The UDP-glucuronosyltransferases: their role in drug metabolism and detoxification.

Background. Phase II biotransformation reactions (also ‘conjugation reactions’) generally serve as a detoxifying step in drug metabolism. Phase II drug metabolising enzymes are mainly transferases. This review covers the major phase II enzymes: UDP-glucuronosyltransferases, sulfotransferases, N-acetyltransferases, glutathione S-transferases and methyltransferases (mainly thiopurine S-methyl transferase and catechol O-methyl transferase). The focus is on the presence of various forms, on tissue and cellular distribution, on the respective substrates, on genetic polymorphism and finally on the interspecies differences in these enzymes. Methods and Results. A literature search using the following databases PubMed, Science Direct and EBSCO for the years, 1969–2010. Conclusions. Phase II drug metabolizing enzymes play an important role in biotransformation of endogenous compounds and xenobiotics to more easily excretable forms as well as in the metabolic inactivation of pharmacologically active compounds. Reduced metabolising capacity of Phase II enzymes can lead to toxic effects of clinically used drugs. Gene polymorphism/ lack of these enzymes may often play a role in several forms of cancer.

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Phase II drug metabolizing enzymes

UDP-glucuronosyltransferases (UGTs) catalyze glucuronidation of a variety of xenobiotics and endobiotics. UGTs are divided into two families, UGT1 and UGT2. The purpose of this study was to estimate the absolute expression levels of each UGT isoform in human liver and to evaluate the interindividual variability. Real-time reverse transcriptase-polymerase chain reaction analysis was performed to determine the copy numbers of nine functional UGT1A isoforms and seven UGT2B isoforms. We noticed that not only primers but also templates as a standard for quantification should prudently be selected. Once we established appropriate conditions, the mRNA levels of each UGT isoform in 25 individual human livers were determined. UGT1A1 (0.9-138.5), UGT1A3 (0.1-66.6), UGT1A4 (0.1-143.3), UGT1A6 (1.0-70.4), UGT1A9 (0.3-132.4), UGT2B4 (0.3-615.0), UGT2B7 (0.2-97.4), UGT2B10 (0.7-253.2), UGT2B15 (0.3-107.8), and UGT2B17 (0.5-157.1) were substantially expressed (x10(4) copy/mug RNA) with large interindividual variability. Abundant isoforms were UGT2B4 and UGT2B10, followed by UGT1A1, UGT2B15, and UGT1A6. The sum of the UGT2B mRNA levels was higher than that of UGT1A mRNA levels. It is interesting to note that the mRNA levels normalized with glyceraldehyde-3-phosphate dehydrogenase mRNA for almost UGT isoforms that are substantially expressed in liver showed significant correlations to each other. Western blot analysis was performed using antibodies specific for UGT1A1, UGT1A4, UGT1A6, or UGT2B7. Correlation between the protein and mRNA levels was observed in only UGT1A1 (r = 0.488; p < 0.01). In conclusion, this study comprehensively determined the absolute values of mRNA expression of each UGT isoform in human livers and found considerable interindividual variability.

https://dmd.aspetjournals.org/content/dmd/early/2009/05/13/dmd.109.027227.full.pdf

Quantitative analysis of UDP-glucuronosyltransferase (UGT) 1A and UGT2B expression levels in human livers.

Relatively few selective substrate and inhibitor probes have been identified for human UDP-glucuronosyltransferases (UGTs). This work investigated the selectivity of trifluoperazine (TFP), as a substrate, and amitriptyline, androsterone, canrenoic acid, hecogenin, phenylbutazone, quinidine, quinine, and sulfinpyrazone, as inhibitors, for human UGTs. Selectivity was assessed using UGTs 1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B7, and 2B15 expressed in HEK293 cells. TFP was confirmed as a highly selective substrate for UGT1A4. However, TFP bound extensively to both HEK293 lysate and human liver microsomes in a concentration-dependent manner (fuinc 0.20-0.59). When corrected for nonspecific binding, Km values for TFP glucuronidation were similar for both UGT1A4 (4.1 microM) and human liver microsomes (6.1+/-1.2 microM) as the enzyme sources. Of the compounds screened as inhibitors, hecogenin, alone, was selective; significant inhibition was observed only for UGT1A4 (IC50 1.5 microM). Using phenylbutazone and quinine as "models," inhibition kinetics were variously described by competitive and noncompetitive mechanisms. Inhibition of UGT2B7 by quinidine was also investigated further, because the effects of this compound on morphine pharmacokinetics (a known UGT2B7 substrate) have been ascribed to inhibition of P-glycoprotein. Quinidine inhibited human liver microsomal and recombinant UGT2B7, with respective Ki values of 335+/-128 microM and 186 microM. In conclusion, TFP and hecogenin represent selective substrate and inhibitor probes for UGT1A4, although the extensive nonselective binding of the former should be taken into account in kinetic studies. Amitriptyline, androsterone, canrenoic acid, hecogenin, phenylbutazone, quinidine, quinine, and sulfinpyrazone are nonselective UGT inhibitors.

https://dmd.aspetjournals.org/content/dmd/early/2005/12/28/dmd.105.007369.full.pdf

Selectivity of substrate (trifluoperazine) and inhibitor (amitriptyline, androsterone, canrenoic acid, hecogenin, phenylbutazone, quinidine, quinine, and sulfinpyrazone) “probes” for human udp-glucuronosyltransferases

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.

The prediction of drug-glucuronidation parameters in humans: UDP-glucuronosyltransferase enzyme-selective substrate and inhibitor probes for reaction phenotyping and in vitro-in vivo extrapolation of drug clearance and drug-drug interaction potential.

Aims
Using the fluconazole–zidovudine (AZT) interaction as a model, to determine whether inhibition of UDP–glucuronosyltransferase (UGT) catalysed drug metabolism in vivo could be predicted quantitatively from in vitro kinetic data generated in the presence and absence bovine serum albumin (BSA).

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Quantitative prediction of in vivo inhibitory interactions involving glucuronidated drugs from in vitro data: the effect of fluconazole on zidovudine glucuronidation

Interactions between the UGT2B7-catalyzed glucuronidation of zidovudine (AZT), 4-methylumbelliferone (4MU), and 1-naphthol (1NP) were analyzed using multisite and empirical kinetic models to explore the existence of multiple substrate and effector binding sites within this important drug metabolizing enzyme. 4MU and 1NP glucuronidation by UGT2B7 exhibit sigmoidal kinetics characteristic of homotropic cooperativity (autoactivation), which may be modeled assuming the existence of two equivalent, interacting substrate binding sites. In contrast, UGT2B7-catalyzed AZT glucuronidation follows hyperbolic (Michaelis-Menten) kinetics. Although 4MU and 1NP decreased the binding affinity of AZT, the kinetics of AZT glucuronidation changed from hyperbolic to sigmoidal in the presence of both modifiers. Data were well described by a generic two-substrate binding site model in which there is no interaction between the sites in the absence of 4MU or 1NP, but heterotropic cooperativity results from the binding of modifier. Inhibition of 4MU and 1NP glucuronidation by AZT and interactions between 4MU and 1NP required more complex three-site models, where the modifier acts via a distinct effector site to alter either substrate binding affinity or Vmax without affecting the homotropic cooperativity characteristic of 4MU and 1NP glucuronidation. It is noteworthy that 1NP inhibited 4MU glucuronidation, whereas 4MU activated 1NP glucuronidation. The results are consistent with the existence of two "catalytic" sites for each substrate within the UGT2B7 active site, along with multiple effector sites. The multiplicity of binding and effector sites results in complex kinetic interactions between UGT2B7 substrates, which potentially complicates inhibition screening studies.

https://molpharm.aspetjournals.org/content/molpharm/early/2008/07/22/mol.108.048645.full.pdf

Kinetic Modeling of the Interactions between 4-Methylumbelliferone, 1-Naphthol, and Zidovudine Glucuronidation by UDP-Glucuronosyltransferase 2B7 (UGT2B7) Provides Evidence for Multiple Substrate Binding and Effector Sites

UDP-glucuronosyltransferases (UGTs) are critical to the detoxification of numerous drugs, environmental pollutants, and endogenous molecules. However, as yet not all of the human UGTs have been cloned and characterized. cDNA clones from the UGT2A3 gene (located on chromosome 4q13 ) were isolated using pooled human liver RNA. Approximately 10% of clones contained a c.1489 A > G nucleotide substitution, yielding proteins with a residue 497 alanine (UGT2A3.2) instead of a threonine (UGT2A3.1). The allele frequency of this polymorphism ( rs 13128286) was 0.13 in a European-American population as determined by direct DNA sequencing. Of 81 structurally diverse glucuronidation substrates tested, UGT2A3 expressed by a baculovirus system selectively glucuronidated bile acids, particularly hyodeoxycholic acid at the 6-hydroxy position. Apparent K m values of UGT2A3.1 and UGT2A3.2 for hyodeoxycholic acid 6-glucuronidation were 69 ± 7 and 44 ± 12 μM, respectively. Of 29 different extrahepatic tissues evaluated by real-time polymerase chain reaction, UGT2A3 mRNA was most highly expressed in small intestine (160% of liver), colon (78% of liver), and adipose tissue (91% of liver). An in silico scan of the proximal UGT2A3 promoter/5′-regulatory region identified transcription factor consensus elements consistent with tissue-selective expression in liver ( HNF1 ) and intestine ( CXD2 ), as well as induction by rifampicin ( pregnane X receptor ). In LS180 human intestinal cells, rifampicin increased UGT2A3 mRNA by more than 4.5-fold compared with vehicle, whereas levels were not significantly affected by the arylhydrocarbon receptor ligand β-naphthoflavone. This is the first report establishing UGT2A3 as a functional enzyme, and it represents significant progress toward the goal of having a complete set of recombinant human UGTs for comparative functional analyses.

https://molpharm.aspetjournals.org/content/molpharm/early/2008/06/03/mol.108.045500.full.pdf

Novel polymorphic human UDP-glucuronosyltransferase 2A3: cloning, functional characterization of enzyme variants, comparative tissue expression, and gene induction.

J. Andrew Williams

Papers

Quantitative prediction of in vivo inhibitory interactions involving glucuronidated drugs from in vitro data: the effect of fluconazole on zidovudine glucuronidation

Kinetic Modeling of the Interactions between 4-Methylumbelliferone, 1-Naphthol, and Zidovudine Glucuronidation by UDP-Glucuronosyltransferase 2B7 (UGT2B7) Provides Evidence for Multiple Substrate Binding and Effector Sites

Novel polymorphic human UDP-glucuronosyltransferase 2A3: cloning, functional characterization of enzyme variants, comparative tissue expression, and gene induction.