Epirubicin glucuronidation is catalyzed by human UDP-glucuronosyltransferase 2B7.

Epirubicin is one of the most active agents for breast cancer. The formation of epirubicin glucuronide by liver UDP-glucuronosyltransferase (UGT) is its main inactivating pathway. This study aimed to investigate epirubicin glucuronidation in human liver microsomes, to identify the specific UGT isoform for this reaction, and to correlate epirubicin glucuronidation with other UGT substrates. Microsomes from human livers were used. UGTs specifically expressed in cellular systems, as well as two UGT2B7 variants, were screened for epirubicin glucuronidation. Epirubicin, morphine, and SN-38 glucuronides were measured by high-pressure liquid chromatography. The mean +/- S.D. formation rate of epirubicin glucuronide in human liver microsomes (n = 47) was 138 +/- 37 pmol/min/mg (coefficient of variation, 24%). This phenotype was normally distributed. We screened commercially available UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A9, UGT2B7, and UGT2B15 for epirubicin glucuronidation. Only UGT2B7 converted epirubicin to its glucuronide. No differences in epirubicin glucuronidation were found in HK293 cells expressing the two UGT2B7 variants at position 268. Catalytic efficiency (V(max)/K(m)) of epirubicin glucuronidation was 1.4 microl/min/mg, a value higher than that observed for morphine, a substrate of UGT2B7. Formation of epirubicin glucuronide was significantly related to that of morphine-3-glucuronide (r = 0.76, p < 0.001) and morphine-6-glucuronide (r = 0.73, p < 0.001). No correlation was found with SN-38, a substrate of UGT1A1 (r = 0.04). UGT2B7 is the major human UGT catalyzing epirubicin glucuronidation, and UGT2B7 is the candidate gene for this phenotype. The reported tyrosine to histidine polymorphism in UGT2B7 does not alter the formation rate of epirubicin glucuronide, and undiscovered genetic polymorphisms in UGT2B7 might change the metabolic fate of this important anticancer agent.     

Involvement of human hepatic UGT1A1, UGT2B4, and UGT2B7 in the glucuronidation of carvedilol.

Carvedilol ((+/-)-1-carbazol-4-yloxy)-3-[[2-(o-methoxyphenoxy)ethyl]amino]-2-propanol) is metabolized primarily into glucuronide conjugates. In the present study, we identified the human UDP-glucuronosyltransferase (UGT) isoforms involved in the glucuronidation of carvedilol by thin-layer chromatography using microsomes from human liver or insect cells expressing recombinant UGT isoforms. We observed two forms of carvedilol glucuronides, namely G1 and G2, in hepatic microsomes. The glucuronidation of carvedilol was catalyzed by at least three recombinant UGT isoforms: UGT1A1, UGT2B4, and UGT2B7. UGT2B4 formed both G1 and G2, whereas UGT1A1 and UGT2B7 were responsible for the formation of glucuronide G2 and G1, respectively. The enzyme kinetics for carvedilol glucuronidation by UGT1A1, UGT2B4, and UGT2B7 in addition to human liver microsomes were examined by Lineweaver-Burk analysis. The values of Km and Vmax for human liver microsomes were 26.6 microM and 106 pmol/min/mg protein for G1, and 46.0 microM and 44.5 pmol/min/mg protein for G2, respectively. The Km values for UGT1A1, UGT2B4, and UGT2B7 for G1 and G2 (22.1-55.1 microM) were comparable to those of the liver microsomes, whereas the Vmax values were in the range of 3.33 to 7.88 pmol/min/mg protein. The Km and Vmax/Km values for UGT2B4 and UGT2B7 for G1 were similar, whereas UGT2B4 had lower Km and higher Vmax/Km values for G2 compared with those of UGT1A1. These results suggest that G1 formation is catalyzed by UGT2B4 and UGT2B7, whereas G2 is formed by UGT2B4 and UGT1A1. These three hepatic UGT isoforms may have important roles in carvedilol metabolism.     

Glucuronidation of etoposide in human liver microsomes is specifically catalyzed by UDP-glucuronosyltransferase 1A1.

A metabolite formed by incubation of human liver microsomes, etoposide, and UDP-glucuronic acid was identified as etoposide glucuronide by liquid chromatography-tandem mass spectrometry analysis. According to the derivatization with trimethylsilylimidazole (Tri-Sil-Z), it was confirmed that the glucuronic acid is linked to an alcoholic hydroxyl group of etoposide and not to a phenolic group. Among nine recombinant human UGT isoforms (UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A8, UGT1A9. UGT1A10, UGT2B7, and UGT2B15), only UGT1A1 exhibited the catalytic activity of etoposide glucuronidation. The enzyme kinetics in pooled human liver microsomes and recombinant UGT1A1 microsomes showed a typical Michaelis-Menten plot. The kinetic parameters of etoposide glucuronidation were K(m) = 439.6 +/- 70.7 microM and V(max) = 255.6 +/- 19.2 pmol/min/mg of protein in human liver microsomes and K(m) = 503.2 +/- 110.2 microM and V(max) = was 266.5 +/- 28.6 pmol/min/mg of protein in recombinant UGT1A1. The etoposide glucuronidation in pooled human liver microsomes was inhibited by bilirubin (IC(50) = 31.7 microM) and estradiol (IC(50) = 34 microM) as typical substrates for UGT1A1. The inhibitory effects of 4-nitrophenol (IC(50) = 121.0 microM) as a typical substrate for UGT1A6 and UGT1A9, imipramine (IC(50) = 393.8 microM) as a typical substrate for UGT1A3 and UGT1A4, and morphine (IC(50) = 109.3 microM) as a typical substrate for UGT2B7 were relatively weak. The interindividual difference in etoposide glucuronidation in 13 human liver microsomes was 78.5-fold (1.4-109.9 pmol/min/mg of protein). The etoposide glucuronidation in 10 to 13 human liver microsomes was significantly correlated with beta-estradiol-3-glucuronidation (r = 0.841, p < 0.01), bilirubin glucuronidation (r = 0.935, p < 0.01), and the immunoquantified UGT1A1 protein content (r = 0.800, p < 0.01). These results demonstrate that etoposide glucuronidation in human liver microsomes is specifically catalyzed by UGT1A1.     

Characterization of rat and human UDP-glucuronosyltransferases responsible for the in vitro glucuronidation of diclofenac.

In the current study, the identification of the rat and human UDP-glucuronosyltransferase (UGT) isoforms responsible for the glucuronidation of diclofenac was determined. Recombinant human UGT1A9 catalyzed the glucuronidation of diclofenac at a moderate rate of 166-pmol/min/mg protein, while UGT1A6 and 2B15 catalyzed the glucuronidation of diclofenac at low rates (<20-pmol/min/mg protein). Conversely, human UGT2B7 displayed a high rate of diclofenac glucuronide formation (>500 pmol/min/mg protein). Recombinant rat UGT2B1 catalyzed the glucuronidation of diclofenac at a rate of 250-pmol/min/mg protein. Rat UGT2B1 and human UGT2B7 displayed a similar, low apparent Km value of <15 microM for both UGT isoforms and high Vmax values 0.3 and 2.8 nmol/min/mg, respectively. Using diclofenac as a substrate, enzyme kinetics in rat and human liver microsomes showed that the enzyme(s) involved in diclofenac glucuronidation had a low apparent Km value of <20 microM and a high Vmax value of 0.9 and 4.3 nmol/min/mg protein, respectively. Morphine is a known substrate for rat UGT2B1 and human UGT2B7 and both total morphine glucuronidation (3-O- and 6-O-glucuronides) and diclofenac glucuronidation reactions showed a strong correlation with one another in human liver microsome samples. In addition, diclofenac inhibited the glucuronidation of morphine in human liver microsomes. These data suggested that rat UGT2B1 and human UGT2B7 were the major UGT isoforms involved in the glucuronidation of diclofenac.     

Identification of human UDP-glucuronosyltransferases involved in N-carbamoyl glucuronidation of lorcaserin

Lorcaserin, a selective serotonin 5-HT(2C) receptor agonist, is a weight management agent in clinical development. Lorcaserin N-carbamoyl glucuronidation governs the predominant excretory pathway of lorcaserin in humans. Human UDP-glucuronosyltransferases (UGTs) responsible for lorcaserin N-carbamoyl glucuronidation are identified herein. Lorcaserin N-carbamoyl glucuronide formation was characterized by the following approaches: metabolic screening using human tissues (liver, kidney, intestine, and lung) and recombinant enzymes, kinetic analyses, and inhibition studies. Whereas microsomes from all human tissues studied herein were found to be catalytically active for lorcaserin N-carbamoyl glucuronidation, liver microsomes were the most efficient. With recombinant UGT enzymes, lorcaserin N-carbamoyl glucuronidation was predominantly catalyzed by three UGT2Bs (UGT2B7, UGT2B15, and UGT2B17), whereas two UGT1As (UGT1A6 and UGT1A9) played a minor role. UGT2B15 was most efficient, with an apparent K(m) value of 51.6 ± 1.9 μM and V(max) value of 237.4 ± 2.8 pmol/mg protein/min. The rank order of catalytic efficiency of human UGT enzymes for lorcaserin N-carbamoyl glucuronidation was UGT2B15 > UGT2B7 > UGT2B17 > UGT1A9 > UGT1A6. Inhibition of lorcaserin N-carbamoyl glucuronidation activities of UGT2B7, UGT2B15, and UGT2B17 in human liver microsomes by mefenamic acid, bisphenol A, and eugenol further substantiated the involvement of these UGT2B isoforms. In conclusion, multiple human UGT enzymes catalyze N-carbamoyl glucuronidation of lorcaserin; therefore, it is unlikely that inhibition of any one of these UGT activities will lead to significant inhibition of the lorcaserin N-carbamoyl glucuronidation pathway. Thus, the potential for drug-drug interaction by concomitant administration of a drug(s) that is metabolized by any of these UGTs is remote.     

UDP-glucuronosyltransferase 1A1 is the principal enzyme responsible for etoposide glucuronidation in human liver and intestinal microsomes: structural characterization of phenolic and alcoholic glucuronides of etoposide and estimation of enzyme kinetics.

Etoposide, an important anticancer agent, undergoes glucuronidation both in vitro and in vivo. In this study, three isomeric glucuronides of etoposide, including one phenolic (EPG) and two alcoholic glucuronides (EAG1 and EAG2), were biosynthesized in vitro with human liver microsomes (HLMs), and identified by liquid chromatography-electrospray ionization-mass spectrometry and confirmed by beta-glucuronidase cleavage. In vitro UDP-glucuronosyltransferase (UGT) reaction screening with 12 recombinant human UGTs demonstrated that etoposide glucuronidation is mainly catalyzed by UGT1A1. Although UGT1A8 and 1A3 also catalyzed the glucuronidation of etoposide, their activities were approximately 10 and 1% of UGT1A1. Enzyme kinetic study indicated that the predominant form of etoposide glucuronide in HLMs and human intestinal microsomes (HIMs) was EPG, whereas EAG1 and EAG2 were the minor metabolites, with approximately an 8 to 10% glucuronidation rate of EPG. For the formation of EPG, the V(max) of HLMs (110 pmol/min/mg protein) was very similar to that of recombinant UGT1A1 (124 pmol/min/mg protein), whereas the V(max) of HIMs (54.4 pmol/min/mg protein) was 2-fold lower than those of HIMs and UGT1A1. The K(m) values of HLMs (530 microM) and HIMs (608 microM) were 2-fold higher than that of UGT1A1 (285 microM). The V(max)/K(m) values for the formation of EPG were 0.21 and 0.09 microl/min/mg protein for HLMs and HIMs, respectively. The data indicated that UGT1A1 is principally responsible for the formation of etoposide glucuronides, mainly in the form of phenolic glucuronide, suggesting that etoposide can be used as a highly selective probe substrate for human UGT1A1 in vitro.     

Identification of UDP-glucuronosyltransferases 1A1, 1A3 and 2B15 as the main contributors to glucuronidation of bakuchiol,a natural biologically active compound

1.?Bakuchiol, one of the main active compounds of Psoralea corylifolia, possesses a variety of pharmacological activities such as anti-tumor and anti-aging effects. Here, we aimed to characterize the glucuronidation of bakuchiol using human liver microsomes (HLM) and expressed UDP-glucuronosyltransferase (UGT) enzymes.

2.?The glucuronide of bakuchiol was confirmed by liquid chromatography–mass spectrometry (LC-MS) and β-glucuronidase hydrolysis assay. Glucuronidation rates and kinetic parameters were derived by enzymatic incubation and model fitting. Activity correlation analyses were performed to identify the main UGT isoforms contributing to hepatic metabolism of bakuchiol.

3.?Among the three UGT enzymes (i.e., UGT1A1, UGT1A3 and UGT2B15) capable of catalyzing bakuchiol glucuronidation, UGT2B15 showed the highest activity with a CL_int value of 100?μl/min/nmol. Bakuchiol glucuronidation was strongly correlated with glucuronidation of 5-hydroxyrofecoxib (r?=?0.933; p?r?=?0.719; p?r?=?0.594; p?4.?In conclusion, UGT1A1, UGT1A3 and UGT2B15 were identified as the main contributors to glucuronidation of bakuchiol.     

Trans-3'-hydroxycotinine O- and N-glucuronidations in human liver microsomes.

Trans-3'-hydroxycotinine is a major metabolite of nicotine in humans and is mainly excreted as O-glucuronide in smoker's urine. Incubation of human liver microsomes with UDP-glucuronic acid produces not only trans-3'-hydroxycotinine O-glucuronide but also N-glucuronide. The formation of N-glucuronide exceeds the formation of O-glucuronide in most human liver microsomes, although N-glucuronide has never been detected in human urine. Trans-3'-hydroxycotinine N-glucuronidation in human liver microsomes was significantly correlated with nicotine and cotinine N-glucuronidations, which are catalyzed mainly by UDP-glucuronosyltransferase (UGT)1A4 and was inhibited by imipramine and nicotine, which are substrates of UGT1A4. Recombinant UGT1A4 exhibited substantial trans-3'-hydroxycotinine N-glucuronosyltransferase activity. These results suggest that trans-3'-hydroxycotinine N-glucuronidation in human liver microsomes would be mainly catalyzed by UGT1A4. In the present study, trans-3'-hydroxycotinine O-glucuronidation in human liver microsomes was thoroughly characterized, since trans-3'-hydroxycotinine O-glucuronide is one of the major metabolites of nicotine. The kinetics were fitted to the Michaelis-Menten equation with a K(m) of 10.0 +/- 0.8 mM and a V(max) of 85.8 +/- 3.8 pmol/min/mg. Among 11 recombinant human UGT isoforms expressed in baculovirus-infected insect cells, UGT2B7 exhibited the highest trans-3'-hydroxycotinine O-glucuronosyltransferase activity (1.1 pmol/min/mg) followed by UGT1A9 (0.3 pmol/min/mg), UGT2B15 (0.2 pmol/min/mg), and UGT2B4 (0.2 pmol/min/mg) at a substrate concentration of 1 mM. Trans-3'-hydroxycotinine O-glucuronosyltransferase activity by recombinant UGT2B7 increased with an increase in the substrate concentration up to 16 mM (10.5 pmol/min/mg). The kinetics by recombinant UGT1A9 were fitted to the Michaelis-Menten equation with K(m) = 1.6 +/- 0.1 mM and V(max) = 0.69 +/- 0.02 pmol/min/mg of protein. Trans-3'-hydroxycotinine O-glucuronosyltransferase activities in 13 human liver microsomes ranged from 2.4 to 12.6 pmol/min/mg and were significantly correlated with valproic acid glucuronidation (r = 0.716, p < 0.01), which is catalyzed by UGT2B7, UGT1A6, and UGT1A9. Trans-3'-hydroxycotinine O-glucuronosyltransferase activity in human liver microsomes was inhibited by imipramine (a substrate of UGT1A4, IC(50) = 55 microM), androstanediol (a substrate of UGT2B15, IC(50) = 169 microM), and propofol (a substrate of UGT1A9, IC(50) = 296 microM). Interestingly, imipramine (IC(50) = 45 microM), androstanediol (IC(50) = 21 microM), and propofol (IC(50) = 41 microM) also inhibited trans-3'-hydroxycotinine O-glucuronosyltransferase activity by recombinant UGT2B7. These findings suggested that trans-3'-hydroxycotinine O-glucuronidation in human liver microsomes is catalyzed by mainly UGT2B7 and, to a minor extent, by UGT1A9.     

Regioselective glucuronidation of denopamine: marked species differences and identification of human udp-glucuronosyltransferase isoform.

Denopamine is one of the oral beta(1)-adrenoceptor-selective partial agonists. Denopamine glucuronide is the most abundant metabolite in human, rat, and dog urine when administered orally. Species differences in denopamine glucuronidation were investigated with liver microsomes obtained from humans and experimental animals. In rat and rabbit, only the phenolic glucuronide was detected, whereas in dog and monkey, not only the phenolic glucuronide but also the alcoholic glucuronide was found. In contrast, in humans, the alcoholic glucuronide was detected exclusively. The kinetics of denopamine glucuronidation in human liver microsomes showed a typical Michaelis-Menten plot. The K(m) and V(max) values accounted for 2.87 +/- 0.17 mM and 7.29 +/- 0.23 nmol/min/mg protein, respectively. With the assessment of denopamine glucuronide formation across a panel of recombinant UDP-glucuronosyltransferase (UGT) isoforms (UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B4, UGT2B7, UGT2B15, and UGT2B17), only UGT2B7 exhibited high denopamine glucuronosyltransferase activity. The K(m) value of denopamine glucuronidation in recombinant UGT2B7 microsomes was close to those in human liver and jejunum microsomes. The formation of denopamine glucuronidation by human liver, jejunum, and recombinant UGT2B7 microsomes was effectively inhibited by diclofenac, a known substrate for UGT2B7. The denopamine glucuronidation activities in seven human liver microsomes were significantly correlated with diclofenac glucuronidation activities (r(2) = 0.685, p < 0.05). These results demonstrate that the denopamine glucuronidation in human liver and intestine is mainly catalyzed by UGT2B7 and that glucuronidation of the alcoholic hydroxyl group, but not the phenolic hydroxyl group, occurs regioselectively in humans.     

Characterization of bropirimine O-glucuronidation in human liver microsomes

1. The antitumour agent bropirimine undergoes significant Phase II conjugation in vivo. Incubation of [14C]bropirimine with human liver microsomes resulted in the formation of a single product peak (M1) using high-performance liquid chromatography with radiochemical detection and was tentatively assigned as bropirimine glucuronide based on sensitivity to beta-glucuronidase and by obtaining the expected mass of 442/444 amu with liquid chromatography/mass spectrometry. Following metabolite isolation, the structure of M1 was established as bropirimine O-glucuronide by 1H-nuclear magnetic spectroscopy. 2. Studies aimed at identifying the human liver UDP-glucuronosyltransferase (UGT) enzyme(s) involved in the glucuronidation of bropirimine were carried out using recombinant human UGTs and it was determined that glucuronidation of bropirimine was catalysed by UGT1A1, UGT1A3 and UGT1A9. Bropirimine O-glucuronidation followed Michaelis-Menten kinetics and the Km and Vmax (mean +/- SD; n = 3) were 1217 +/- 205 microM and 667 +/- 188 pmol min(-1) mg(-1), respectively. 3. The activity of bropirimine O-glucuronidation by human liver microsomes was inhibited by bilirubin (40%) and with mefenamic acid (80%). Although buprenorphine extensively inhibited the activity of bropirimine O-glucuronidation by UGT1A3, the inhibition profile did not parallel that observed in HLMs. 4. The results demonstrate that UGT1A9 and to a lesser extent UGT1A1 are responsible for the majority of bropirimine O-glucuronidation in man.     

The UDP-glucuronosyltransferase 2B7 isozyme is responsible for gemfibrozil glucuronidation in the human liver.

Gemfibrozil, a fibrate hypolipidemic agent, is eliminated in humans by glucuronidation. A gemfibrozil glucuronide has been reported to show time-dependent inhibition of cytochrome P450 2C8. Comprehensive assessment of the drug interaction between gemfibrozil and cytochrome P450 2C8 substrates requires a clear understanding of gemfibrozil glucuronidation. However, the primary UDP-glucuronosyltransferase (UGT) isozymes responsible for gemfibrozil glucuronidation remain to be determined. Here, we identified the main UGT isozymes involved in gemfibrozil glucuronidation. Evaluation of 12 recombinant human UGT isozymes shows gemfibrozil glucuronidation activity in UGT1A1, UGT1A3, UGT1A9, UGT2B4, UGT2B7, and UGT2B17, with UGT2B7 showing the highest activity. The kinetics of gemfibrozil glucuronidation in pooled human liver microsomes (HLMs) follows Michaelis-Menten kinetics with high and low affinity components. The high affinity K(m) value was 2.5 microM, which is similar to the K(m) value of gemfibrozil glucuronidation in recombinant UGT2B7 (2.2 microM). In 16 HLMs, a significant correlation was observed between gemfibrozil glucuronidation and both morphine 3-OH glucuronidation (r = 0.966, p < 0.0001) and flurbiprofen glucuronidation (r = 0.937, p < 0.0001), two reactions mainly catalyzed by UGT2B7, whereas no significant correlation was observed between gemfibrozil glucuronidation and either estradiol 3beta-glucuronidation and propofol glucuronidation, two reactions catalyzed by UGT1A1 and UGT1A9, respectively. Flurbiprofen and mefenamic acid inhibited gemfibrozil glucuronidation in HLMs with similar IC(50) values to those reported in recombinant UGT2B7. These results suggest that UGT2B7 is the main isozyme responsible for gemfibrozil glucuronidation in humans.     

Troglitazone glucuronidation in human liver and intestine microsomes: high catalytic activity of UGT1A8 and UGT1A10.

Troglitazone glucuronidation in human liver and intestine microsomes and recombinant UDP-glucuronosyltransferases (UGTs) were thoroughly characterized. All recombinant UGT isoforms in baculovirus-infected insect cells (UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B7, and UGT2B15) exhibited troglitazone glucuronosyltransferase activity. Especially UGT1A8 and UGT1A10, which are expressed in extrahepatic tissues such as stomach, intestine, and colon, showed high catalytic activity, followed by UGT1A1 and UGT1A9. The kinetics of the troglitazone glucuronidation in the recombinant UGT1A10 and UGT1A1 exhibited an atypical pattern of substrate inhibition when the substrate concentration was over 200 micro M. With a Michaelis-Menten equation at 6 to 200 micro M troglitazone, the K(m) value was 11.1 +/- 5.8 micro M and the V(max) value was 33.6 +/- 3.7 pmol/min/mg protein in recombinant UGT1A10. In recombinant UGT1A1, the K(m) value was 58.3 +/- 29.2 micro M and the V(max) value was 12.3 +/- 2.5 pmol/min/mg protein. The kinetics of the troglitazone glucuronidation in human liver and jejunum microsomes also exhibited an atypical pattern. The K(m) value was 13.5 +/- 2.0 micro M and the V(max) value was 34.8 +/- 1.2 pmol/min/mg for troglitazone glucuronidation in human liver microsomes, and the K(m) value was 8.1 +/- 0.3 micro M and the V(max) was 700.9 +/- 4.3 pmol/min/mg protein in human jejunum microsomes. When the intrinsic clearance was estimated with the in vitro kinetic parameter, microsomal protein content, and weight of tissue, troglitazone glucuronidation in human intestine was 3-fold higher than that in human livers. Interindividual differences in the troglitazone glucuronosyltransferase activity in liver microsomes from 13 humans were at most 2.2-fold. The troglitazone glucuronosyltransferase activity was significantly (r = 0.579, p < 0.05) correlated with the beta-estradiol 3-glucuronosyltransferase activity, which is mainly catalyzed by UGT1A1. The troglitazone glucuronosyltransferase activity in pooled human liver microsomes was strongly inhibited by bilirubin (IC(50) = 1.9 micro M), a typical substrate of UGT1A1. These results suggested that the troglitazone glucuronidation in human liver would be mainly catalyzed by UGT1A1. Interindividual differences in the troglitazone glucuronosyltransferase activity in S-9 samples from five human intestines was 8.2-fold. The troglitazone glucuronosyltransferase activity in human jejunum microsomes was strongly inhibited by emodin (IC(50) = 15.6 micro M), a typical substrate of UGT1A8 and UGT1A10, rather than by bilirubin (IC(50) = 154.0 micro M). Therefore, it is suggested that the troglitazone glucuronidation in human intestine might be mainly catalyzed by UGT1A8 and UGT1A10.     

Stereoselective conjugation of oxazepam by human UDP-glucuronosyltransferases (UGTs): S-oxazepam is glucuronidated by UGT2B15, while R-oxazepam is glucuronidated by UGT2B7 and UGT1A9.

(R,S)-Oxazepam is a 1,4-benzodiazepine anxiolytic drug that is metabolized primarily by hepatic glucuronidation. In previous studies, S-oxazepam (but not R-oxazepam) was shown to be polymorphically glucuronidated in humans. The aim of the present study was to identify UDP-glucuronosyltransferase (UGT) isoforms mediating R- and S-oxazepam glucuronidation in human liver, with the long term objective of elucidating the molecular genetic basis for this drug metabolism polymorphism. All available recombinant UGT isoforms were screened for R- and S-oxazepam glucuronidation activities. Enzyme kinetic parameters were then determined in representative human liver microsomes (HLMs) and in UGTs that showed significant activity. Of 12 different UGTs evaluated, only UGT2B15 showed significant S-oxazepam glucuronidation. Furthermore, the apparent K(m) for UGT2B15 (29-35 microM) was similar to values determined for HLMs (43-60 microM). In contrast, R-oxazepam was glucuronidated by UGT1A9 and UGT2B7. Although apparent K(m) values for HLMs (256-303 microM) were most similar to UGT2B7 (333 microM) rather than UGT1A9 (12 microM), intrinsic clearance values for UGT1A9 were 10 times higher than for UGT2B7. A common genetic variation results in aspartate (UGT2B15*1) or tyrosine (UGT2B15*2) at position 85 of the UGT2B15 protein. Microsomes from human embryonic kidney (HEK)-293 cells overexpressing UGT2B15*1 showed 5 times higher S-oxazepam glucuronidation activity than did UGT2B15*2 microsomes. Similar results were obtained for other substrates, including eugenol, naringenin, 4-methylumbelliferone, and androstane-3alpha-diol. In conclusion, S-oxazepam is stereoselectively glucuronidated by UGT2B15, whereas R-oxazepam is glucuronidated by multiple UGT isoforms. Allelic variation associated with the UGT2B15 gene may explain polymorphic S-oxazepam glucuronidation in humans.     

Involvement of multiple UDP-glucuronosyltransferase 1A isoforms in glucuronidation of 5-(4'-hydroxyphenyl)-5-phenylhydantoin in human liver microsomes.

In humans, orally administered phenytoin, 5,5-diphenylhydantoin, is mainly excreted as 5-(4'-hydroxyphenyl)-5-phenylhydantoin (4'-HPPH) O-glucuronide. Phenytoin is oxidized to 4'-HPPH by CYP2C9 and to a minor extent by CYP2C19, and then 4'-HPPH is metabolized to 4'-HPPH O-glucuronide by UDP-glucuronosyltransferase (UGT). In the present study, 4'-HPPH O-glucuronidation in human liver microsomes was investigated. The metabolite formed by incubation with human liver microsomes, 4'-HPPH, and UDP-glucuronic acid was identified as 4'-HPPH O-glucuronide by liquid chromatography-tandem mass spectrometry analysis. The 4'-HPPH O-glucuronosyltransferase activity in human liver microsomes was not saturated at concentrations up to 500 microM of 4'-HPPH. Any commercially available recombinant human UGTs (UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A9, UGT2B7, and UGT2B15) expressed in baculovirus-infected insect cells did not show detectable 4'-HPPH O-glucuronide. The 4'-HPPH O-glucuronidation in pooled human liver microsomes was inhibited by beta-estradiol as a typical substrate for UGT1A1 (IC(50) = 21.1 microM) and imipramine as a typical substrate for UGT1A4 (IC(50) = 57.7 microM). The inhibitory effects of propofol as a specific substrate for UGT1A9 (IC(50) = 167.1 microM) and emodin as a substrate for UGT1A8 and UGT1A10 (IC(50) = 287.6 microM) were not prominent. The interindividual difference in the 4'-HPPH O-glucuronidation in 14 human liver microsomes was 28.5-fold (0.023-0.656 nmol/min/mg of protein). The 4'-HPPH O-glucuronosyltransferase activity in 11 human liver microsomes was significantly (r = 0.609, P < 0.05) correlated with the 4-nitrophenol glucuronosyltransferase activity, which is catalyzed by UGT1A6 and UGT1A9. These results suggest that multiple UGT1As such as UGT1A1, UGT1A4, UGT1A6, and UGT1A9 are involved in 4'-HPPH O-glucuronidation in human liver microsomes, although the percentage contribution of each UGT1A could not be estimated. Large interindividual differences in the glucuronidation of 4'-HPPH might be responsible for the nonlinearity of the phenytoin plasma concentration or adverse reactions in humans.     

Glucuronidation of anti-HIV drug candidate bevirimat: identification of human UDP-glucuronosyltransferases and species differences.

Bevirimat [BVM, PA-457, 3-O-(3',3'-dimethylsuccinyl)-betulinic acid], a new anti-human immunodeficiency virus drug candidate, is metabolized to two monoglucuronides [mono-BVMG (I) and mono-BVMG (II)] and one diglucuronide (di-BVMG) both in vivo and in vitro. UDP-glucuronosyltransferase (UGT) reaction screening, enzyme kinetics, and species differences for the glucuronidation of BVM in vitro were investigated with pooled human liver microsomes (HLMs) and human intestinal microsomes (HIMs), animal liver microsomes, and 12 recombinant human UGT isoforms. Glucuronidation of BVM with HLMs predominantly involved the formation of mono-BVMG (I) (V(max) = 61 pmol/min/mg protein, K(m) = 27 microM) and mono-BVMG (II) (V(max) = 48 pmol/min/mg protein, K(m) = 16 microM). Di-BVMG was also observed but was a minor metabolite. HIMs mainly revealed glucuronidation to form mono-BVMG (II) (V(max) = 90 pmol/min/mg of protein, K(m) = 8.3 microM). UGT1A3 predominantly formed mono-BVMG (I) (V(max) = 65 pmol/min/mg of protein, K(m) = 13 microM), whereas UGT1A4 is a less active isoform (V(max) = 1.8 pmol/min/mg of protein, K(m) = 5.6 microM). UGT2B7 was involved in the formation of both mono-BVMG (I) (V(max) = 6.1 pmol/min/mg of protein, K(m) = 6.0 microM) and mono-BVMG (II) (V(max) = 6.5 pmol/min/mg of protein, K(m) = 7.8 microM). Among the animal liver microsomes examined, all species (rat, mouse, dog, and marmoset) demonstrated conjugation to form both mono-BVMG (I) and mono-BVMG (II), with dog liver microsomes exhibiting a higher formation rate for mono-BVMG (I), whereas marmoset liver microsomes showed a higher formation rate for mono-BVMG (II). The data suggest a primary role of UGT1A3 for the glucuronidation of BVM.     

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.     

In vitro metabolism of eupatilin by multiple cytochrome P450 and UDP-glucuronosyltransferase enzymes

Eupatilin, a pharmacologically active flavone derived from Artemisia plants, is extensively metabolized to eupatilin glucuronide, 4-O-desmethyleupatilin and 4-O-desmethyleupatilin glucuronide in human liver microsomes. This study characterized the human liver cytochrome P450 (CYP) and UDP-glucuronosyltransferase (UGT) enzymes responsible for the metabolism of eupatilin. The specific CYPs responsible for O-demethylation of eupatilin to the major metabolite, 4-O-desmethyleupatilin were identified using a combination of correlation analysis, immuno-inhibition, chemical inhibition in human liver microsomes and metabolism by human cDNA-expressed CYP enzymes. UGT enzymes involved in the eupatilin glucuronidation were identified using pooled human liver microsomes and human cDNA-expressed UGT enzymes. Eupatilin was predominantly metabolized by CYP1A2 and, to a lesser extent, CYP2C8 mediated O-demethylation of eupatilin to 4-O-desmethyleupatilin. Eupatilin glucuronidation was catalysed by UGT1A1, UGT1A3, UGT1A7, UGT1A8, UGT1A9, and UGT1A10.     

Glucosidation of hyodeoxycholic acid by UDP-glucuronosyltransferase 2B7

Previous studies have shown that several endogenous compounds, such as bilirubin and certain bile acids, are glucosidated in human liver. In this work, we have identified human UDP-glucuronosyltransferase 2B7 (UGT2B7) as the isoform that catalyzes the glucosidation of hyodeoxycholic acid (HDCA). The glucosidation by UGT2B7 was specific for HDCA and was not observed with the other bile acids examined, lithocholic acid, chenodeoxycholic acid, and ursodeoxycholic acid. The kinetics of HDCA glucuronidation and glucosidation by UGT2B7 were characterized. The K(m) values for glucuronidation and glucosidation of HDCA were 11.6 and 17.9 microM, respectively, with V(max) values of 4.15 nmol/min/mg protein for glucuronidation and 3.28 nmol/min/mg for glucosidation. At a fixed concentration of HDCA, the apparent K(m) for UDP-glucuronic acid was 89 microM with a V(max) of 3.53 nmol/min/mg. The corresponding parameters for UDP-glucose were 442 microM and 1.98 nmol/min/mg, respectively. UGT2B7 catalyzed the addition of the glucose and glucuronic acid moieties to an hydroxyl group on HDCA and also possessed some capacity to use UDP-xylose as sugar donor. The two polymorphic variants of UGT2B7, UGT2B7(*)1 and UGT2B7(*)2 could both glucosidate HDCA. This is the first report that identifies UGT2B7 as the enzyme responsible for the glucosidation of the bile acid, HDCA.     

Almokalant glucuronidation in human liver and kidney microsomes: evidence for the involvement of UGT1A9 and 2B7

1. Almokalant, a class III antiarrythmic drug, is metabolized to form isomeric glucuronides identified in human urine. Synthesis of the total glucuronide was studied in human liver and kidney microsomes. Recombinant UDP-glucuronosyltransferases (UGTs) were screened for activity and kinetic analysis was performed to identify the isoform(s) responsible for the formation of almokalant glucuronide in man. 2. From a panel of recombinant isoforms used, both UGT1A9 and 2B7 catalysed the glucuronidation of almokalant. The Km values in both instances were similar with 1.06 mM for the 1A9 and 0.97 mM for the 2B7. Vmax for 1A9 was fourfold higher than that measured for UGT2B7, 92 compared with 21 pmol min(-1) mg(-1), respectively, but UGT1A9 was expressed at approximately twofold higher level than the UGT2B7 in the recombinant cell lines. Therefore, the contribution of UGT2B7 to almokalant glucuronidation could be as significant as that of UGT1A9 in man. 3. Liver and kidney microsomes displayed similar Km values to the cloned expressed UGTs, with the liver and kidney microsomes at 1.68 and 1.06 mM almost identical to the 1A9. 4. The results suggest a significant role for UGT1A9 and 2B7 in the catalysis of almokalant glucuronidation.     

Mechanistic studies on the reversible metabolism of rofecoxib to 5-hydroxyrofecoxib in the rat: evidence for transient ring opening of a substituted 2-furanone derivative using stable isotope-labeling techniques.

Rofecoxib is a potent and highly selective cyclooxygenase-2 inhibitor used for the treatment of osteoarthritis and pain. Following administration of [4-(14)C]rofecoxib to intact rats, the plasma C(max) (at approximately 1 h) was followed by a secondary C(max) (at approximately 10 h), which was not observed in bile duct-cannulated rats. Following administration of [4-(14)C]5-hydroxyrofecoxib to intact or bile duct-cannulated rats, radiolabeled rofecoxib was detected in plasma, and once again a secondary C(max) for rofecoxib was observed (at approximately 10 h), which occurred only in the intact animals. These results indicate that reversible metabolism of rofecoxib to 5-hydroxyrofecoxib occurs in the rat and that the process is dependent upon an uninterrupted bile flow. Studies on the contents of the gastrointestinal tract of rats showed that conversion of 5-hydroxyrofecoxib to parent compound occurs largely in the lower intestine. Treatment of rats with [5-(18)O]5-hydroxyrofecoxib, followed by liquid chromatography-tandem mass spectrometry analyses of plasma samples, confirmed that 5-hydroxyrofecoxib undergoes metabolism to the parent drug, yielding [1-(18)O]rofecoxib, [2-(18)O]rofecoxib, and unlabeled rofecoxib. Similarly, treatment with [1,2-(18)O(2)]rofecoxib afforded the same three isotopic variants of rofecoxib. These findings are consistent with a metabolic sequence involving 5-hydroxylation of rofecoxib, biliary elimination of the corresponding glucuronide, and deconjugation of the glucuronide in the lower gastrointestinal tract. Reduction of the 5-hydroxyrofecoxib thus liberated yields a hydroxyacid that cyclizes spontaneously to regenerate rofecoxib, which is reabsorbed and enters the systemic circulation. This sequence represents a novel form of enterohepatic recycling and reflects the susceptibility of 5-hydroxyrofecoxib, as well as rofecoxib itself, to reversible 2-furanone ring opening under in vivo conditions.