Human liver UDP-glucuronosyltransferase isoforms involved in the glucuronidation of 7-ethyl-10-hydroxycamptothecin

1. The human liver UDP-glucuronosyltransferase (UGT) isoforms involved in the glucuronidation of 7-ethyl-10-hydroxycamptothecin (SN-38), the active metabolite of irinotecan (CPT-11), have been studied using microsomes from human liver and insect cells expressing human UGTs (1A1, 1A3, 1A4, 1A6, 1A9, 2B7, 2B15). 2. The glucuronidation of SN-38 was catalysed by UGT1A1, UGT1A3, UGT1A6 and UGT1A9 as well as by liver microsomes. Among these UGT isoforms, UGT1A1 showed the highest activity of SN-38 glucuronidation at both low (1 µM) and high (200 µM) substrate concentrations. The ranking in order of activity at low and high substrate concentrations was UGT1A1 > UGT1A9 > UGT1A6> UGT1A3 and UGT1A1 > UGT1A3 > UGT1A6 ≥ UGT1A9, respectively. 3. The enzyme kinetics of SN-38 glucuronidation were examined by means of Lineweaver-Burk analysis. The activity of the glucuronidation in liver microsomes exhibits a monophasic kinetic pattern, with an apparent K m and V max of 35.9 µM and 134pmol?min -1?mg -1 protein, respectively. The UGT isoforms involved in SN-38 glucuronidation could be classified into two types: low- K m types such as UGT1A1 and UGT1A9, and high- K m types such as UGT1A3 and UGT1A6, in terms of affinity toward substrate. UGT1A1 had the highest V max followed by UGT1A3. V max of UGT1A6 and UGT1A9 were approximately 1/9 to 1/12 of that of UGT1A1. 4. The activity of SN-38 glucuronidation by liver microsomes and UGT1A1 was effectively inhibited by bilirubin. Planar and bulky phenols substantially inhibited the SN-38 glucuronidation activity of liver microsomes and UGT1A9, and/or UGT1A6. Although cholic acid derivatives strongly inhibited the activity of SN-38 glucuronidation by UGT1A3, the inhibition profile did not parallel that in liver microsomes. 5. These results demonstrate that at least four UGT1A isoforms are responsible for SN-38 glucuronidation in human livers, and suggest that the role and contribution of each differ substantially.     

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.     

Acyl glucuronidation of fluoroquinolone antibiotics by the UDP-glucuronosyltransferase 1A subfamily in human liver microsomes.

Acyl glucuronidation is an important metabolic pathway for fluoroquinolone antibiotics. However, it is unclear which human UDP-glucuronosyltransferase (UGT) enzymes are involved in the glucuronidation of the fluoroquinolones. The in vitro formation of levofloxacin (LVFX), grepafloxacin (GPFX), moxifloxacin (MFLX), and sitafloxacin (STFX) glucuronides was investigated in human liver microsomes and cDNA-expressed recombinant human UGT enzymes. The apparent Km values for human liver microsomes ranged from 1.9 to 10.0 mM, and the intrinsic clearance values (calculated as Vmax/Km) had a rank order of MFLX > GPFX > STFX > > LVFX. In a bank of human liver microsomes (n = 14), the glucuronidation activities of LVFX, MFLX, and STFX correlated highly with UGT1A1-selective beta-estradiol 3-glucuronidation activity, whereas the glucuronidation activity of GPFX correlated highly with UGT1A9-selective propofol glucuronidation activity. Among 12 recombinant UGT enzymes, UGT1A1, 1A3, 1A7, and 1A9 catalyzed the glucuronidation of these fluoroquinolones. Results of enzyme kinetics studies using the recombinant UGT enzymes indicated that UGT1A1 most efficiently glucuronidates MFLX, and UGT1A9 most efficiently glucuronidates GPFX. In addition, the glucuronidation activities of MFLX and STFX in human liver microsomes were potently inhibited by bilirubin with IC50 values of 4.9 microM and 4.7 microM, respectively; in contrast, the glucuronidation activity of GPFX was inhibited by mefenamic acid with an IC50 value of 9.8 microM. These results demonstrate that UGT1A1, 1A3, and 1A9 enzymes are involved in the glucuronidation of LVFX, GPFX, MFLX, and STFX in human liver microsomes, and that MFLX and STFX are predominantly glucuronidated by UGT1A1, whereas GPFX is mainly glucuronidated by UGT1A9.     

Differential rates of glucuronidation for 7-ethyl-10-hydroxy-camptothecin (SN-38) lactone and carboxylate in human and rat microsomes and recombinant UDP-glucuronosyltransferase isoforms.

7-ethyl-10-hydroxy-camptothecin (SN-38), the active metabolite of the anti-cancer agent irinotecan, contains a lactone ring that equilibrates with a carboxylate form. Since SN-38 lactone is the active and toxic form, it is prudent to examine whether the more soluble carboxylate is a surrogate for SN-38 lactone conjugation. Therefore, relative rates of glucuronidation and isoform specificity of SN-38 lactone and carboxylate were characterized. The stability of SN-38 lactone and carboxylate in incubation mixtures of microsomes and UDP-glucuronosyltransferase (UGT) isoforms was used to determine optimal incubation times. Microsomal incubations were conducted using rat and human intestinal and hepatic microsomes and human and rat recombinant UGT1A isoforms. Where estimates of lactone and carboxylate glucuronidation rates could not be established due to short incubation times and detection limits, kinetic modeling was used to recover these rate constants. The stability experiments revealed that the lactone was stabilized by rat microsomes, however, the opposite was observed in human microsomes and recombinant isoforms. For all tissues and most UGT isoforms examined, the lactone consistently had catalytic rates up to 6-fold greater than the carboxylate. The rank order of glucuronidation for both SN-38 lactone and carboxylate was 1A7 > 1A1 > 1A9 > 1A8 and 1A7 > 1A8 > 1A1 for human and rat isoforms, respectively. This study provides further support that SN-38 lactone and carboxylate may be considered pharmacokinetically distinct agents. The in vivo impact of this conjugation difference is unknown, since variations in protein binding and transport proteins may affect intracellular concentrations of the lactone or carboxylate.     

Stereoselective glucuronidation of propranolol in human and cynomolgus monkey liver microsomes: role of human hepatic UDP-glucuronosyltransferase isoforms, UGT1A9, UGT2B4 and UGT2B7

The stereoselective glucuronidation of propranolol (PL) in human and cynomolgus monkey liver microsomes, and the roles of human hepatic UDP-glucuronosyltransferase (UGT) isoforms involved in the enantiomeric glucuronidation of PL using recombinant UGT enzymes were investigated. In Michaelis-Menten plots, R- and S-PL glucuronidation by human liver microsomes showed sigmoidal kinetics whereas the kinetics of enantiomeric PL glucuronidation by cynomolgus monkey liver microsomes was monophasic. The Km, Vmax and CLint values of cynomolgus monkey liver microsomes were generally higher than the S50, Vmax and CLmax values of human liver microsomes in R- and S-PL glucuronidation. The glucuronidation of R- and S-PL was catalyzed by at least 3 UGT isoforms: UGT1A9, UGT2B4 and UGT2B7. Michaelis-Menten plots for R- and S-PL glucuronidation by UGT1A9 were monophasic, whereas the kinetics of UGT2B7 showed sigmoidal curves. Enantiomeric R-PL glucuronidation by UGT2B4 showed sigmoidal kinetics, whereas S-PL glucuronidation displayed monophasic kinetics. UGT1A9 showed remarkable stereoselectivity in Vmax and CLint values of R-PL < S-PL. These findings demonstrate that the profiles of enantiomeric PL glucuronidation in human and cynomolgus monkey liver microsomes are largely different and suggest that the human hepatic UGT isoforms UGT1A9, UGT2B4 and UGT2B7 play distinctive roles in enantiomeric PL glucuronidation.     

Evaluation of 5-hydroxytryptophol and other endogenous serotonin (5-hydroxytryptamine) analogs as substrates for UDP-glucuronosyltransferase 1A6.

Serotonin is a specific in vitro substrate for human UDP-glucuronosyltransferase (UGT) 1A6. In this study, the contribution of UGT1A6 to the glucuronidation of endogenous structural analogs of serotonin, including 5-hydroxytryptophol, N-acetylserotonin, and 6-hydroxymelatonin, was evaluated using available recombinant human UGT isoforms, human liver microsomes, and liver microsomes from animals that do not express functional UGT1A6 (Gunn rats and cats). Only UGT1A6 and UGT1A9 were found to glucuronidate 5-hydroxytryptophol at a concentration of 2 mM, although the glucuronidation rate with UGT1A6 was over 10 times that of UGT1A9. K(m) values for human liver microsomes (156, 141, and 134 microM) were most similar to that of expressed UGT1A6 (135 microM) but vastly different from that of UGT1A9 (3674 microM). 5-Hydroxytryptophol glucuronidation by human liver microsomes (n = 54) correlated well with serotonin glucuronidation (R(s) = 0.83) and UGT1A6 protein content (R(s) = 0.85). 5-Hydroxytryptophol also competitively inhibited serotonin glucuronidation by human liver microsomes (K(i) = 291 microM) and UGT1A6 (K(i) = 200 microM). N-acetylserotonin was glucuronidated most extensively by UGT1A6, although UGT1A9 and UGT1A10 showed moderate catalysis. 6-Hydroxymelatonin was glucuronidated largely by UGT1A9 and UGT1A10 but not at all by UGT1A6. Gunn rat liver glucuronidation rates for serotonin, 5-hydroxytryptophol, N-acetylserotonin, and 6-hydroxymelatonin were 11, 5, 32, and 3%, respectively, of that of normal rat liver. Cat liver microsomes did not glucuronidate serotonin, whereas relatively low activities were observed for the other indole substrates. In conclusion, these results indicate that human UGT1A6 plays a predominant role in the glucuronidation of 5-hydroxytryptophol and N-acetylserotonin, whereas 6-hydroxymelatonin is not a substrate for this enzyme.     

Evaluation of 3,3',4'-trihydroxyflavone and 3,6,4'-trihydroxyflavone (4'-O-glucuronidation) as the in vitro functional markers for hepatic UGT1A1

Identifying uridine 5'-diphospho-(UDP)-glucuronosyltransferase (UGT)-selective probes (substrates that are primarily glucuronidated by a single isoform) is complicated by the enzymes' large overlapping substrate specificity. Here, regioselective glucuronidation of two flavonoids, 3,3',4'-trihydroxyflavone (3,3',4'-THF) and 3,6,4'-trihydroxyflavone (3,6,4'-THF), is used to probe the activity of hepatic UGT1A1. The glucuronidation kinetics of 3,3',4'-THF and 3,6,4'-THF was determined using 12 recombinant human UGT isoforms and pooled human liver microsomes (pHLM). The individual contribution of main UGT isoforms to the metabolism of the two flavonoids in pHLM was estimated using the relative activity factor approach. UGT1A1 activity correlation analyses using flavonoids-4'-O-glucuronidation vs β-estradiol-3-glucuronidation (a well-recognized marker for UGT1A1) or vs SN-38 glucuronidation were performed using a bank of HLMs (n = 12) including three UGT1A1-genotyped HLMs (i.e., UGT1A1*1*1, UGT1A1*1*28, and UGT1A1*28*28). The results showed that UGT1A1 and 1A9, followed by 1A7, were the main isoforms for glucuronidating the two flavonoids, where UGT1A1 accounted for 92 ± 7% and 91 ± 10% of 4'-O-glucuronidation of 3,3',4'-THF and 3,6,4'-THF, respectively, and UGT1A9 accounted for most of the 3-O-glucuronidation. Highly significant correlations (R(2) > 0.944, p < 0.0001) between the rates of flavonoids 4'-O-glucuronidation and that of estradiol-3-glucuronidation or SN-38 glucuronidation were observed across 12 HLMs. In conclusion, UGT1A1-mediated 4'-O-glucuronidation of 3,3',4'-THF and 3,6,4'-THF was highly correlated with the glucuronidation of estradiol (3-OH) and SN-38. This study demonstrated for the first time that regioselective glucuronidation of flavonoids can be applied to probe hepatic UGT1A1 activity in vitro.     

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.     

Glucuronidation of trans-resveratrol by human liver and intestinal microsomes and UGT isoforms

Resveratrol (trans-resveratrol, trans-3,5,4'-trihydroxystilbene) is a naturally occurring stilbene analogue found in high concentrations in red wine. There is considerable research interest to determine the therapeutic potential of resveratrol, as it has been shown to have tumour inhibitory and antioxidant properties. This study was performed to investigate the glucuronidation of resveratrol and possible drug interactions via glucuronidation. Two glucuronide conjugates, resveratrol 3-O-glucuronide and resveratrol 4'-O-glucuronide, were formed by human liver and intestinal microsomes. UGT1A1 and UGT1A9 were predominantly responsible for the formation of the 3-O-glucuronide (Km = 149 microM) and 4'-O-glucuronide (Km = 365 microM), respectively. The glucuronide conjugates were formed at higher levels (up to 10-fold) by intestinal rather than liver microsomes. Resveratrol was co-incubated with substrates of UGT1A1 (bilirubin and 7-ethyl-10-hydroxycamptothecin (SN-38)) and UGT1A9 (7-hydroxytrifluoromethyl coumarin (7-HFC)). No major changes were noted in bilirubin glucuronidation in the presence of resveratrol. Resveratrol significantly inhibited the glucuronidation of SN-38 (Ki = 6.2 +/- 2.1 microM) and 7-HFC (Ki = 0.6 +/- 0.2 microM). Hence, resveratrol has the potential to inhibit the glucuronidation of concomitantly administered therapeutic drugs or dietary components that are substrates of UGT1A1 and UGT1A9.     

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.     

The specificity of glucuronidation of entacapone and tolcapone by recombinant human UDP-glucuronosyltransferases.

The COMT inhibitors entacapone and tolcapone are rapidly metabolized in vivo, mainly by glucuronidation. In this work, the main UGT isoforms responsible for their glucuronidation in vitro were characterized by using a subset of representative cloned and expressed human UGT isoforms. Entacapone in particular was seen to be an exceptionally good substrate for UGT1A9 with an even higher reaction velocity value at 500 microM substrate concentration compared with that of the commonly used substrate, propofol (1.3 and 0.78 nmol min(-1) mg(-1), respectively). Neither entacapone nor tolcapone was glucuronidated by UGT1A6. Tolcapone was not detectably glucuronidated by UGT1A1, and the rate of glucuronidation of entacapone was also low by this isoform. However, UGT1A1 was the only UGT capable of catalyzing the formation of two glucuronides of the catecholic entacapone. Both COMT inhibitors were glucuronidated at low rates by the representative members of the UGT2B family, UGT2B7 and UGT2B15. Michaelis-Menten parameters were determined for entacapone and tolcapone using recombinant human UGT isoforms and human liver microsomes to compare the kinetic properties of the two COMT inhibitors. The kinetic data illustrates that UGT1A9 exhibited a much greater rate of glucuronidation and a far lower K(m) value for both entacapone and tolcapone than UGT2B15 and UGT2B7 whose contribution is minor by comparison. Entacapone showed a 3 to 4 times higher V(max) value and a 4 to 6 times lower K(m) value compared with those of tolcapone both in UGT1A9 cell lysates and in human liver microsomes.     

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.     

Carboxyl-glucuronidation of mitiglinide by human UDP-glucuronosyltransferases

Mitiglinide (MGN) is a new potassium channel antagonist for the treatment of type 2 diabetes mellitus. In the present study, a potential metabolic pathway of MGN, via carboxyl-linked glucuronic acid conjugation, was found. MGN carboxyl-glucuronide was isolated from a reaction mixture consisting of MGN and human liver microsomes fortified with UDP-glucuronic acid (UDPGA) and identified by a hydrolysis reaction with beta-glucuronidase and HPLC-MS/MS. Kinetic analysis indicated that MGN from four species had the highest affinity for the rabbit liver microsomal enzyme (K(m)=0.202 mM) and the lowest affinity for the dog liver microsomal enzyme (K(m)=1.164 mM). The metabolic activity (V(max)/K(m)) of MGN to the carboxyl-glucuronidation was in the following order: rabbit>dog>rat>human. With the assessment of MGN glucuronide formation across a panel of recombinant UDP-glucuronosyltransferase (UGT) isoforms (UGT1A3, UGT1A4, UGT1A6, UGT1A9, and UGT2B7), only UGT1A3 and UGT2B7 exhibited high MGN glucuronosyltransferase activity. The K(m) values of MGN glucuronidation in recombinant UGT1A3 and UGT2B7 microsomes were close to those in human liver microsomes. The formation of MGN glucuronidation by human liver microsomes was effectively inhibited by quercetin (substrate for UGT1A3) and diclofenac (substrate for UGT2B7), respectively. The MGN glucuronidation activities in 15 human liver microsomes were significantly correlated with quercetin (r(2)=0.806) and diclofenac glucuronidation activities (r(2)=0.704), respectively. These results demonstrate that UGT1A3 and UGT2B7 are catalytic enzymes in MGN carboxyl-glucuronidation in human liver.     

Identification of human UDP-glucuronosyltransferase isoform(s) responsible for the glucuronidation of 2-(4-chlorophenyl)- 5-(2-furyl)-4-oxazoleacetic acid (TA-1801A)

We characterized the hepatic and intestinal UDP-glucuronosyltransferase (UGT) isoform(s) responsible for the glucuronidation of 2-(4-chlorophenyl)-5-(2-furyl)-4-oxazoleacetic acid (TA-1801A) in humans through several in vitro mechanistic studies. Assessment of a panel of recombinant UGT isoforms revealed the TA-1801A glucuronosyltransferase activity of UGT1A1, UGT1A3, UGT1A7, UGT1A9, and UGT2B7. Kinetic analyses of the TA-1801A glucuronidation by recombinant UGT1A1, UGT1A3, UGT1A9, and UGT2B7 showed that the K(m) value for UGT2B7 was apparently consistent with those in human liver and jejunum microsomes. The TA-1801A glucuronosyltransferase activity in human liver microsomes was inhibited by bilirubin (typical substrate for UGT1A1), propofol (typical substrate for UGT1A9), diclofenac (substrate for UGT1A9 and UGT2B7), and genistein (substrate for UGT1A1, UGT1A3, and UGT1A9). The inhibition by bilirubin, propofol, and diclofenac of the TA-1801A glucuronidation was less pronounced in jejunum microsomes than liver microsomes, suggesting that the contribution of UGT1A1, UGT1A9, and UGT2B7 to the TA-1801A glucuronidation is smaller in the intestine than the liver. In contrast, genistein strongly inhibited the TA-1801A glucuronosyltransferase activity in both human liver and jejunum microsomes. These results suggest that the glucuronidation of TA-1801A is mainly catalyzed by UGT1A1, UGT1A9, and UGT2B7 in the liver, and by UGT1A1, UGT1A3, and UGT2B7 in the intestine in humans.     

Glucuronidation of HMR1098 in human microsomes: evidence for the involvement of UGT1A1 in the formation of S-glucuronides.

HMR1098, a novel KATP-blocking agent, is metabolized to form an S-glucuronide in rat and dog bile. Synthesis of the S-glucuronide metabolite was studied in human liver and kidney microsomes. Recombinant UPD-glucuronosyltransferases (UGTs) were screened for activity, and kinetic analysis was performed to identify the isoform or isoforms responsible for the formation of this novel S-glucuronide in humans. S-Glucuronidation is relatively rare, but from this study it appears that S-glucuronides are not generated exclusively by a single UGT isoform. From the panel of recombinant isoforms used, both UGT1A1 and UGT1A9 catalyzed the glucuronidation of HMR1098. The Vmax values in both instances were similar, but the Km for UGT1A1 was substantially lower than that measured for UGT1A9, 82 microM compared with 233 microM, respectively. Liver and kidney microsomes displayed similar Km values, but the Vmax in kidney was more than 20-fold less than in liver microsomes, which is suggestive of a significant role for the bilirubin UGT in catalysis of HMR1098, although other UGTs may play a secondary role.     

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.     

Common human UGT1A polymorphisms and the altered metabolism of irinotecan active metabolite 7-ethyl-10-hydroxycamptothecin (SN-38)

7-Ethyl-10-hydroxycamptothecin (SN-38) is the pharmacologically active metabolite of irinotecan, in addition to being responsible for severe toxicity. Glucuronidation is the main metabolic pathway of SN-38 and has been shown to protect against irinotecan-induced gastrointestinal toxicity. The purpose of this study was to determine whether common polymorphic UDP-glucuronosyltransferase (UGT) affects SN-38 glucuronidation. First, kinetic characterization of SN-38-glucuronide (SN-38-G) formation was assessed for all known human UGT1A and UGT2B overexpressed in human embryonic kidney 293 cells. To assess the relative activity of UGT isoenzymes for SN-38, rates of formation of SN-38-G were monitored by liquid chromatography/mass spectrometry analysis and normalized by level of UGT cellular expression. Determination of intrinsic clearances predicts that hepatic UGT1A1 and UGT1A9 and the extrahepatic UGT1A7 are major components in SN-38-G formation, whereas a minor role is suggested for UGT1A6, UGT1A8, and UGT1A10. In support of the involvement of UGT1A9, a strong coefficient of correlation was observed in the glucuronidation of SN-38 and a substrate, mainly glucuronidate, by UGT1A9 (flavopiridol) by human liver microsomes (coefficient of correlation, 0.905; p = 0.002). In vitro functional experiments revealed a negative impact of the UGT1A1 allelic variants. Residual activities of 49, 7, 8, and 11% were observed for UGT1A1*6 (G(71)R), UGT1A1*27 (P(229)Q), UGT1A1*35 (L(233)R), and UGT1A1*7 (Y(486)D), respectively. Common variants of UGT1A7, UGT1A7*3 (N(129)K;R(131)K;W(208)R), and UGT1A7*4 (W(208)R), displayed residual activities of 41 and 28% compared with the UGT1A7*1 allele. Taken together, these data provide the evidence that molecular determinants of irinotecan response may include the UGT1A polymorphisms studied herein and common genetic variants of the hepatic UGT1A9 isoenzyme yet to be described.     

Substrate-dependent modulation of UDP-glucuronosyltransferase 1A1 (UGT1A1) by propofol in recombinant human UGT1A1 and human liver microsomes

Our previous study has shown that propofol, a probe substrate for human UDP-glucuronosyltransferase (UGT) 1A9, activated the glucuronidation of 4-methylumbelliferone (4-MU) by recombinant UGT1A1 in a concentration-dependent manner. In the present study, we investigated the mechanism of activation, and whether the stimulatory effect occurs when another substrate is used with human liver microsomes. The glucuronidation of 4-MU followed Michaelis-Menten kinetics with a K(m) value of 101 microM in the absence of propofol. In the presence of 200 microM propofol, a concentration that causes heterotopic activation of 4-MU glucuronidation (4-MUG), the V(max) value increased to 1.5-fold, while the K(m) value decreased to 0.53-fold. In order to assess whether propofol activates UGT1A1 activity for a substrate other than 4-MU, the effect of propofol on oestradiol 3beta-glucuronidation by recombinant UGT1A1 and in human liver microsomes was evaluated. In contrast to 4-MUG activity, propofol inhibited UGT1A1-catalysed oestradiol 3beta-glucuronidation in recombinant UGT1A1 as well as in human liver microsomes with IC(50) values of 59 and 228 microM, respectively. In addition, a known UGT1A1 modulator, 17alpha-ethynyloestradiol, stimulated oestradiol 3beta-glucuronidation slightly at a concentration of 5 microM, while it inhibited 4-MUG in recombinant UGT1A1 at all concentrations tested (5-100 microM). These findings indicate that the modulation of UGT1A1 by propofol is substrate-dependent, and thus care should be taken when extrapolating the stimulatory effects of drugs for one glucuronidation substrate.     

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.