Glucuronidation Reactions Research Articles

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.

Bioanalytical Aspects in Characterization and Quantification of Glucuronide Conjugates in Various Biological Matrices

The occurrence of phase II metabolism via conjugation pathway, has been observed for not only several endogenous molecules such as bilirubin, bile acids, steroid hormones (androgens and estrogens), retinoids, thyroid hormones, leukotriene B4, hydroxy metabolites of arachidonic and linoleic acids, several phenolic neurotransmitters etc. but also for a number of xenobiotics belonging to diversified chemical classes. Although topic of focus in this review is pertaining to glucuronidation, other conjugation phase II reactions are known to occur via sulfation, acetylation, methylation or simply via direct addition of glycine or glutathione adducts. The review covers the importance of glucuronidation while accounting for various types of known glucuronidation reactions. The formation of acyl glucuronides and the liability of acyl glucuronides to form chemically reactive species via acyl migration mechanism are also covered in this review. A concise review of the literature on bioanalytical aspects of the analysis of glucuronides revealed that application of HPLC with UV, fluorescence, coulometric or mass spectral detections have been generally practiced for the determination of several types of glucuronides in biological fluids. Several examples are provided in this review for the estimation of glucuronides under various sub-heading such as direct estimation of nonchiral glucuronides, direct estimation of diastereomeric glucuronides, estimation followed by derivatization, estimation by using ion-pairing agent, estimation of mixed glucuronides by HPLC and/or LC-MS/MS. Additionally, several aspects pertaining to the development of sound bioanalytical methodology including aspects for handling procedures and a validation framework for establishing glucuronide assay are presented.

Tissue-specific, Inducible, and Hormonal Control of the Human UDP-Glucuronosyltransferase-1 (UGT1) Locus

The human UDP-glucuronosyltransferase 1 (UGT1) locus spans nearly 200 kb on chromosome 2 and encodes nine UGT1A proteins that play a prominent role in drug and xenobiotic metabolism. Transgenic UGT1 (Tg-UGT1) mice have been created, and it has been demonstrated that tissue-specific and xenobiotic receptor control of the UGT1A genes is influenced through circulating humoral factors. In Tg-UGT1 mice, the UGT1A proteins are differentially expressed in the liver and gastrointestinal tract. Gene expression profiles confirmed that all of the UGT1A genes can be targeted for regulation by the pregnane X receptor activator pregnenolone-16alpha-carbonitrile (PCN) or the Ah receptor ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In addition, the selective induction of glucuronidation activity toward lamotrigine, ethinyl estradiol, chenodeoxycholic acid, and lithocholic acid by either PCN or TCDD in small intestine from Tg-UGT1 mice corresponded to expression of the locus in this tissue. Induction of UGT1A1 by PCN and TCDD is believed to be highly dependent upon glucocorticoids, because submicromolar concentrations of dexamethasone actively promote PCN and TCDD induction of UGT1A1 in Tg-UGT1 primary hepatocytes. The role of hormonal control of the UGT1 locus was further verified in pregnant and nursing Tg-UGT1 mice. In maternal 14-day post-conception Tg-UGT1mice, liver UGT1A1, UGT1A4, and UGT1A6 were induced, with the levels returning to near normal by birth. However, maternal liver UGT1A4 and UGT1A6 were dramatically elevated and maintained after birth, indicating that these proteins may play a critical role in maternal metabolism during lactation. With expression of the UGT1 locus confirmed in a variety of mouse tissues, these results suggested that the Tg-UGT1 mice will be a useful model to examine the regulatory and functional properties of human glucuronidation.

Hepatic expression of the UGT1A9 gene is governed by hepatocyte nuclear factor 4alpha.

UDP-glucuronosyltransferase (UGT) enzymes catalyze the glucuronidation reaction, which is a major pathway in the catabolism and elimination of numerous endo- and xenobiotics. Among the UGT enzyme family members, the UGT1A7, UGT1A8, UGT1A9, and UGT1A10 isoforms are issued from a single gene through differential splicing. However, these enzymes display distinct tissue-specific expression patterns. Indeed, UGT1A7, UGT1A8, and UGT1A10 are exclusively expressed in extrahepatic tissues, whereas UGT1A9 transcripts are found at high concentrations in liver. In the present study, we report that the liver-enriched hepatocyte nuclear factor 4 (HNF4)-alpha controls the hepatic expression of the UGT1A9 enzyme. Liver-specific disruption of the HNF4alpha gene in mice drastically decreases liver UGT1A9 mRNA levels. Furthermore, an HNF4alpha response element (HNF4alpha RE) was identified in the promoter of human UGT1A9 at position -372 to -360 base pairs by transient transfection, electrophoretic mobility shift assays, and chromatin immunoprecipitation experiments. It is interesting that this response element is absent in the proximal UGT1A7, UGT1A8, and UGT1A10 gene promoters. In conclusion, the present study identifies HNF4alpha as a major factor for the control of UGT1A9 hepatic expression and suggests that the absence of UGT1A7, UGT1A8, and UGT1A10 expression in the liver is caused by, at least in part, a few base pair changes in their promoter sequences in the region corresponding to the HNF4alpha RE of the UGT1A9 gene.

Glucuronidation of aliphatic alcohols in human liver microsomes in vitro

The glucuronidation of several short-chained aliphatic alcohols in vitro, with the use of human liver microsomes (HLM) as catalyst, was performed, and the kinetics were studied. The concentrations of the glucuronides were determined by gas chromatography–mass spectrometry after derivatization to the trimethylsilyl compounds. Alcohols from ethanol to pentanols were found to couple with activated glucuronic acid in HLM to a widely varying amount. For analytic reasons the glucuronide of methanol, which is formed after methanol consumption in human beings in vivo, could not be determined. The length of the alkyl chain played the decisive role in the maximum turnover rate of the glucuronidation. The affinity of the alcohols to UDP-glucuronosyltransferase (UDPGT), which catalyzes the glucuronidation reaction, was shown to depend strongly on their structure. Alcohols with a very short alkyl chain and secondary alcohols were glucuronidated much more slowly in comparison with findings for the longer chain primary alcohols and showed less affinity to UDPGT. The alcohols mutually inhibited glucuronidation. However, ethanol inhibited the glucuronidation of isopentanol or n-pentanol only in high concentrations, whereas the two pentanols inhibited each other to a high degree. The glucuronidation of aliphatic alcohols is probably catalyzed by only one of several very similar enzymes of the UDPGT. This finding was indicated by the fact that the Michaelis–Menten constants of the alcohols—with the use of different lots of the HLM from different liver donors—had nearly the same values.

An assessment of udp-glucuronosyltransferase induction using primary human hepatocytes.

Uridine diphosphate glucuronosyltransferases (UGTs) catalyze the glucuronidation of a wide range of xenobiotics and endogenous substrates. However, there is a lack of information concerning the response of human UGTs to inducers, and this observation prompted the current investigation. The glucuronidation of estradiol (3- and 17-positions), naphthol, propofol, and morphine (3- and 6-positions) was assessed against a battery of recombinant human UGTs to determine selective glucuronidation reactions for induction studies. The potential induction of the glucuronidation of estradiol at the 3-position, naphthol, propofol, and morphine at the 3-position was subsequently investigated in cultured primary human hepatocytes against a range of prototypic inducers including dexamethasone, 3-methylcholanthrene (3-MC), phenobarbital, rifampicin, and omeprazole. Treatment with 3-MC induced estradiol-3-glucuronidation (up to 2.5-fold) in four of five donors investigated. Statistically significant increases in naphthol glucuronidation (up to 1.7-fold) were observed following treatment with carbamazepine. UGT1A9-mediated propofol glucuronidation was induced by phenobarbital (up to 2.2-fold) and rifampicin (up to 1.7-fold). However, treatment with alpha-naphthoflavone and tangeretin resulted in a decrease in propofol glucuronidation (30% of control values). Statistically significant induction of morphine-3-glucuronidation was observed in at least three donors following treatment with phenobarbital, rifampicin, and carbamazepine. Each UGT isoform investigated displayed a distinct induction profile. Although statistically significant increases in glucuronidation were observed for each reaction studied, the level of induction was less than that observed for CYP1A2 or CYP3A4 and exhibited a large interdonor variability. The clinical relevance of the induction responses obtained in this study is unclear.

Characterization of metabolites of hydroxycinnamates in the in vitro model of human small intestinal epithelium caco-2 cells.

Hydroxycinnamic acids are antioxidant phenolic compounds which are widespread in plant foods, contribute significantly to total polyphenol intakes, and are absorbed by humans. The extent of their putative health benefit in vivo depends largely on their bioavailability. However, the mechanisms of absorption and metabolism of these phenolic compounds have not been described. In this study, we used the in vitro Caco-2 model of human small intestinal epithelium to investigate the metabolism of the major dietary hydroxycinnamates (ferulate, sinapate, p-coumarate, and caffeate) and of diferulates. The appearance of metabolites in the medium versus time was monitored, and the various conjugates and derivatives produced were identified by HPLC-DAD, LC/MS, and enzyme treatment with beta-glucuronidase or sulfatase. Enterocyte-like differentiated Caco-2 cells have extra- and intracellular esterases able to de-esterify hydroxycinnamate and diferulate esters. In addition, intracellular UDP-glucuronosyltransferases and sulfotransferases existing in Caco-2 cells are able to form the sulfate and the glucuronide conjugates of methyl ferulate, methyl sinapate, methyl caffeate, and methyl p-coumarate. However, only the sulfate conjugates of the free acids, ferulic acid, sinapic acid, and p-coumaric acid, were detected after 24 h. The O-methylated derivatives, ferulic and isoferulic acid, were the only metabolites detected following incubation of Caco-2 cells with caffeic acid. These results show that the in vitro model system differentiated Caco-2 cells have the capacity to metabolize dietary hydroxycinnamates, including various phase I (de-esterification) and phase II (glucuronidation, sulfation, and O-methylation) reactions, and suggests that the human small intestinal epithelium plays a role in the metabolism and bioavailability of these phenolic compounds.

Specific Protein-1 Is a Universal Regulator of UDP-glucose Dehydrogenase Expression: ITS POSITIVE INVOLVEMENT IN TRANSFORMING GROWTH FACTOR-β SIGNALING AND INHIBITION IN HYPOXIA

UDP-glucose dehydrogenase (UGDH) is a key enzyme of the unique pathway for the synthesis of UDP-glucuronate, the substrate for the numerous glucuronosyl transferases, which act on the synthesis of glycosaminoglycans and glucuronidation reaction of xeno- and endobiotics. Using the bacterial artificial chromosome approach, we have cloned and characterized the human UGDH promoter. The core promoter of -644 nucleotides conferred reporter gene activity in transient transfection assay of a variety of cell types, including MRC5 fibroblasts and the HepG2 hepatoma cell line. The minimal promoter of -100 nucleotides contains a functional inverted TATA box. No consensus CAAT sequence was found up to -2133 nucleotides. The expression of UGDH was up- and down-regulated by transforming growth factor (TGF)-beta and hypoxia, respectively. TGF-beta enhanced the activity of all the deletion constructs, except the minimal promoter. Hypoxia slightly increased the activity of the short promoter-containing constructs but decreased that of the -374 nucleotides and core promoter constructs. The core promoter contained numerous GC-rich sequences for the binding of Sp1 transcription factor. Bisanthracycline, an anti-Sp1 compound, decreased UGDH mRNA expression and inhibited the core promoter constructs activity. Gel mobility shift and supershift assays after TGF-beta stimulation demonstrated an increased DNA binding of the nuclear extract proteins to the two Sp1 sequences located in the -374-bp promoter. By contrast, nuclear extract proteins from hypoxia-treated cells demonstrated a decreased binding of the consensus Sp1 sequence. These results indicate that numerous Sp1 cis-acting sequences of the UGDH core promoter are responsible for up- and down-regulation of the gene after TGF-beta stimulation and in hypoxic conditions, respectively.

The UDP-glucuronosyltransferase 1A9 Enzyme Is a Peroxisome Proliferator-activated Receptor α and γ Target Gene

Peroxisome proliferator-activated receptor (PPAR) alpha and gamma are ligand-activated transcription factors belonging to the nuclear receptor family. PPAR alpha mediates the hypolipidemic action of the fibrates, whereas PPAR gamma is a receptor for the antidiabetic glitazones. In the present study, the UDP-glucuronosyltransferase (UGT) 1A9 enzyme is identified as a PPAR alpha and PPAR gamma target gene. UGTs catalyze the glucuronidation reaction, which is a major pathway in the catabolism and elimination of numerous endo- and xenobiotics. Among the UGT1A family enzymes, UGT1A9 metabolizes endogenous compounds, including catecholestrogens, and xenobiotics, such as fibrates and to a lesser extent troglitazone. Treatment of human hepatocytes and macrophages and murine adipocytes with activators of PPAR alpha or PPAR gamma resulted in an enhanced UGT1A9 expression and activity. In addition, disruption of the PPAR alpha gene in mice completely abolished the PPAR alpha agonist-induced UGT1A9 mRNA and activity levels. A PPAR response element was identified in the promoter of UGT1A9 at positions -719 to -706 bp by transient transfection and electromobility shift assays. Considering the role of UGT1A9 in catecholestrogen metabolism, PPAR alpha and PPAR gamma activation may contribute to the protection against genotoxic catecholestrogens by stimulating their inactivation in glucuronide derivatives. Furthermore, since UGT1A9 is involved in the catabolism of fibrates, these results suggest that PPAR alpha and PPAR gamma may control the intracellular level of active fibrates.

The effect of valproic acid on drug and steroid glucuronidation by expressed human UDP-glucuronosyltransferases

Valproic acid glucuronidation kinetics were carried out with three human UGT isoforms: UGT1A6, UGT1A9, and UGT2B7 as well as human liver and kidney microsomes. The glucuronidation of valproic acid was typified by high K m values with microsomes and expressed UGTs (2.3–5.2 mM). The ability of valproic acid to interact with the glucuronidation of drugs, steroids and xenobiotics in vitro was investigated using the three UGT isoforms known to glucuronidate valproic acid. In addition to this the effect of valproic acid was investigated using two other UGT isoforms: UGT1A1 and UGT2B15 which do not glucuronidate valproic acid. Valproic acid inhibited UGT1A9 catalyzed propofol glucuronidation in an uncompetitive manner and UGT2B7 catalyzed AZT glucuronidation competitively ( K i =1.6±0.06 mM). Valproate significantly inhibited UGT2B15 catalyzed steroid and xenobiotic glucuronidation although valproate was not a substrate for this UGT isoform. No significant inhibition of UGT1A1 or UGT1A6 by valproic acid was observed. These data indicate that valproic acid inhibition of glucuronidation reactions is not always due to simple competitive inhibition of substrates.

Pharmacogenomics of human UDP-glucuronosyltransferase enzymes.

UDP-glucuronosyltransferase (UGT) enzymes comprise a superfamily of key proteins that catalyze the glucuronidation reaction on a wide range of structurally diverse endogenous and exogenous chemicals. Glucuronidation is one of the major phase II drug-metabolizing reactions that contributes to drug biotransformation. This biochemical process is also involved in the protection against environmental toxicants, carcinogens, dietary toxins and participates in the homeostasis of numerous endogenous molecules, including bilirubin, steroid hormones and biliary acids. Over the years, significant progress was made in the field of glucuronidation, especially with regard to the identification of human UGTs, study of their tissue distribution and substrate specificities. More recently, the degree of allelic diversity has also been revealed for several human UGT genes. Some polymorphic UGTs have demonstrated a significant pharmacological impact in addition to being relevant to drug-induced adverse reactions and cancer susceptibility. This review focuses on human UGTs, the description of the nature of polymorphic variations and their functional impact. The pharmacogenomic implication of polymorphic UGTs is presented, more specifically the role of UGT polymorphisms in modifying cancer risk and their impact on individual risk to drug-induced toxicities.

Glucuronidation of 4-methylumbelliferone and 4-hydroxybiphenyl and in vitro induction of UDP-glucuronosyltransferase 2B12-mRNA in precision-cut rat liver slices

Fresh rat liver slices were used to demonstrate the glucuronidation of the model substrates 4-methylumbelliferone (MU) and 4-hydroxybiphenyl (HB). Both glucuronidation reactions proved to be more stable than cytochrome P450-dependent monooxygenations. After an incubation time of 48 h there was no decrease in MU glucuronidation rate, whereas HB glucuronidation was stable until 24 h, and then decreased by about 50% until 48 h. The technique of quantitative competitive RT-PCR was used to determine the expression of UDP-glucuronosyltransferase 2B12-mRNA (UGT2B12-mRNA) in precision-cut rat liver slices. Constitutive levels of UGT2B12-mRNA were measurable. Following 24 h culture of rat liver slices in the presence of phenobarbital, the level of UGT2B12-mRNA increased about twofold, which corresponds to the inducibility in vivo. The addition of beta-naphthoflavone had no influence. The results show that precision-cut liver slices are not only suitable for the detection of an in vitro induction of cytochrome P450-mRNAs, which is characterized by high induction factors, but also of poor induction effects, e.g. on UGT2B12-mRNA, provided that the respective mRNA is exactly quantified.

Acyl glucuronidation and glucosidation of a new and selective endothelin ET(A) receptor antagonist in human liver microsomes.

Compound A [(+)-(5S,6R,7R)-2-isopropylamino-7-[4-methoxy-2-((2R)-3-methoxy-2-methylpropyl)-5-(3,4-methylenedioxyphenyl) cyclopenteno [1,2-b] pyridine 6-carboxylic acid] is a new and selective endothelin ET(A) receptor antagonist. It underwent significant acyl glucuronidation and acyl glucosidation in human liver microsomes supplemented with UDP-glucuronic acid (UDPGA) and UDP-glucose (UDPG). These two conjugations were observed in a panel of human liver microsomal samples (n = 16) that gave rise to varying activities but with no significant correlation with each other in the native and activator-treated microsomal preparations (r(2) <or= 0.4, p > 0.05). The lack of correlation may be explained by the involvement of multiple UDP-glucuronosyltransferases (UGTs; UGT1A1, 1A3, 1A9, 2B7 and 2B15) in the glucuronidation but essentially solely UGT2B7 in the glucosidation. Both reactions conformed to monophasic Michaelis-Menten kinetics in human liver microsomes. The glucuronidation reaction exhibited apparent K(m) values (mean +/- S.E.) for compound A and UDPGA of 8.4 +/- 0.6 and 605 +/- 35 microM, respectively, whereas the values for the glucosidation reaction were 10.2 +/- 1.5 and 670 +/- 120 microM, respectively. In both pooled human liver microsomes and expressed UGT2B7, UDPG and UDPGA competitively inhibited their counterpart conjugations with K(i) values close to their K(m) values, indicating a comparable affinity of the enzyme toward these two nucleotide sugars. We herein report a drug acyl glucoside formed in human liver microsomes at a considerable turnover rate and provide the evidence for a UGT isoform (UGT2B7) capable of transferring both glucuronic acid and glucose from UDPGA and UDPG to an aglycone.

Modulation of phase II drug metabolizing enzyme activities by N-heterocycles.

Drugs and other chemicals that contain one or more heterocyclic nitrogen atoms are most widely known for their ability to both inhibit and induce cytochrome p450s. Their ability to affect a wide range of Phase II drug metabolizing enzyme activities has received much less attention and exposure. For some N-heterocycles, induction of Phase II metabolism occurs in the absence of induction effects on cytochrome p450, a property with potential utility in reducing drug and chemical toxicity (chemoprotection). The mechanism of this induction, and an accounting of the Phase II selectivity, has not been rigorously investigated. This review gathers and documents existing information on the simultaneous inductive effects of N-heterocycles on glucuronidation, sulfation (sulfonation) and glutathione conjugation reactions, as well epoxide hydrolase and quinone oxidoreductase activities. Basic information on cytochrome p450 induction is provided for comparative purposes. The review will serve as a base for any future interest in the phenomenon. The existing information on enzyme changes derives largely from laboratory animal studies. Chemicals investigated range from both simple and complex pyridines and imidazoles to benzoquinolines and phenanthrolines. Isomeric differences can have a major impact on induction characteristics and Phase II selectivity. Promising indications for the effectiveness of N-heterocycle elicited enzyme changes in chemoprotection in vivo awaits investigation.

Genetic polymorphisms of UDP-glucuronosyltransferases and their functional significance

UDP-Glucuronosyltransferase (UGT), the microsomal enzyme responsible for glucuronidation reactions, exists as a superfamily of enzymes. Genetic polymorphism has been described for 6 of the 16 functional human UGT genes characterised to date, namely UGT 1A1, 1A6, 1A7, 2B4, 2B7 and 2B15. Since glucuronidation is an essential pathway for the elimination of a myriad of xenobiotics and endogenous compounds, genetic polymorphism of UGT is potentially of toxicological and physiological importance. However, functional significance has only been convincingly demonstrated for genetic polymorphism of UGT1A1. Apart from impaired bilirubin glucuronidation, the mutations responsible for Gilbert syndrome also affect the elimination of a limited number of xenobiotics. It has been proposed on the basis of altered catalytic activity of mutants of UGT 1A6, 1A7 and 2B15 that genetic polymorphism of these forms may be of toxicological significance, but this is yet to be proven.

Inhibition and active sites of UDP-glucuronosyltransferases 2B7 and 1A1.

Two human UDP-glucuronosyltransferases (UGTs), UGT2B7 and UGT1A1, catalyze the glucuronidation of many endo- and xenobiotics. Although UGT1A1 uniquely catalyzes the glucuronidation of the endobiotic, bilirubin, and UGT2B7 uniquely catalyzes the glucuronidation of morphine to both the 3-0 glucuronide and the 6-0 glucuronide, both catalyze the glucuronidation of the mixed opioid agonist/antagonist buprenorphine with high efficiency. Etonitazenyl, a mu opioid receptor antagonist, was found to inhibit competitively opioid, steroid, and other substrate glucuronidation reactions catalyzed by UGT2B7. Data showing several benzodiazepines and alternative substrates interacting competitively support previous work, which indicates a single binding domain within UGT2B7. Etonitazenyl also competitively inhibited the glucuronidation of buprenorphine catalyzed by UGT1A1. However, neither etonitazenyl nor buprenorphine inhibited bilirubin glucuronidation except at very high concentrations. Therefore, it is unlikely that buprenorphine therapy for opioid or other drug addiction would influence bilirubin glucuronidation and lead to hyperbilirubenmia. Anthraflavic acid and catechol estrogen glucuronidation, catalyzed by UGT1A1, was also not inhibited by etonitazenyl or buprenorphine. Reactions catalyzed by UGT1A6 were not affected by etonitazenyl. These studies indicate that UGT2B7 has one binding site and that UGT1A1 has two or more binding sites for xenobiotics and endobiotics.

Glucuronidation of olanzapine by cDNA-expressed human UDP-glucuronosyltransferases and human liver microsomes.

Olanzapine is a widely used, newer antipsychotic agent, which is metabolized by various pathways: hydroxylation and N-demethylation by cytochrome P450, N-oxidation by flavin monooxygenase and direct glucuronidation. In vivo studies have pointed towards the latter pathway as being of major importance. Accordingly, the glucuronidation reaction was studied in vitro using cDNA-expressed human UDP-glucuronosyltransferase (UGT) enzymes and a pooled human liver microsomal preparation (HLM). Glucuronidated olanzapine was determined by HPLC after acid or enzymatic hydrolysis. The following UGT-isoenzymes were screened for their ability to glucuronidate olanzapine: 1A1, 1A3, 1A4, 1A6, 1A9, 2B7 and 2B15. Only UGT1A4 was able to glucuronidate olanzapine obeying saturation kinetics. The K(m) value was 227 micromol/l (SE 43), i.e. of the same order of magnitude as for other psychotropic drugs, and the V(max) value was 2370 pmol/(min mg) (SE 170). Glucuronidation was also mediated by the HLM preparation, but a saturation level was not reached. The olanzapine glucuronidation reaction was inhibited by several drugs known as substrates for UGT1A4, e.g. amitriptyline, trifluoperazine and lamotrigine. Thus, competition for glucuronidation by UGT1A4 represents a possibility for drug-drug interactions in subjects receiving several of these psychotropic drugs at the same time. Whether such possible interactions are of any clinical importance may await further studies in patients.

Excretion of the N(2)-glucuronide conjugate of 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine in urine and its relationship to CYP1A2 and NAT2 activity levels in humans.

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a mutagenic and carcinogenic heterocyclic aromatic amine formed in meat products during cooking. The genotoxity of PhIP requires an initial cytochrome P450-mediated N-oxidation followed by N-O-esterification catalyzed generally by N-acetyltransferases and sulfotransferases. This study examined the urinary excretion of N(2)-(beta-1-glucos-iduronyl)-2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine-the major human urinary N-oxidation metabolite of PhIP-and determined its relationship to individual activity levels of cytochrome P4501A2 (CYP1A2) and N-acetyltransferase (NAT2). The subjects (33 males and 33 females) in the dietary study were phenotyped for their CYP1A2 and NAT2 activity prior to consumption of meat-based diet, and urine collections were obtained 0-12 and 12-24 h after ingestion of the meal. Acidic hydrolysis of N(2)-(beta-1-glucosiduronyl)-2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine and its d(3)-analog to form their respective deaminated products 2-hydroxy-1-methyl-6-phenylimidazo[4,5-b]pyridine (2-OH-PhIP) was used in the assay. The products after derivatization were analyzed by capillary gas chromatography-negative ion chemical ionization mass spectrometry with selective ion monitoring. The amount of N(2)-(beta-1-glucosiduronyl)-2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine measured as the acid hydrolysis product 2-OH-PhIP in the 0-12 h urine was 20.2 +/- 8.0% (mean +/- SD) of the ingested dose; the median was 18.8% and the range varied from 5.4 to 39.6% within the group. In a subset (n = 18) of samples from individual urine collected from the 12-24 h period, an average value of 4.4 +/- 2.5% (+/- SD) of the dose was recovered. The excretion of N(2)-(beta-1-glucosiduronyl)-2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine in the 0-12 h urine was significantly related to the quantity of PhIP ingested for all subjects (r = 0.52, P <0.0001). Linear regression analysis of the relationship between the excretion level of N(2)-(beta-1-glucosiduronyl)-2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine, adjusted for meat intake and CYP1A2 activity in the combined group of males and females showed a low association (r = 0.25, P = 0.05). There was no association between the amount of N(2)-(beta-1-glucosiduronyl)-2-hydroxyamino-1-methyl-6-phenylimid-azo[4,5-b]pyridine in urine and NAT2 activity levels of the subjects nor with the age of the subjects. N(2)-(beta-1-glucosi-duronyl)-2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine comprised a significant proportion of the ingested dose in some individuals; however, considerable variation was found within the group. The results indicate that interindividual differences in the rates of N-oxidation of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, as well as phase II glucuronidation reactions regulate the formation of this metabolite in humans.

In vitro characterization of hepatic flavopiridol metabolism using human liver microsomes and recombinant UGT enzymes.

To assess the contribution of drug metabolism to the variability on flavopiridol glucuronidation observed in cancer patients, and to determine the ability of all known human UDP-glucuronosyltransferase (UGT) isoforms to glucuronidate flavopiridol. Inter-individual variation in flavopiridol glucuronidation was determined by HPLC using hepatic microsomes from 62 normal liver donors. Identification of enzymes capable of glucuronidating flavopiridol was determined by LC/MS using human embryonic kidney 293 (HEK293) cells stably expressing all sixteen known human UGTs. The major product of the flavopiridol glucuronidation reaction in human liver microsomes was FLAVO-7-G. High variability (coefficient of variation = 49%) was observed in the glucuronidation of flavopiridol by human liver microsomes. In vitro formation of FLAVO-7-G and FLAVO-5-G was mainly catalyzed by UGT1A9 and UGT1A4, respectively. Similar catalytic efficiencies (Vmax/Km) were observed for human liver microsomes (1.6 microl/min/mg) and UGT1A9 (1.5 microl/min/mg). UGT1A9 is the major UGT involved in the hepatic glucuronidation of flavopiridol in humans. The data suggests that hepatic glucuronidation may be a major determinant of the variable systemic glucuronidation of flavopiridol in cancer patients. The large variability in flavopiridol glucuronidation may be due to differences in liver metabolism among individuals, as a result of genetic differences in UGT1A9.

Importance of histidine residues for the function of the human liver UDP-glucuronosyltransferase UGT1A6: evidence for the catalytic role of histidine 370.

The human UDP-glucuronosyltransferase isoform UGT1A6 catalyzes the nucleophilic attack of phenolic xenobiotics on glucuronic acid, leading to the formation of water-soluble glucuronides. Based on the irreversible inhibition of the enzyme activity by the histidyl-selective reagent diethyl pyrocarbonate (DEPC), histidine was suggested to play a key role in the glucuronidation reaction. Therefore, the role of four strictly conserved histidine residues (His38, His361, His370, and His485) in the glucuronidation of 4-methylumbelliferone, as reporter substrate, was examined using site-directed mutagenesis. For this purpose, stable heterologous expression of wild-type and mutant UGT1A6 was achieved in the yeast Pichia pastoris. Replacement of histidine residues by alanine or glutamine led to fully inactive H38A, H38Q, and H485A mutants. Substitution of His361 by alanine affected the interaction of the enzyme with the cosubstrate, as indicated by a 4-fold increase in the K(m) value toward UDP-glucuronic acid. Interestingly, H370A mutant presented a severely impaired catalytic efficiency (with a V(max) value approximately 5% that of the wild-type), whereas conservative substitution of His370 by glutamine (H370Q) led to a significant restoration of activity. Whereas H361A was inactivated by DEPC as the wild-type enzyme, this chemical reagent only produced a minor effect on either H370Q or H370A mutant, providing evidence that His370 is probably the reactive histidine residue targeted by DEPC. The dramatic changes in catalytic efficiency on substitution of His370 by alanine and the ability of glutamine to function in place of histidine along with a weak sensitivity of these mutants to DEPC strongly suggest that His370 plays a catalytic role in the glucuronidation reaction.