Biomedical subjects

Peter I Mackenzie

Publications and source records attributed to Peter I Mackenzie.

At least 19 recordsLinked to original sources

PubMed2006

Amino terminal domains of human UDP-glucuronosyltransferases (UGT) 2B7 and 2B15 associated with substrate selectivity and autoactivation.

Despite the important role of UDP-glucuronosyltransferases (UGT) in the metabolism of drugs, environmental chemicals and endogenous compounds, the structural features of these enzymes responsible for substrate binding and selectivity remain poorly understood. Since UGT2B7 and UGT2B15 exhibit distinct, but overlapping, substrate selectivities, UGT2B7-UGT2B15 chimeras were constructed here to identify substrate binding domains. A UGT2B7-15-7 chimera that incorporated amino acids 61-194 of UGT2B15 glucuronidated the UGT2B15 substrates testosterone and phenolphthalein, but not the UGT2B7 substrates zidovudine and 11alpha-hydroxyprogesterone. Derived apparent K(m) values for testosterone and phenolphthalein glucuronidation by UGT2B7-15((61-194))-7 were similar in magnitude to those determined for UGT2B15. Moreover, glucuronidation of the non-selective substrate 4-methylumbelliferone (4MU) by UGT2B7-15((61-194))-7 and UGT2B15 followed Michaelis-Menten and weak substrate inhibition kinetics, respectively, whereas 4MU glucuronidation by UGT2B7 exhibited sigmoidal kinetics characteristic of autoactivation. Six UGT2B7-15-7 chimeras that incorporated smaller domains of UGT2B15 were subsequently generated. Of these, UGT2B7-15((61-157))-7, UGT2B7-15((91-157))-7 and UGT2B7-15((61-91))-7 glucuronidated 4MU, but activity towards the other substrates investigated here was not detected. Like UGT2B7, the UGT2B7-15((61-157))-7, UGT2B7-15((91-157))-7 and UGT2B7-15((61-91))-7 chimeras exhibited sigmoidal 4MU glucuronidation kinetics. The sigmoidal 4MU kinetic data were well modelled using both the Hill equation and the expression for a two-site model that assumes the simultaneous binding of two substrate molecules at equivalent sites. It may be concluded that residues 61-194 of UGT2B15 are responsible for substrate binding and for conferring the unique substrate selectivity of UGT2B15, while residues 158-194 of UGT2B7 appear to facilitate the binding of multiple 4MU molecules within the active site.

Amino Acid Motifs↗

PubMed2006

Isolation of the UDP-glucuronosyltransferase 1A3 and 1A4 proximal promoters and characterization of their dependence on the transcription factor hepatocyte nuclear factor 1alpha.

The UGT1A3-1A5 genes are a highly related UDP-glucuronosyltransferase (UGT) cluster exhibiting high levels of coding and regulatory region homology. However, the ensuing proteins have both differing substrate specificities and differing expression patterns. The expression profile of each enzyme also varies considerably from one individual to the next. Differences in UGT expression have been predicted to contribute to an individual's response to pharmaceuticals and to predisposition toward cancer in the event of carcinogen exposure. Therefore, it is desirable to elucidate the mechanisms that drive the transcription of UGT genes and identify the factors responsible for their variable expression. To this end, we have isolated the UGT1A3, UGT1A4, and UGT1A5 proximal promoters and begun to investigate the regulatory elements necessary for activity in vitro. We have established that the nucleotide sequence upstream of the UGT1A5 exon 1 is an ineffective promoter, correlating with the lack of substantial expression of this UGT in human tissues. In contrast, the UGT1A3 and UGT1A4 proximal promoters are both highly active in hepatic and colonic cell lines, with maximal activity being encoded by the proximal 500 base pairs. However, the UGT1A3 and UGT1A4 promoters exhibit low activity in the human embryonic kidney cell line HEK293, unless coexpressed with hepatocyte nuclear factor (HNF) 1alpha. Furthermore, mutation of the consensus-like HNF1-binding site in the UGT1A3 promoter abolishes promoter function in all cell types. This study suggests an important role for HNF1alpha in the transcriptional regulation of the human UGT1A3 and UGT1A4 genes.

Caco-2 Cells↗

PubMed2006

Sulfinpyrazone C-glucuronidation is catalyzed selectively by human UDP-glucuronosyltransferase 1A9.

The uricosuric agent sulfinpyrazone (SFZ) is metabolized via C-glucuronidation, an uncommon metabolic pathway, in humans. The present study aimed to characterize SFZ glucuronidation by human liver microsomes (HLMs) and identify the hepatic forms of UDP-glucuronosyltransferase responsible for this pathway. Incubations of SFZ with HLMs formed a single glucuronide that was resistant to beta-glucuronidase and acid hydrolysis, consistent with formation of a C-glucuronide. Mass spectral analysis confirmed the identity of the metabolite as SFZ glucuronide (sulfinpyrazone beta-D-glucuronide; SFZG). SFZ C-glucuronidation by HLMs exhibited Michaelis-Menten kinetics, with mean (+/- S.D.) Km and Vmax values of 51 +/- 21 microM and 2.6 +/- 0.6 pmol/min . mg, respectively. Fifteen recombinant human UDP-glucuronosyltransferases (UGTs), expressed in HEK293 cells, were screened for their capacity to catalyze SFZ C-glucuronidation. Of the hepatically expressed enzymes, only UGT1A9 formed SFZG. UGTs 1A7 and 1A10, which are expressed in the gastrointestinal tract, also metabolized SFZ, but rates of metabolism were low compared with UGT1A9. SFZ glucuronidation by UGT1A9 exhibited "weak" negative cooperative kinetics, which was modeled by the Hill equation (S50 16 microM). The data indicate that UGT1A9 is the enzyme responsible for hepatic SFZ C-glucuronidation and that SFZ may be used as a substrate "probe" for UGT1A9 activity in HLMs.

Catalysis↗

PubMed2006

Fatty acyl-CoA as an endogenous activator of UDP-glucuronosyltransferases.

The acyl-CoA-dependent modulation of hepatic microsomal UDP-glucuronosyltransferase (UGT) function in rats was studied. Oleoyl- and palmitoyl-CoAs inhibited UGT activity toward 4-methylumbelliferone in the presence of Brij 58. However, acyl-CoAs enhanced UGT activity in untreated microsomes. A maximum activation of about 8-fold over the control was observed at 15 microM oleoyl-CoA, whereas 50 microM or more oleoyl-CoA had an inhibitory effect on UGT function. Medium- and long-chain acyl-CoAs also exhibited similar effects. On the basis of resistance to tryptic digestion of UGTs, oleoyl-CoA at 15 microM has no ability to change the permeability of the endoplasmic reticulum (ER) membrane, although perturbation of the membrane occurred with 50 microM oleoyl-CoA. N-Ethylmaleimide and 5,5'-dithiobis(2-nitrobenzoic acid) abolished the oleoyl-CoA (15 microM)-dependent activation of microsomal UGT. These results suggest that: (1) acyl-CoAs play a role as an endogenous activator of UGTs, and (2) a sulfhydryl group is required for the activation of UGT by physiological concentrations of acyl-CoAs.

Acyl Coenzyme A↗

PubMed2006

Inhibition of UDP-glucuronosyltransferase 2b7-catalyzed morphine glucuronidation by ketoconazole: dual mechanisms involving a novel noncompetitive mode.

Glucuronidation of morphine in humans is predominantly catalyzed by UDP-glucuronosyltransferase 2B7 (UGT2B7). Since our recent research suggested that cytochrome P450s (P450s) interact with UGT2B7 to affect its function [Takeda S et al. (2005) Mol Pharmacol 67:665-672], P450 inhibitors are expected to modulate UGT2B7-catalyzed activity. To address this issue, we investigated the effects of P450 inhibitors (cimetidine, sulfaphenazole, erythromycin, nifedipine, and ketoconazole) on the UGT2B7-catalyzed formation of morphine-3-glucuronide (M-3-G) and morphine-6-glucuronide (M-6-G). Among the inhibitors tested, ketoconazole was the most potent inhibitor of both M-3-G and M-6-G formation by human liver microsomes. The others were less effective except that nifedipine exhibited an inhibitory effect on M-6-G formation comparable to that by ketoconazole. Neither addition of NADPH nor solubilization of liver microsomes affected the ability of ketoconazole to inhibit morphine glucuronidation. In addition, ketoconazole had an ability to inhibit morphine UGT activity of recombinant UGT2B7 freed from P450. Kinetic analysis suggested that the ketoconazole-produced inhibition of morphine glucuronidation involves a mixed-type mechanism. Codeine potentiated inhibition of morphine glucuronidation by ketoconazole. In contrast, addition of another substrate, testosterone, showed no or a minor effect on ketoconazole-produced inhibition of morphine UGT. These results suggest that 1) metabolism of ketoconazole by P450 is not required for inhibition of UGT2B7-catalyzed morphine glucuronidation; and 2) this drug exerts its inhibitory effect on morphine UGT by novel mechanisms involving competitive and noncompetitive inhibition.

Analgesics, Opioid↗

PubMed2006

In vitro characterization of lamotrigine N2-glucuronidation and the lamotrigine-valproic acid interaction.

Studies were performed to investigate the UDP-glucuronosyltransferase enzyme(s) responsible for the human liver microsomal N2-glucuronidation of the anticonvulsant drug lamotrigine (LTG) and the mechanistic basis for the LTG-valproic acid (VPA) interaction in vivo. LTG N2-glucuronidation by microsomes from five livers exhibited atypical kinetics, best described by a model comprising the expressions for the Hill (1869 +/- 1286 microM, n = 0.65 +/- 0.16) and Michaelis-Menten (Km 2234 +/- 774 microM) equations. The UGT1A4 inhibitor hecogenin abolished the Michaelis-Menten component, without affecting the Hill component. LTG N2-glucuronidation by recombinant UGT1A4 exhibited Michaelis-Menten kinetics, with a Km of 1558 microM. Although recombinant UGT2B7 exhibited only low activity toward LTG, inhibition by zidovudine and fluconazole and activation by bovine serum albumin (BSA) (2%) strongly suggested that this enzyme was responsible for the Hill component of microsomal LTG N2-glucuronidation. VPA (10 mM) abolished the Hill component of microsomal LTG N2-glucuronidation, without affecting the Michaelis-Menten component or UGT1A4-catalyzed LTG metabolism. Ki values for inhibition of the Hill component of LTG N2-glucuronidation by VPA were 2465 +/- 370 microM and 387 +/- 12 microM in the absence and presence, respectively, of BSA (2%). Consistent with published data for the effect of fluconazole on zidovudine glucuronidation by human liver microsomal UGT2B7, the Ki value generated in the presence of BSA predicted the magnitude of the LTG-VPA interaction reported in vivo. These data indicate that UGT2B7 and UGT1A4 are responsible for the Hill and Michaelis-Menten components, respectively, of microsomal LTG N2-glucuronidation, and the LTG-VPA interaction in vivo arises from inhibition of UGT2B7.

Anticonvulsants↗

PubMed2006

In vitro-in vivo correlation for drugs and other compounds eliminated by glucuronidation in humans: pitfalls and promises.

Enzymes of the UDP-glucuronosyltransferase (UGT) superfamily are responsible for the metabolism of many drugs, environmental chemicals and endogenous compounds. Identification of the UGT(s) involved in the metabolism of a given compound ('reaction phenotyping') currently relies on multiple confirmatory approaches, which may be confounded by the dependence of UGT activity on enzyme source, incubation conditions, and the occurrence of atypical glucuronidation kinetics. However, the increasing availability of substrate and inhibitor 'probes' for the individual UGTs provides the prospect for reliable phenotyping of glucuronidation reactions using human liver microsomes or hepatocytes, thereby providing data directly relevant to drug metabolism in humans. While the feasibility of computational prediction of UGT substrate selectivity has been demonstrated, the development of easily interpretable and generalisable models requires further improvement in the datasets available for analysis. Quantitative prediction of the hepatic clearance of glucuronidated drugs and the magnitude of inhibitory interactions based on in vitro kinetic data is more problematic. Intrinsic clearance (CL(int)) values generated using human liver microsomes under-predict in vivo hepatic clearance, typically by an order of magnitude. In vivo clearances of glucuronidated drugs are also generally under-predicted by CL(int) values from human hepatocytes, but to a lesser extent than observed with the microsomal model. While it is anticipated that systematic analysis of the potential causes of under-prediction may provide more reliable in vitro-in vivo scaling strategies, mechanistic interpretation of in vitro-in vivo correlation more broadly awaits further advances in our understanding of the structural and cellular determinants of UGT activity.

Glucuronides↗

PubMed2006

Relationship between hyperbilirubinaemia and UDP-glucuronosyltransferase 1A1 (UGT1A1) polymorphism in adult HIV-infected Thai patients treated with indinavir.

OBJECTIVES: To investigate the association between the UGT1A1*6 (G71R) and UGT1A1*28 (promoter (TA)7-repeat) genotypes and hyperbilirubinaemia in Thai patients treated with indinavir, and characterize the inhibition of human UGTs by indinavir in vitro. METHODS: Ninety-six Thai HIV patients receiving indinavir, 800 mg t.i.d. or 800 mg b.i.d. "boosted" with ritonavir (100 mg b.i.d.), had serum bilirubin levels measured to 24 weeks post-treatment and were genotyped for UGT1A1*6 and UGT1A1*28. The inhibition selectivity and kinetics of indinavir were determined using a panel of recombinant human UGTs. RESULTS: UGT1A1*6 and UGT1A1*28 frequencies in the Thai patients were 10.4% and 15.6%, respectively. Total, conjugated (direct) and unconjugated (indirect) serum bilirubin concentrations increased significantly at 24 weeks of indinavir treatment for all four genotypes, with a trend towards higher levels depending on the number of UGT1A1 mutant alleles; *6/*28 > *6 > *28 > reference. The hazards ratio (HR) for serious hyperbilirubinaemia (total bilirubin > 2.5 mg/dl) at week 24 was statistically significant only in those patients carrying the UGT1A1*6 (HR 2.87) and UGT1A1*6/*28 (HR 11.42) genotypes. The Ki values for indinavir inhibition of UGT1A1 and UGT1A1*6 were 4.1 and 10.7 mumol/l respectively. However, indinavir was also shown to inhibit other human UGTs, notably UGT1A3 and UGT1A7. CONCLUSIONS: In contrast to Caucasian HIV-infected patients treated with indinavir, the promoter polymorphism (UGT1A1*28) is of less significance than the coding region (UGT1A1*6) mutation as a risk factor for hyperbilirubinaemia. The Ki values determined for indinavir inhibition of UGT1A1 are consistent with an interaction in vivo, with an additive effect in patients with already impaired bilirubin glucuronidation activity.

Adult↗

PubMed2006

The caudal-related homeodomain protein Cdx2 and hepatocyte nuclear factor 1alpha cooperatively regulate the UDP-glucuronosyltransferase 2B7 gene promoter.

The gastrointestinal tract, contains several UDP-glucuronosyltransferases (UGTs) of the UGT1A and UGT2B subfamilies. UGT2B7 is one particular enzyme expressed throughout the gastrointestinal tract that possesses broad substrate specificity towards orally administered drugs. Because the caudal-related homeodomain protein 2 (Cdx2) regulates many gastrointestinal properties, we sought to determine whether it could regulate the UGT2B7 promoter in the colon-derived cell line Caco-2. Levels of Cdx2 and UGT2B7 were measured in differentiated and non-differentiated Caco-2 cells by the quantitative polymerase chain reaction. The capacity of the UGT2B7 gene promoter to drive expression of the luciferase reporter gene was assessed by transfection into Caco-2 cells, with transcription factor expression plasmids. Mutation of putative transcription factor binding sites and electrophoretic mobility shift assays were used to define important regulatory regions of the UGT2B7 gene promoter. The levels of Cdx2 and UGT2B7 mRNAs were co-ordinately increased in differentiated Caco2 cells compared to non-differentiated cells. Cdx2 activates the UGT2B7 proximal promoter by binding to two adjacent sites. Promoter activation requires Cdx2 binding to both sites wherein these proteins interact to form a putative functional dimer. Dimerization was shown to be dependent on redox state using extracts depleted of dithiothreitol. In addition, Cdx2 was shown to cooperatively activate the UGT2B7 promoter in conjunction with hepatocyte nuclear factor 1alpha (HNF1alpha), a mechanism previously observed to regulate other intestine-specific genes. The present study is the first to define transcription factors involved in the control of intestinal UGT2B expression. The demonstration that Cdx2 and HNF1alpha are important regulators of UGT2B7 expression will aid in defining pathways for coordinate control of drug metabolism in the gastrointestinal tract.

Biotransformation↗

PubMed2006

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

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). METHODS: Kinetic constants for AZT glucuronidation were generated using human liver microsomes (HLM) and recombinant UGT2B7, the principal enzyme responsible for AZT glucuronidation, as the enzyme sources with and without fluconazole. K(i) values were used to estimate the decrease in AZT clearance in vivo. RESULTS: Addition of BSA (2%) to incubations decreased the K(m) values for AZT glucuronidation by 85-90% for the HLM (923 +/- 357 to 91 +/- 9 microm) and UGT2B7 (478-70 microm) catalysed reactions, with little effect on V(max). Fluconazole, which was shown to be a selective inhibitor of UGT2B7, competitively inhibited AZT glucuronidation by HLM and UGT2B7. Like the K(m), BSA caused an 87% reduction in the K(i) for fluconazole inhibition of AZT glucuronidation by HLM (1133 +/- 403 to 145 +/- 36 microm) and UGT2B7 (529 to 73 microm). K(i) values determined for fluconazole using HLM and UGT2B7 in the presence (but not absence) of BSA predicted an interaction in vivo. The predicted magnitude of the interaction ranged from 41% to 217% of the reported AUC increase in patients, depending on the value of the in vivo fluconazole concentration employed in calculations. CONCLUSIONS: K(i) values determined under certain experimental conditions may quantitatively predict inhibition of UGT catalysed drug glucuronidation in vivo.

Alamethicin↗

PubMed2005

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

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.

Cell Line↗

PubMed2005

Human UDP-glucuronosyltransferase 1A5: identification, expression, and activity.

The human UDP-glucuronosyltransferase (UGT) subfamily 1A includes nine genes. The expression of all the UGT1A isoforms, apart from UGT1A5, has been reported previously. We have now detected a low basal level of UGT1A5 expression in cultured human hepatocytes, and treatment with rifampicin or 3-methylcholanthrene increased the level of UGT1A5 mRNA. Low-level UGT1A5 expression was also found in HepG2 and Caco-2 cells as well as human liver. Furthermore, UGT1A5 expression has been detected in various segments of the intestine from human donors, revealing high interindividual variability in its level and distribution along the intestine. Full-length UGT1A5 cDNA was isolated from Caco-2 cells that had been transfected with the pregnane X receptor and treated with rifampicin. Recombinant UGT1A5, expressed in baculovirus-infected insect cells, exhibited very low rates of 4-methylumbelliferone and scopoletin glucuronidation, whereas 1-hydroxypyrene was a much better substrate for it. UGT1A5 did not glucuronidate 4-aminobiphenyl, a good substrate for the highly homologous enzymes UGT1A4 and UGT1A3. However, replacing the first 110 amino acids of UGT1A5, a region that may be involved in substrate binding, with the counterpart segment from UGT1A4 did not increase the 4-aminobiphenyl glucuronidation activity. Collectively, this work demonstrates for the first time that the human UGT1A5 is expressed in several tissues, is inducible, and is catalytically active.

Adult↗

PubMed2005

Identification and characterization of functional hepatocyte nuclear factor 1-binding sites in UDP-glucuronosyltransferase genes.

The hepatocyte nuclear factor 1 (HNF1) transcription factor family is composed of two closely related homeodomain proteins with similar but distinct expression profiles. Homodimers and heterodimers of these transcription factors, HNF1alpha and HNF1beta, increase transcription from target genes through direct physical interaction with one or more elements of sufficient similarity to a 13 nucleotide-inverted dyad consensus-binding sequence. Potential HNF1-binding sites have been found in the proximal upstream regulatory regions of most known human UDP-glucuronosyltransferase (UGT) genes. As the liver and gastrointestinal tract are both important sites of glucuronidation and express significant levels of one or both HNF1 proteins, it is thought that these homeoproteins may play a role in transcriptional regulation of UGTs. This chapter explores the current evidence that HNF1 transcription factors are explicitly involved in the transcription of mammalian UGT genes. Most data supporting this hypothesis come from in vitro reporter assays, site-directed mutagenesis, and electrophoretic mobility-shift assays, for which methods are detailed. However, as in vitro functionality of transcription factors does not necessarily imply significance in vivo, some of the limitations of these techniques are also examined. In addition, available in vivo data are discussed, with particular attention given to contributions made by HNF1alpha knockout mouse models and microarray studies of human tissue. Finally, possible scenarios in which HNF1-mediated regulation of UGT expression may be clinically relevant are suggested.

Animals↗

PubMed2005

Nomenclature update for the mammalian UDP glycosyltransferase (UGT) gene superfamily.

Several novel UDP glycosyltransferase (UGT) genes, mainly UDP glucuronosyltransferases, have been identified in the human, mouse and rat genomes and in other mammalian species. This review provides an update of the UGT nomenclature to include these new genes and prevent the confusion that arises when the same gene is given different names. The new genes are named following previously established recommendations, taking into consideration evolutionary relatedness and the names already in general usage in the literature. The mammalian UGT gene superfamily currently has 117 members that can be divided into four families, UGT1, UGT2, UGT3 and UGT8. The 5-exon genes of the UGT1 family each contain a unique first exon, plus four exons that are shared between the genes; the exons 1 appear to have evolved by a process of duplication, leading to the synthesis of proteins with identical carboxyl-terminal and variable amino-terminal domains. Exon-sharing is also seen with the 6-exon UGT2A1 and UGT2A2 genes. However, UGT2A3 and those of the UGT2B (six exons), UGT3 (seven exons) and UGT8 gene families (five or six exons) do not share exons and most likely were derived by a process of duplication of all exons in the gene. Most UGT1 and UGT8 enzymes have been characterized in detail; however, the catalytic functions of the UGT3A enzymes and several UGT2 enzymes remain to be characterized.

Animals↗

PubMed2005

S-Naproxen and desmethylnaproxen glucuronidation by human liver microsomes and recombinant human UDP-glucuronosyltransferases (UGT): role of UGT2B7 in the elimination of naproxen.

AIMS: To characterize the kinetics of S-naproxen ('naproxen') acyl glucuronidation and desmethylnaproxen acyl and phenolic glucuronidation by human liver microsomes and identify the human UGT isoform(s) catalysing these reactions. METHODS: Naproxen and desmethylnaproxen glucuronidation were investigated using microsomes from six and five livers, respectively. Human recombinant UGTs were screened for activity towards naproxen and desmethylnaproxen. Where significant activity was observed, kinetic parameters were determined. Naproxen and desmethylnaproxen glucuronides were measured by separate high-performance liquid chromatography methods. RESULTS: Naproxen acyl glucuronidation by human liver microsomes followed biphasic kinetics. Mean apparent K(m) values (+/-SD, with 95% confidence interval in parentheses) for the high- and low-affinity components were 29 +/- 13 microm (16, 43) and 473 +/- 108 microm (359, 587), respectively. UGT 1A1, 1A3, 1A6, 1A7, 1A8, 1A9, 1A10 and 2B7 glucuronidated naproxen. UGT2B7 exhibited an apparent K(m) (72 microm) of the same order as the high-affinity human liver microsomal activity, which was inhibited by the UGT2B7 selective 'probe' fluconazole. Although data for desmethylnaproxen phenolic glucuronidation by human liver microsomes were generally adequately fitted to either the single- or two-enzyme Michaelis-Menten equation, model fitting was inconclusive for desmethylnaproxen acyl glucuronidation. UGT 1A1, 1A7, 1A9 and 1A10 catalysed both the phenolic and acyl glucuronidation of desmethylnaproxen, while UGT 1A3, 1A6 and 2B7 formed only the acyl glucuronide. Atypical glucuronidation kinetics were variably observed for naproxen and desmethylnaproxen glucuronidation by the recombinant UGTs. CONCLUSION: UGT2B7 is responsible for human hepatic naproxen acyl glucuronidation, which is the primary elimination pathway for this drug.

Glucuronosyltransferase↗

PubMed2005

Modulation of UDP-glucuronosyltransferase 2B7 function by cytochrome P450s in vitro: differential effects of CYP1A2, CYP2C9 and CYP3A4.

Effects of cytochrome P450 isoforms, CYP1A2, CYP2C9 and CYP3A4, on the catalytic activity of UDP-glucuronosyltransferase 2B7 (UGT2B7) expressed in COS cell microsomal membranes were investigated using morphine as a substrate. When detergent-untreated COS cell microsomes were used as the enzyme source, the activity of morphine-3-glucuronide formation by UGT2B7 was reduced by addition of purified CYP1A2 and CYP2C9 in a concentration-dependent manner. The effect of CYP1A2 was greater than that of CYP2C9. In contrast, exogenous CYP3A4 had little effect on morphine glucuronidation activity. These results suggest that CYP1A2 and CYP2C9 have ability to modify UGT2B7 function. However, the mechanism(s) underlying the modulation of UGT2B7 function by these P450s seems to differ from that by CYP3A4.

Animals↗

PubMed2004

Modulation of UDP-glucuronosyltransferase function by cytochrome P450: evidence for the alteration of UGT2B7-catalyzed glucuronidation of morphine by CYP3A4.

Modulation of UDP-glucuronosyltransferase 2B7 (UGT2B7)-catalyzed morphine glucuronidation by cytochrome P450 (P450) was studied. The effects of P450 isozymes on the kinetic parameters of UGT2B7-catalyzed glucuronidation of the morphine 3-hydroxyl group were examined by simultaneous expression of UGT2B7 and either CYP3A4, -1A2, or -2C9 in COS-1 cells. Although coexpression of CYP3A4 with UGT2B7 had little effect on Vmax, the Km was increased by about 9.8-fold compared with the UGT2B7 single expression system. The other P450 isozymes (CYP1A2 and CYP2C9) had some effects on Km and Vmax values. Immunoprecipitation of UGT from solubilized human liver microsomes resulted in coprecipitation of CYP3A4 with UGT2B7. The protein-protein interaction between CYP3A4 and UGT2B7 was further confirmed by overlay assay using glutathione S-transferase-CYP3A4 fusion protein. Addition of CYP3A4 to untreated COS microsomes expressing UGT2B7 had no or minor effects on morphine glucuronidation. In contrast, the formation of morphine-3-glucuronide by detergent-treated microsomes from COS-1 cells expressing UGT2B7 was reduced by CYP3A4, whereas the formation of the 6-glucuronide was enhanced. These results strongly suggest that 1) the glucuronidation activity of UGT2B7 toward morphine is specifically modulated by interaction with CYP3A4 in microsomal membranes and that 2) CYP3A4 alters UGT2B7 regioselectivity so that the ratio of morphine activation/detoxication is increased. This study provides the first evidence that P450 is not only involved in oxidation of drugs but also modulates the function of UGTs.

Animals↗

PubMed2004

Regulation of UDP glucuronosyltransferases in the gastrointestinal tract.

The UDP glucuronosyltransferases (UGT) of the gastrointestinal (GI) tract have a crucial role in protection against the toxic effects of lipophilic chemicals in the environment. UGTs such as UGT1A7, UGT1A8, and UGT1A10 are exclusively expressed in gastrointestinal tissues, each with a unique tissue distribution pattern that is subject to interindividual variation. The factors regulating this tissue-specific expression and that contribute to variability are beginning to be elucidated. Studies on the UGT1A7, 1A8, 1A9, and 1A10 gene promoters in Caco-2 cells, an in vitro model of enterocytes of the gastrointestinal tract, have identified the caudal homeodomain transcription factor, Cdx2, as an important regulator of the UGT1A8 and 1A10 gene proximal promoters. This transcription factor is found exclusively in the small intestine and colon: it is absent in the gastric epithelium and the esophagus. Cdx2 regulates the UGT1A8 and 1A10 promoters in cooperation with hepatocyte nuclear factor 1alpha (HNF1alpha). It is noteworthy that UGT1A7 is not expressed in gastrointestinal tissue distal to the gastric mucosa and does not contain a Cdx2 binding site in its proximal promoter. Transcription factors, including Sp1, which differentially bind to the initiator regions of the UGT1A8, 1A9, and 1A10 promoters, also contribute to the differences in expression of these UGTs in Caco-2 cells. The identification of important regulatory regions of UGT genes expressed in the gastrointestinal tract, and the transcription factors that bind to these regions, will aid in the elucidation of factors that contribute to interindividual differences in gastrointestinal UGT expression. In turn, this will lead to further understanding of interindividual variation in the capacity of the GI tract to metabolize lipophilic chemicals and to act as a barrier to dietary toxins and orally administered drugs.

Animals↗