Udp-glucuronosyltransferase 2b7 is the major enzyme responsible for gemcabene glucuronidation in human liver microsomes.

The predominant metabolic pathway of gemcabene in humans is glucuronidation. The principal human UDP-glucuronosyltransferases (UGTs) involved in the glucuronidation of gemcabene were determined in this study. Glucuronidation of gemcabene was catalyzed by recombinant UGT1A3, recombinant UGT2B7, and recombinant UGT2B17, as well as by human liver microsomes (HLM). Gemcabene glucuronidation in recombinant UGTs and HLM followed non-Michaelis-Menten kinetics consistent with homotropic activation, but pharmacokinetics in humans were linear over the dose range tested (total plasma C(max), 0.06-0.88 mM). Gemcabene showed similar affinity (S(50)) for recombinant UGTs (0.92-1.45 mM) and HLM (1.37 mM). S-Flurbiprofen was identified as a more selective inhibitor of recombinant UGT2B7-catalyzed gemcabene glucuronidation (>23-fold lower IC(50)) when compared with recombinant UGT1A3- or recombinant UGT2B17-catalyzed gemcabene glucuronidation. The IC(50) for S-flurbiprofen inhibition of gemcabene glucuronidation was similar in HLM (60.6 microM) compared with recombinant UGT2B7 (27.4 microM), consistent with a major role for UGT2B7 in gemcabene glucuronidation in HLM. In addition, 5,6,7,3',4',5'-hexamethoxyflavone inhibited recombinant UGT1A3 and recombinant UGT2B17-catalyzed gemcabene glucuronidation (with 4-fold greater potency for recombinant UGT1A3) but did not inhibit gemcabene glucuronidation in HLM, suggesting that UGT1A3 and UGT2B17 do not contribute significantly to gemcabene glucuronidation. Reaction rates for gemcabene glucuronidation from a human liver bank correlated well (r(2)=0.722, P<0.0001; n=24) with rates of glucuronidation of the UGT2B7 probe substrate 3'-azido-3'-deoxythymidine. In conclusion, using the three independent experimental approaches typically used for cytochrome P450 reaction phenotyping, UGT2B7 is the major enzyme contributing to gemcabene glucuronidation in human liver microsomes.     

The glucuronidation of Delta4-3-Keto C19- and C21-hydroxysteroids by human liver microsomal and recombinant UDP-glucuronosyltransferases (UGTs): 6alpha- and 21-hydroxyprogesterone are selective substrates for UGT2B7.

The stereo- and regioselective glucuronidation of 10 Delta(4)-3-keto monohydroxylated androgens and pregnanes was investigated to identify UDP-glucuronosyltransferase (UGT) enzyme-selective substrates. Kinetic studies were performed using human liver microsomes (HLMs) and a panel of 12 recombinant human UGTs as the enzyme sources. Five of the steroids, which were hydroxylated in the 6beta-, 7alpha-, 11beta- or 17alpha-positions, were not glucuronidated by HLMs. Of the remaining compounds, comparative kinetic and inhibition studies indicated that 6alpha- and 21-hydroxyprogesterone (OHP) were glucuronidated selectively by human liver microsomal UGT2B7. 6alpha-OHP glucuronidation by HLMs and UGT2B7 followed Michaelis-Menten kinetics, whereas 21-OHP glucuronidation by these enzyme sources exhibited positive cooperativity. UGT2B7 was also identified as the enzyme responsible for the high-affinity component of human liver microsomal 11alpha-OHP glucuronidation. In contrast, UGT2B15 and UGT2B17 were the major forms involved in human liver microsomal testosterone 17beta-glucuronidation and the high-affinity component of 16alpha-OHP glucuronidation. Activity of UGT1A subfamily enzymes toward the hepatically glucuronidated substrates was generally low, although UGT1A4 and UGT1A9 contribute to the low-affinity components of microsomal 16alpha- and 11alpha-OHP glucuronidation, respectively. Interestingly, UGT1A10, which is expressed only in the gastrointestinal tract, exhibited activity toward most of the glucuronidated substrates. The results indicate that 6alpha- and 21-OHP may be used as selective "probes" for human liver microsomal UGT2B7 activity and, taken together, provide insights into the regio- and stereoselectivity of hydroxysteroid glucuronidation by human UGTs.     

A high throughput assay for the glucuronidation of 7-hydroxy-4-trifluoromethylcoumarin by recombinant human UDP-glucuronosyltransferases and liver microsomes

Abstract

1.?UDP-glucuronosyltransferases (UGTs) are versatile and important conjugation enzymes in the metabolism of drugs and other xenobiotics.

2.?We have developed a convenient quantitative multi-well plate assay to measure the glucuronidation rate of 7-hydroxy-4-trifluoromethylcoumarin (HFC) for several UGTs.

3.?We have used this method to screen 11 recombinant human UGTs for HFC glucuronidation activity and studied the reaction kinetics with the most active enzymes. We have also examined the HFC glucuronidation activity of liver microsomes from human, pig, rabbit and rat.

4.?At a substrate concentration of 20?µM, the most active HFC glucuronidation catalysts were UGT1A10 followed by UGT1A6 >UGT1A7 >UGT2A1, whereas at 300?µM UGT1A6 was about 10 times better catalyst than the other recombinant UGTs. The activities of UGTs 1A3, 1A8, 1A9, 2B4 and 2B7 were low, whereas UGT1A1 and UGT2B17 exhibited no HFC glucuronidation activity. UGT1A6 exhibited a significantly higher V_max and Km values toward both HFC and UDP-glucuronic acid than the other UGTs.

5.?Human, pig and rabbit, but not rat liver microsomes, catalyzed HFC glucuronidation at high rates.

6.?This new method is particularly suitable for fast activity screenings of UGTs 1A6, 1A7, 1A10 and 2A1 and HFC glucuronidation activity determination from various samples.     

Characterization of hepatic and intestinal glucuronidation of magnolol: application of the relative activity factor approach to decipher the contributions of multiple UDP-glucuronosyltransferase isoforms

Magnolol is a food additive that is often found in mints and gums. Human exposure to this compound can reach a high dose; thus, characterization of magnolol disposition in humans is very important. Previous studies indicated that magnolol can undergo extensive glucuronidation in humans in vivo. In this study, in vitro assays were used to characterize the glucuronidation pathway in human liver and intestine. Assays with recombinant human UDP-glucuronosyltransferase enzymes (UGTs) revealed that multiple UGT isoforms were involved in magnolol glucuronidation, including UGT1A1, -1A3, -1A7, -1A8, -1A9, -1A10, and -2B7. Magnolol glucuronidation by human liver microsomes (HLM), human intestine microsomes (HIM), and most recombinant UGTs exhibited strong substrate inhibition kinetics. The degree of substrate inhibition was relatively low in the case of UGT1A10, whereas the reaction catalyzed by UGT1A9 followed biphasic kinetics. Chemical inhibition studies and the relative activity factor (RAF) approach were used to identify the individual UGTs that played important roles in magnolol glucuronidation in HLM and HIM. The results indicate that UGT2B7 is mainly responsible for the reaction in HLM, whereas UGT2B7 and UGT1A10 are significant contributors in HIM. In summary, the current study clarifies the glucuronidation pathway of magnolol and demonstrates that the RAF approach can be used as an efficient method for deciphering the roles of individual UGTs in a given glucuronidation pathway in the native tissue that is catalyzed by multiple isoforms with variable and atypical kinetics.     

Characterization of UDP-glucuronosyltransferases (UGTS) involved in the metabolism of troglitazone in rats and humans

UDP-glucuronosyltransferases (UGTs) involved in troglitazone glucuronidation in rats and humans have been characterized to support the previous toxicity study on troglitazone in Gunn rats and to examine whether the UGT polymorphism or inhibition of bilirubin metabolism is related to the clinically reported rare cases of liver failure. The experiments using Gunn rats revealed that UGT1 enzymes are not involved in troglitazone glucuronidation and that the responsible enzyme in rats was suggested to be UGT2B2, an androsterone UGT, by inhibition studies. In humans, contribution of UGT1A1 was estimated to be about 30% of the total troglitazone glucuronidation by UGTs, using human liver microsomes and recombinant UGTs. Other UGT1 and UGT2 enzymes seem to be responsible for the rest of the troglitazone glucuronidation in humans. The multiplicity of UGTs involved in troglitazone glucuronidation in humans may allow even patients lacking bilirubin UGT (UGT1A1) activity to produce troglitazone glucuronide. These observations suggest that the polymorphism of UGT is not the reason behind the liver failure induced by the troglitazone treatment, and troglitazone does not inhibit bilirubin glucuronidation in clinical treatment. In addition, the increased bilirubin level in the blood of patients who have troglitazone-induced liver failure is a consequence of liver injury and not due to inhibition of bilirubin glucuronidation by troglitazone.     

Prominent but reverse stereoselectivity in propranolol glucuronidation by human UDP-glucuronosyltransferases 1A9 and 1A10.

Propranolol is a nonselective beta-adrenergic blocker used as a racemic mixture in the treatment of hypertension, cardiac arrhythmias, and angina pectoris. For study of the stereoselective glucuronidation of this drug, the two propranolol glucuronide diastereomers were biosynthesized, purified, and characterized. A screen of 15 recombinant human UDP-glucuronosyltransferases (UGTs) indicated that only a few isoforms catalyze propranolol glucuronidation. Analysis of UGT2B4 and UGT2B7 revealed no significant stereoselectivity, but these two enzymes differed in glucuronidation kinetics. The glucuronidation kinetics of R-propranolol by UGT2B4 exhibited a sigmoid curve, whereas the glucuronidation of the same substrate by UGT2B7 was inhibited by substrate concentrations above 1 mM. Among the UGTs of subfamily 1A, UGT1A9 and UGT1A10 displayed high and, surprisingly, opposite stereoselectivity in the glucuronidation of propranolol enantiomers. UGT1A9 glucuronidated S-propranolol much faster than R-propranolol, whereas UGT1A10 exhibited the opposite enantiomer preference. Nonetheless, the Km values for the two enantiomers, both for UGT1A9 and for UGT1A10, were in the same range, suggesting similar affinities for the two enantiomers. Unlike UGT1A9, the expression of UGT1A10 is extrahepatic. Hence, the reverse stereoselectivity of these two UGTs may signify specific differences in the glucuronidation of propranolol enantiomers between intestine and liver microsomes. Subsequent experiments confirmed this hypothesis: human liver microsomes glucuronidated S-propranolol faster than R-propranolol, whereas human intestine microsomes glucuronidated S-propranolol faster. These findings suggest a contribution of intestinal UGTs to drug metabolism, at least for UGT1A10 substrates.     

Glucuronidation of the aspirin metabolite salicylic acid by expressed UDP-glucuronosyltransferases and human liver microsomes.

Acetylsalicylic acid (aspirin) is a common nonsteroidal anti-inflammatory drug used for treatment of pain and arthritis. In the body, acetylsalicylic acid is rapidly deacetylated to form salicylic acid. Both compounds have been proposed as anti-inflammatory agents. Major metabolites of salicylic acid are its acyl and phenolic glucuronide conjugates. Formation of these conjugates, catalyzed by UDP-glucuronosyltransferases (UGTs), decreases the amount of pharmacologically active salicylic acid present. We aimed to identify the UGTs catalyzing the glucuronidation of salicylic acid using both heterologously expressed enzymes and pooled human liver microsomes (HLMs) and to develop a liquid chromatography-tandem mass spectrometry method to quantify glucuronidation activity of UGTs 1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B15, and 2B17 Supersomes. All UGTs tested, except 1A4, 2B15, and 2B17, catalyzed salicylic acid phenolic and acyl glucuronidation. Ratios of salicylic acid phenolic to acyl glucuronide formation varied more than 12-fold from 0.5 for UGT1A6 to 6.1 for UGT1A1. These results suggest that all UGTs except 1A4, 2B15, and 2B17 might be involved in the glucuronidation of salicylic acid in vivo. From comparisons of apparent Km values determined in pooled HLMs and in expressed UGTs, UGT2B7 was suggested as a likely catalyst of salicylic acid acyl glucuronidation, whereas multiple UGTs were suggested as catalysts of phenolic glucuronidation. The results of this UGT screening may help target future evaluation of the effects of UGT polymorphisms on response to aspirin in clinical and population-based studies.     

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

Valproic acid glucuronidation kinetics were carried our 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 (2.3-5.2mM). 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+/-0.06mM). 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.     

Inhibition of paracetamol glucuronidation by tyrosine kinase inhibitors

AIMS

We aimed to investigate the effects of tyrosine kinase inhibitors (TKIs) on paracetamol (acetaminophen) glucuronidation.

METHODS

The inhibition of nine small molecule TKIs on paracetamol glucuronidation was investigated in human liver microsomes (HLMs) and recombinant human UDP-glucuronosyltransferases (UGTs).

RESULTS

Sorafenib, dasatinib and imatinib exhibited mixed inhibition against paracetamol glucuronidation in pooled HLMs, and potent inhibition in UGT1A9 and UGT2B15. Dasatinib and imatinib also inhibited UGT1A1-mediated paracetamol glucuronidation. Axitinib, erlotinib, gefitinib, lapatinib, nilotinib and vandetanib exhibited weak inhibition of paracetamol glucuronidation activity in HLMs.

CONCLUSIONS

The inhibition of paracetamol glucuronidation by TKIs might be of particular concern when they are co-administered.     

Glucuronidation of anabolic androgenic steroids by recombinant human UDP-glucuronosyltransferases.

A multidimensional study on the glucuronidation of anabolic androgenic steroids and their phase I metabolites by 11 recombinant human UDP-glucuronosyltransferases (UGTs) was carried out using liquid chromatographic-tandem mass spectrometric analyses. Large differences between the enzymes with respect to the conjugation profiles of the 11 tested aglycones were detected. Two UGTs, 1A6 and 1A7, did not exhibit measurable activity toward any of the aglycones that were examined in this study. Regioselectivity was demonstrated by UGTs 1A8, 1A9, and 2B15 that preferentially catalyzed hydroxyl glucuronidation at the 17beta-position. Most of the other enzymes glucuronidated hydroxyl groups at both the 3alpha- and the 17beta-positions. Clear stereoselectivity was observed in glucuronidation of diastereomeric nandrolone metabolites (5alpha-estran-3alpha-ol-17-one and 5beta-estran-3alpha-ol-17-one), whereas such specificity was not seen when analogous methyltestosterone metabolites were assayed. UGTs 1A1, 1A3, 1A4, 1A8, 1A9, 1A10, 2B4, 2B7, and 2B15 readily glucuronidated 5alpha-androstane-3alpha,17beta-diol, but none of them exhibited methyltestosterone glucuronidation activity. In agreement with the latter observations, we found that the methyltestosterone glucuronidation activity of human liver microsomes is extremely low, whereas in induced rat liver microsomes it was significantly higher. The homology among UGTs 1A7 to 1A10 at the level of amino acid sequence is very high, and it was thus surprising to find large differences in their activity toward this set of aglycones. Furthermore, the high activity of UGT1A8 and 1A10 toward some of the substrates indicates that extrahepatic enzymes might play a role in the metabolism of anabolic androgenic steroids.     

Bisphenol-A glucuronidation in human liver and breast: identification of UDP-glucuronosyltransferases (UGTs) and influence of genetic polymorphisms

1.?Bisphenol-A is a ubiquitous environmental contaminant that is primarily metabolized by glucuronidation and associated with various human diseases including breast cancer. Here we identified UDP-glucuronosyltransferases (UGTs) and genetic polymorphisms responsible for interindividual variability in bisphenol-A glucuronidation in human liver and breast.

2.?Hepatic UGTs showing the highest bisphenol-A glucuronidation activity included UGT2B15 and UGT1A9. Relative activity factor normalization indicated that UGT2B15 contributes?>80% of activity at bisphenol-A concentrations under 5?μM, while UGT1A9 contributes up to 50% of activity at higher concentrations.

3.?Bisphenol-A glucuronidation by liver microsomes (46 donors) ranged from 0.25 to 4.3 nmoles/min/mg protein. Two-fold higher glucuronidation (p?=?0.018) was observed in UGT1A9 *22/*22 livers compared with *1/*1 and *1/*22 livers. However, no associations were observed for UGT2B15*2 or UGT1A1*28 genotypes.

4.?Bisphenol-A glucuronidation by breast microsomes (15 donors) ranged from <0.2 to 56 fmoles/min/mg protein. Breast mRNA expression of UGTs capable of glucuronidating bisphenol-A was highest for UGT1A1, followed by UGT2B4, UGT1A9, UGT1A10, UGT2B7 and UGT2B15. Bisphenol-A glucuronidation was over 10-fold lower in breast tissues with the UGT1A1*28 allele compared with tissues without this allele (p?=?0.006).

5.?UGT2B15 and UGT1A9 contribute to glucuronidation variability in liver, while UGT1A1 is important in breast.     

Kinetics of acetaminophen glucuronidation by UDP-glucuronosyltransferases 1A1, 1A6, 1A9 and 2B15. Potential implications in acetaminophen-induced hepatotoxicity

The importance of uridine 5'-diphosphate-glucuronosyltranferases (UGT) 2B15 and other UGT enzymes (1A1, 1A6, and 1A9) in glucuronidating acetaminophen (APAP) is demonstrated. The kinetics and contributions of various UGTs in glucuronidating APAP are presented using clinically and toxicologically relevant concentrations of the substrate. UGT 1A9 and UGT 2B15 contribute significantly toward glucuronidating APAP when incubations were conducted in either phosphate or Tris-HCl buffers at 0.1 and 1.0 mM substrate concentrations. At 10 mM APAP, UGT 1A9 is a significant enzyme responsible for metabolizing APAP in either one of the buffers. UGT 1A1 is the next most important enzyme in glucuronidating APAP at this high substrate concentration. The contribution of UGT 1A6 at 10 mM APAP concentration became obscured by similar relative activities exhibited by UGTs 1A7, 1A8, and 2B7. These observations may reflect the differences in kinetic parameters for APAP glucuronidation by the individual UGTs. UGT 1A1 demonstrated Hill kinetics while UGT 1A9 displayed Michaelis-Menten kinetics. Substrate inhibition kinetics is observed with UGT 1A6, UGT 2B15, and human liver microsomes. The substrate inhibition is confirmed by employing stable isotope-labeled APAP as the substrate, while APAP glucuronide is used to test for inhibition of d4-APAP glucuronide. The in vitro hepatotoxicity caused by APAP in combination with phenobarbital or phenytoin is demonstrated in this study. The inhibition of APAP glucuronidation by phenobarbital leads to an increase in APAP-mediated toxicity in human hepatocytes. The toxicity to hepatocytes was further increased by coadministering APAP with phenytoin and phenobarbital. This synergistic increase in toxicity is postulated to be due to inhibition of UGTs (1A6, 1A9, and 2B15) responsible for detoxifying APAP through the glucuronidation pathway.     

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 microM) and high (200 microM) substrate concentrations. The ranking in order of activity at low and high substrate concentrations was UGT1A1 > UGT1A9 > UGT1A6 > UGT1A3 and UGT1A1 > UGT1A3 > UGT1A6 > or = 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 Km and Vmax of 35.9 microM and 134 pmol min(-1) mg(-1) protein, respectively. The UGT isoforms involved in SN-38 glucuronidation could be classified into two types: low-Km types such as UGT1A1 and UGT1A9, and high-Km types such as UGT1A3 and UGT1A6, in terms of affinity toward substrate. UGT1A1 had the highest Vmax followed by UGT1A3. Vmax 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 UMT1A9, 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.     

Glucuronidation of the oxidative cytochrome P450-mediated phenolic metabolites of the endocrine disruptor pesticide: methoxychlor by human hepatic UDP-glucuronosyl transferases.

Methoxychlor, a currently used pesticide, is a proestrogen exhibiting estrogenic activity in mammals in vivo. Methoxychlor undergoes oxidative metabolism by cytochromes P450, yielding 1,1,1-trichloro-2-(4-hydroxyphenyl)-2-(4-methoxyphenyl)ethane (mono-OH-M) and 1,1,1-trichloro-2,2-bis(4-hydroxyphenyl)ethane (bis-OH-M) as main metabolites. Since humans may be exposed to these estrogenic metabolites, which are potential substrates of UDP-glucuronosyltransferases (UGTs), their glucuronide conjugation was investigated with human liver preparations and individual UGTs. Incubation of both mono-OH-M and bis-OH-M with human liver microsomes formed monoglucuronides. The structures of the glucuronides were identified by liquid chromatography/tandem mass spectometry. Examination of cDNA-expressed recombinant human hepatic UGTs revealed that several catalyze glucuronidation of both compounds. Among the cDNA-expressed UGT1A enzymes, UGT1A9 seemed to be the main catalyst of formation of mono-OH-M-glucuronide, whereas UGT1A3 seemed to be the most active in bis-OH-M-glucuronide formation. Furthermore, the chiral selectivity of mono-OH-M glucuronidation was examined. The results of the incubation of single enantiomers generally agreed with the chiral analyses of mono-OH-M derived from the glucuronidase digestion of the glucuronides of the racemic mono-OH-M. There was a relatively slight but consistent enantioselective preference of individual UGT1A1, UGT1A3, UGT1A9, and UGT2B15 enzymes for glucuronidation of the S- over the R-mono-OH-M, whereas in human liver microsomes differences were observed among donors in generating the respective R/S-mono-OH-M ratio. Since it was previously shown that human liver microsomes demethylate methoxychlor mainly into S-mono-OH-M, the observation that UGT1A isoforms preferentially glucuronidate the S-mono-OH-M suggests a suitable mechanism for eliminating this major enantiomer. This enantiomeric preference, however, is not extended to all samples of human liver microsomes that we tested.     

The human UDP-glucuronosyltransferase UGT1A3 is highly selective towards N2 in the tetrazole ring of losartan, candesartan, and zolarsartan

Losartan, candesartan, and zolarsartan are AT(1) receptor antagonists that inhibit the effect of angiotensin II. We have examined their glucuronidation by liver microsomes from several animals and by recombinant human UDP-glucuronosyltransferases (UGTs). Large differences in the production of different glucuronide regioisomers of the three sartans were observed among liver microsomes from human (HLM), rabbit, rat, pig, moose, and bovine. However, all the liver microsomes produced one or two N-glucuronides in which either N1 or N2 of the tetrazole ring were conjugated. O-Glucuronides were also detected, including acyl glucuronides of zolarsartan and candesartan. Examination of individual human UGTs of subfamilies 1A and 2B revealed that N-glucuronidation activity is widespread, along with variable regioselectivity with respect to the tetrazole nitrogens of these sartans. Interestingly, UGT1A3 exhibited a strong regioselectivity towards the N2 position of the tetrazole ring in all three sartans. Moreover, the tetrazole-N2 of zolarsartan was only conjugated by UGT1A3, whereas the tetrazole-N1 of this aglycone was accessible to other enzymes, including UGT1A5. Zolarsartan O-glucuronide was mainly produced by UGTs 1A10 and 2B7. UGT2B7, alongside UGT1A3, glucuronidated candesartan at the tetrazole-N2 position, whereas UGTs 1A7-1A10 mainly yielded candesartan O-glucuronide. In the case of losartan, no O-glucuronide was generated by any tested human enzyme. Nevertheless, UGTs 1A1, 1A3, 1A10, 2B7, and 2B17 glucuronidated losartan at the tetrazole-N2, while UGT1A10 also yielded the respective N1-glucuronide. Kinetic analyses revealed that the main contributors to losartan glucuronidation in HLM are UGT1A1 and UGT2B7. The results provide ample new data on substrate specificity in drug glucuronidation.     

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.     

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.     

N-glucuronidation of the platelet-derived growth factor receptor tyrosine kinase inhibitor 6,7-(dimethoxy-2,4-dihydroindeno[1,2-C]pyrazol-3-yl)-(3-fluoro-phenyl)-amine by human UDP-glucuronosyltransferases.

The potential cancer therapeutic agent, 6,7-(dimethoxy-2, 4-dihydroindeno[1,2-c]pyrazol-3-yl)-(3-fluoro-phenyl)-amine (JNJ-10198409), formed three N-glucuronides that were positively identified by liquid chromatography-tandem mass spectrometry and NMR as N-amine-glucuronide (Glu-A), 1-N-pyrazole-glucuronide (Glu-B), and 2-N-pyrazole-glucuronide (Glu-C). All three N-glucuronides were detected in rat liver microsomes, whereas only Glu-A and -B were found in monkey and human liver microsomes. In contrast to common glucuronides, Glu-B was completely resistant to beta-glucuronidase. Kinetic analyses revealed that glucuronidation of JNJ-10198409 in human liver microsomes exhibited atypical kinetics that may be described by a two-site binding model. For the high affinity binding, K(m) values were 1.2 and 5.0 microM, and V(max) values were 2002 and 2,403 nmol min(-1) mg(-1) for Glu-A and Glu-B, respectively. Kinetic constants of low affinity binding were not determined due to low solubility of the drug. Among the human UDP-glucuronosyltransferases (UGTs) tested, UGT1A9, 1A8, 1A7, and 1A4 were the most active isozymes to produce Glu-A; for the formation of Glu-B, UGT1A9 was the most active enzyme, followed by UGT1A3, 1A7, and 1A4. Glucuronidation of JNJ-10198409 by those UGT1A enzymes followed classic Michaelis-Menten kinetics. In contrast, no glucuronides were formed by all UGT2B isozymes tested, including UGT2B4, 2B7, 2B15, and 2B17. Collectively, these results suggested that glucuronidation of JNJ-10198409 in human liver microsomes is catalyzed by multiple UGT1A enzymes. Since UGT1A enzymes are widely expressed in various tissues, it is anticipated that both hepatic and extrahepatic glucuronidation will likely contribute to the elimination of the drug in humans. Additionally, conjugation at the nitrogens of the pyrazole ring represents a new structural moiety for UGT1A-mediated reactions.     

Enantiomer Selective Glucuronidation of the Non‐Steroidal Pure Anti‐Androgen Bicalutamide by Human Liver and Kidney: Role of the Human UDP‐Glucuronosyltransferase (UGT)1A9 Enzyme

Bicalutamide (Casodex^®) is a non‐steroidal pure anti‐androgen used in the treatment of localized prostate cancer. It is a racemate drug, and its activity resides in the (R)‐enantiomer, with little in the (S)‐enantiomer. A major metabolic pathway for bicalutamide is glucuronidation catalysed by UDP‐glucuronosyltransferase (UGT) enzymes. While (S)bicalutamide is directly glucuronidated, (R)bicalutamide requires hydroxylation prior to glucuronidation. The contribution of human tissues and UGT isoforms in the metabolism of these enantiomers has not been extensively investigated. In this study, both (R) and/or (S)bicalutamide were converted into glucuronide (‐G) derivatives after incubation of pure and racemic solutions with microsomal extracts from human liver and kidney. Intestinal microsomes exhibited only low reactivity with these substrates. Km values of liver and kidney samples for (S)bicalutamide glucuronidation were similar, and lower than values obtained with the (R)‐enantiomer. Among the 16 human UGTs tested, UGT1A8 and UGT1A9 were able to form both (S) and (R)bicalutamide‐G from pure or racemic substrates. UGT2B7 was also able to form (R)bicalutamide‐G. Kinetic parameters of the recombinant UGT2B7, UGT1A8 and UGT1A9 enzymes support a predominant role of the UGT1A9 isoform in bicalutamide metabolism. Accordingly, (S)bicalutamide inhibited the ability of human liver and kidney microsomes to glucuronidate the UGT1A9 probe substrate, propofol. In conclusion, the present study provides the first comprehensive analysis of in vitro bicalutamide glucuronidation by human tissues and UGTs and identifies UGT1A9 as a major contributor for (R) and (S) glucuronidation in the human liver and kidney.     

Identification and characterization of human UDP-glucuronosyltransferases responsible for xanthotoxol glucuronidation

1.?Xanthotoxol is a furanocoumarin that possesses many pharmacological activities and in this study its in vitro glucuronidation was studied.

2.?Xanthotoxol can be rapidly metabolized to a mono-glucuronide in both human intestine microsomes (HIM) and human liver microsomes (HLM); the structure of the metabolite was confirmed by NMR spectroscopy.

3.?Reaction phenotyping with 12 commercial recombinant human UGTs, as well as with the Helsinki laboratory UGT1A10 that carry a C-terminal His-tag (UGT1A10-H), revealed that UGT1A10-H catalyzes xanthotoxol glucuronidation at the highest rate, followed by UGT1A8. The other enzymes, namely UGT1A3, UGT1A1, UGT1A6, UGT1A10 (commercial), and UGT2B7 displayed moderate-to-low reaction rates.

4.?In kinetic analyses, HIM exhibited much higher affinity for xanthotoxol, along with high V_max and mild substrate inhibition, whereas the kinetics in HLM was biphasic. UGT1A1 (high K_m value), UGT1A10-H (low K_m value), and UGT1A8 exhibited mild substrate inhibition.

5.?Considering the above findings and the current knowledge on UGTs expression in HIM, it is likely that UGT1A10 is mainly responsible for xanthotoxol glucuronidation in the human small intestine, with some contribution from UGT1A1. In the liver, this reaction is mainly catalyzed by UGT1A1 and UGT2B7.

6.?Glucuronidation appears to be the major metabolic pathway of xanthotoxol in human.