4, 2B15, and 2B17 metabolized 1NP. However, kinetic models varied with substrate (for the same isoform) and from isoform to isoform (with the same substrate). Hyperbolic (Michaelis-Menten), substrate inhibition, and sigmoidal kinetics were variably observed for both 4MU and 1NP glucuronidation by the various UGTs. K_m or S₅₀ (sigmoidal kinetics) and V_max values varied 525- (8–4204 μM) and 5535-fold, respectively, for 4MU glucuronidation, and 1360- (1.3–1768 μM) and 560-fold, respectively, for 1NP glucuronidation. The use of a two-site model proved useful for those reactions exhibiting non-Michaelis-Menten glucuronidation kinetics. The organic solvents generally had a relatively minor effect on UGT isoform activity. UGT 1A6, 2B15, and 2B17 were most susceptible to the presence of solvent, although solvent-selective inhibition was occasionally observed with other isoforms. Diclofenac and probenecid inhibited all isoforms, precluding the use of these compounds for the reaction phenotyping of xenobiotic glucuronidation pathways in human tissues. Diclofenac and probenecid K_i values, determined for selected isoforms, ranged from 11 to 60 μM and 96 to 2790 μM, respectively. Overall, the results emphasize the need for the careful design and interpretation of kinetic and inhibition studies with human UGTs.

CYP2C9 is the most abundant CYP2C subfamily enzyme in human liver and the most important contributor from this subfamily to drug metabolism. Polymorphisms resulting in decreased enzyme activity are common in the CYP2C9 gene and this, combined with narrow therapeutic indices for several key drug substrates, results in some important issues relating to drug safety and efficacy. CYP2C9 substrate selectivity is detailed and, based on crystal structures for the enzyme, we describe how CYP2C9 catalyzes these reactions. Factors relevant to clinical response to CYP2C9 substrates including inhibition, induction and genetic polymorphism are discussed in detail. In particular, we consider the issue of ethnic variation in pattern and frequency of genetic polymorphisms and clinical implications. Warfarin is the most well studied CYP2C9 substrate; recent work on use of dosing algorithms that include CYP2C9 genotype to improve patient safety during initiation of warfarin dosing are reviewed and prospects for their clinical implementation considered. Finally, we discuss a novel approach to cataloging the functional capabilities of rare ‘variants of uncertain significance’, which are increasingly detected as more exome and genome sequencing of diverse populations is conducted.

Major advances in the characterization of uridine diphosphate (UDP)-glucuronosyltransferase (UGT) enzyme substrate and inhibitor selectivities and the development of experimental paradigms to investigate xenobiotic glucuronidation in vitro now permit the prediction of a range of drug-glucuronidation parameters in humans. In particular, the availability of substrate and inhibitor “probes” for the major hepatic drug metabolizing UGTs together with batteries of recombinant enzymes allow the reaction phenotyping of drug glucuronidation reactions. Additionally, in vitro experimental approaches and scaling strategies have been successfully applied to the quantitative prediction of in vivo clearance via glucuronidation and drug-drug interaction potential.

Cytochrome P450 (CYP) and UDP-glucuronosyltransferase (UGT), which both exist as enzyme “superfamilies,” are together responsible for the metabolism of most hepatically cleared drugs. There is currently intense interest in the development of techniques that permit identification of the CYP and UGT isoform(s) involved in the metabolism of a newly discovered drug, and hence prediction of factors likely to alter elimination in vivo. In addition, the quantitative scaling of kinetic parameters for a metabolic pathway assumes importance for identifying newly discovered drugs with undesirable in vivo pharmacokinetic properties. Although qualitative and quantitative in vitro–in vivo correlation based on data generated using human liver tissue or recombinant enzymes have been applied successfully to many drugs eliminated by CYP, these strategies have proved less definitive for glucuronidated compounds. Computational (in silico) modeling techniques that potentially provide a facile and economic alternative to the in vitro methods are now emerging. This review assesses the utility of in vitro and in silico approaches for the qualitative and quantitative prediction of drug glucuronidation parameters and the challenges facing the development of generalizable models.