Species and tissue differences in serotonin glucuronidation

1.?Serotonin is a UGT1A6 substrate that is mainly found in the extrahepatic tissues where some UGT1As are expressed. The aim of the present study was to characterize serotonin glucuronidation in various tissues of humans and rodents.

2.?Serotonin glucuronidation in the human liver and kidney fitted to the Michaelis–Menten model, and the K_m values were similar to that of recombinant UGT1A6. However, serotonin glucuronidation in the human intestine fitted to the Hill equation, indicating that it is likely catalyzed not only by UGT1A6, but also by another UGT1A isoform. Serotonin glucuronidation in the rat liver, intestine and kidney fitted well to the Michaelis–Menten model and exhibited monophasic kinetics in the kidney, but biphasic kinetics in the liver and intestine. Furthermore, serotonin glucuronidation in the rat brain fitted best to the Hill equation. Serotonin glucuronidation in the mouse tissues fitted to the Michaelis–Menten model and exhibited monophasic kinetics in the liver and intestine microsomes, but biphasic kinetics in the kidney and brain microsomes.

3.?In conclusion, we clarified that tissue and species differences exist in serotonin glucuronidation. It is necessary to take these potential differences into account when considering the pharmacodynamics and pharmacokinetics of serotonin.     

In vitro inhibition of human UGT isoforms by ritonavir and cobicistat

1.?Ritonavir and cobicistat are pharmacokinetic boosting agents used to increase systemic exposure to other antiretroviral therapies. The manufacturer’s data suggests that cobicistat is a more selective CYP3A4 inhibitor than ritonavir. However, the inhibitory effect of ritonavir and cobicistat on human UDP glucuronosyltransferase (UGT) enzymes in Phase II metabolism is not established. This study evaluated the inhibition of human UGT isoforms by ritonavir versus cobicistat.

2.?Acetaminophen and ibuprofen were used as substrates to evaluate the metabolic activity of the principal human UGTs. Metabolite formation rates were determined by HPLC analysis of incubates following in vitro incubation of index substrates with human liver microsomes (HLMs) at different concentrations of ritonavir or cobicistat. Probenecid and estradiol served as positive control inhibitors.

3.?The 50% inhibitory concentrations (IC₅₀) of cobicistat and ritonavir were at least 50?µM, which substantially exceeds usual clinical plasma concentrations. Probenecid inhibited the glucuronidation of acetaminophen (IC₅₀ 0.7?mM), but not glucuronidation of ibuprofen. At relatively high concentrations, estradiol inhibited ibuprofen glucuronidation (IC₅₀ 17?µM).

4.?Ritonavir and cobicistat are unlikely to produce clinically important drug interactions involving drugs metabolized to glucuronide conjugates by UGT1A1, 1A3, 1A6, 1A9, 2B4 and 2B7.     

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.     

Raloxifene glucuronidation in liver and intestinal microsomes of humans and monkeys: contribution of UGT1A1, UGT1A8 and UGT1A9

1.?Raloxifene is an antiestrogen that has been marketed for the treatment of osteoporosis, and is metabolized into 6- and 4′-glucuronides by UDP-glucuronosyltransferase (UGT) enzymes. In this study, the in vitro glucuronidation of raloxifene in humans and monkeys was examined using liver and intestinal microsomes and recombinant UGT enzymes (UGT1A1, UGT1A8 and UGT1A9).

2.?Although the K_m and CL_int values for the 6-glucuronidation of liver and intestinal microsomes were similar between humans and monkeys, and species differences in V_max values (liver microsomes, humans?>?monkeys; intestinal microsomes, humans?<?monkeys) were observed, no significant differences were noted in the K_m or S₅₀, V_max and CL_int or CL_max values for the 4′-glucuronidation of liver and intestinal microsomes between humans and monkeys.

3.?The activities of 6-glucuronidation in recombinant UGT enzymes were UGT1A1?>?UGT1A8?>UGT1A9 for humans, and UGT1A8?>?UGT1A1?>?UGT1A9 for monkeys. The activities of 4′-glucuronidation were UGT1A8?>?UGT1A1?>?UGT1A9 in humans and monkeys.

4.?These results demonstrated that the profiles for the hepatic and intestinal glucuronidation of raloxifene by microsomes were moderately different between humans and monkeys.     

Molecular cloning and functional analysis of minipig UDP-glucuronosyltransferase 1A6

Abstract

1.?UDP-glucuronosyltransferase 1A6 (UGT1A6) plays important roles in the glucuronidation of numerous drugs, environmental pollutants, and endogenous substances. Minipigs have been used as experimental animals in pharmacological and toxicological studies because many of their physiological characteristics are similar to those of humans. The aim of the present study was to examine similarities and differences in the enzymatic properties of UGT1A6 between humans and minipigs.

2.?Minipig UGT1A6 (mpUGT1A6) cDNA was cloned by the RACE method, and the corresponding proteins were expressed in insect cells. The enzymatic function of mpUGT1A6 was analyzed by the kinetics of serotonin glucuronidation.

3.?Amino acid homology between human UGT1A6 (hUGT1A6) and mpUGT1A6 was 79.9%. The kinetics of serotonin glucuronidation by recombinant hUGT1A6 and mpUGT1A6 enzymes fit the Michaelis–Menten equation. The K_m, V_max, and CL_int values of hUGT1A6 were 10.5?mM, 4.04?nmol/min/mg protein, and 0.39?µL/min/mg protein, respectively. The K_m value of mpUGT1A6 was similar to that of hUGT1A6, whereas the V_max and CL_int values of mpUGT1A6 were approximately 2-fold higher than those of hUGT1A6.

4.?These results suggest that the enzymatic properties of UGT1A6 enzymes are moderately different between humans and minipigs.     

Glucuronidation of icaritin by human liver microsomes,human intestine microsomes and expressed UDP-glucuronosyltransferase enzymes: identification of UGT1A3, 1A9 and 2B7 as the main contributing enzymes

1.?Icaritin is a natural flavonoid with anti-osteoporosis activity. This study aimed to characterize icaritin glucuronidation by pooled human liver microsomes (HLM) and pooled human intestine microsomes (HIM), and to determine the contribution of individual UDP-glucuronosyltrans-ferase (UGT) enzyme to icaritin glucuronidation.

2.?Glucuronidation rates were determined by incubating icaritin with uridine diphosphate glucuronic acid (UDPGA)-supplemented microsomes. Kinetic parameters were derived by appropriate model fitting. Relative activity factors and activity correlation analysis were performed to identify main UGT isoforms.

3.?UGT1A3, 1A7, 1A8, 1A9 and 2B7 were mainly responsible for catalyzing the formation of two glucuronides (G1 and G2). Icaritin 3-O-glucuronidation (G1) was significantly correlated with Chenodeoxycholic acid (CDCA) glucuronidation (r?=?0.787, p?=?0.002), propofol glucuronidation (r?=?0.661, p?=?0.019) and Zidovudine (AZT) glucuronidation (r?=?0.805, p?=?0.002). Similarly, icaritin 7-O-glucuronidation (G2) was also correlated with CDCA glucuronidation (r?=?0.640, p?=?0.025), propofol glucuronidation (r?=?0.592, p?=?0.043) and AZT glucuronidation (r?=?0.661, p?=?0.019). In addition, UGT1A3, 1A9 and 2B7 contributed 37.5, 33.8 and 21.3% for G1 in pooled HLM, respectively. Also, UGT1A3, 1A9 and 2B7 contributed 34.3, 20.0 and 8.6% for G2 in pooled HLM, respectively.

4.?Icaritin was subjected to significant glucuronidation, wherein UGT1A3, 1A7, 1A8, 1A9 and 2B7 were main contributing enzymes.     

Structure- and isoform-specific glucuronidation of six curcumin analogs

1.?In the present study, we aimed to characterize the glucuronidation of six curcumin analogs (i.e. RAO-3, RAO-8, RAO-9, RAO-18, RAO-19, and RAO-23) derived from galangal using human liver microsomes (HLM) and twelve expressed UGT enzymes.

2.?Formation of glucuronide was confirmed using high-resolution mass spectrometry. Single glucuronide metabolite was generated from each of six curcumin analogs. The fragmentation patterns were analyzed and were found to differ significantly between alcoholic and phenolic glucuronides.

3.?All six curcumin analogs except one (RAO-23) underwent significant glucuronidation in HLM and expressed UGT enzymes. In general, the methoxy group (close to the phenolic hydroxyl group) enhanced the glucuronidation liability of the curcumin analogs.

4.?UGT1A9 and UGT2B7 were primarily responsible for the glucuronidation of two alcoholic analogs (RAO-3 and RAO-18). By contrast, UGT1A9 and four UGT2Bs (UGT2B4, 2B7, 2B15 and 2B17) played important roles in conjugating three phenolic analogs (RAO-8, RAO-9, and RAO-19). Interestingly, the conjugated double bonds system (in the aliphatic chain) was crucial to the substrate selectivity of gastrointestinal UGTs (i.e. UGT1A7, 1A8 and 1A10).

5.?In conclusion, glucuronidation of six curcumin analogs from galangal were structure- and isoform-specific. The knowledge should be useful in identifying a curcumin analog with improved metabolic property.     

Human UGT2B7 is the major isoform responsible for the glucuronidation of clopidogrel carboxylate

Clopidogrel is predominantly hydrolyzed to clopidogrel carboxylic acid (CCA) by carboxylesterase 1, and subsequently CCA is glucuronidated to clopidogrel acyl glucuronide (CAG) by uridine diphosphate‐glucuronosyltransferases (UGTs); however, the UGT isoenzymes glucuronidating CCA remain unidentified to date. In this study, the glucuronidation of CCA was screened with pooled human liver microsomes (HLMs) and 7 human recombinant UGT (rUGT) isoforms. Results indicated that rUGT2B7 exhibited the highest catalytical activity for the CCA glucuronidation as measured with a mean V_max value of 120.9 pmol/min/mg protein, 3‐ to 12‐fold higher than that of the other rUGT isoforms tested. According to relative activity factor approach, the relative contribution of rUGT2B7 to CCA glucuronidation was estimated to be 58.6%, with the minor contributions (3%) from rUGT1A9. Moreover, the glucuronidation of CCA followed Michaelis‐Menten kinetics with a mean K_m value of 372.9 μM and 296.4 μM for pooled HLMs and rUGT2B7, respectively, showing similar affinity for both. The formation of CAG was significantly inhibited by azidothymidine and gemfibrozil (well‐characterized UGT2B7 substrates) in a concentration‐dependent manner, or by fluconazole (a typical UGT2B7‐selective inhibitor) in a time‐dependent manner, for both HLMs and rUGT2B7, respectively. In addition, CCA inhibited azidothymidine glucuronidation (catalyzed almost exclusively by UGT2B7) by HLMs and rUGT2B7 in a concentration‐dependent manner, indicating that CCA is a substrate of UGT2B7. These results reveal that UGT2B7 is the major enzyme catalyzing clopidogrel glucuronidation in the human liver, and that there is the potential for drug‐drug interactions between clopidogrel and the other substrate drugs of UGT2B7.     

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 K_m values in both instances were similar with 1.06?mM for the 1A9 and 0.97?mM for the 2B7. V_max 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 K_m 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.     

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.     

CYP450 1A2 and multiple UGT1A isoforms are responsible for jatrorrhizine metabolism in human liver microsomes

Jatrorrhizine, one of the protoberberine alkaloids derived from the plant Coptis chinensis, is expected to be developed as a new gastric prokinetic drug, but its metabolic characteristics in humans remain unknown. This study characterized the phase I and phase II metabolites, metabolic kinetics, and cytochrome P450 (CYP) and UDP‐glucuronosyltransferase (UGT) enzymes responsible for the metabolism of jatrorrhizine in human liver microsomes (HLMs). Chemical inhibition in HLMs and metabolism by recombinant human CYP or UGT enzymes were employed to determine the key metabolic enzyme subtypes. In HLMs, demethyleneberberine (demethylated product) and jatrorrhizine glucuronide were identified as the phase I and phase II metabolites, respectively. The enzyme kinetics for both demethylation and glucuronidation were fitted to the Michaelis–Menten equation. Demethylation was inhibited significantly by furafylline and predominantly catalysed by recombinant CYP1A2, whereas glucuronidation was inhibited by silibinin, quercetin, as well as 1‐naphthol and catalysed by recombinant UGT1A1, UGT1A3, UGT1A7, UGT1A8, UGT1A9 and UGT1A10. These results showed that jatrorrhizine is metabolized by human CYP1A2 and multiple UGT1A isoforms. Copyright © 2013 John Wiley & Sons, Ltd.     

In vitro glucuronidation of aprepitant: a moderate inhibitor of UGT2B7

Abstract

1.?Aprepitant, an oral antiemetic, commonly used in the prevention of chemotherapy-induced nausea and vomiting, is primarily metabolized by CYP3A4. Aprepitant glucuronidation has yet to be evaluated in humans. The contribution of human UDP-glucuronosyltransferase (UGT) isoforms to the metabolism of aprepitant was investigated by performing kinetic studies, inhibition studies and correlation analyses. In addition, aprepitant was evaluated as an inhibitor of UGTs.

2.?Glucuronidation of aprepitant was catalyzed by UGT1A4 (82%), UGT1A3 (12%) and UGT1A8 (6%) and K_ms were 161.6?±?15.6, 69.4?±?1.9 and 197.1?±?28.2?µM, respectively. Aprepitant glucuronidation was significantly correlated with both UGT1A4 substrates anastrazole and imipramine (r_s?=?0.77, p?<?0.0001 for both substrates; n?=?44), and with the UGT1A3 substrate thyroxine (r_s?=?0.58, p?<?0.0001; n?=?44).

3.?We found aprepitant to be a moderate inhibitor of UGT2B7 with a K_i of ～10?µM for 4-MU, morphine and zidovudine. Our results suggest that aprepitant can alter clearance of drugs primarily eliminated by UGT2B7. Given the likelihood for first-pass metabolism by intestinal UGT2B7, this is of particular concern for oral aprepitant co-administered with oral substrates of UGT2B7, such as zidovudine and morphine.     

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.     

Glucuronidation of macelignan by human liver microsomes and expressed UGT enzymes: identification of UGT1A1 and 2B7 as the main contributing enzymes

Macelignan is a natural phenolic compound that possesses many types of health benefits such as antiinflammation. This study aimed to characterize the metabolism of macelignan via the glucuronidation pathway and to identify the main UGT enzymes involved in macelignan glucuronidation. The rates of glucuronidation were determined by incubating macelignan with UDPGA‐supplemented microsomes. Kinetic parameters were derived by fitting an appropriate model to the data. Reaction phenotyping, the relative activity factor (RAF) approach and activity correlation analysis were employed to identify the main UGT enzymes contributing to the hepatic metabolism of macelignan. Glucuronidation of macelignan in pooled human liver microsomes (pHLM) was rather efficient with a high CL_int (the intrinsic clearance) value of 13.90 ml/min/mg. All UGT enzymes, except UGT1A4, 1A6 and 2B10, showed metabolic activities toward macelignan. UGT1A1 and 2B7 were the enzymes with the highest activities; the CL_int values were 4.92 and 2.13 ml/min/mg, respectively. Further, macelignan glucuronidation was significantly correlated with 3‐O‐glucuronidation of β‐estradiol (r = 0.69; p < 0.01) and glucuronidation of zidovudine (r = 0.60; p < 0.05) in a bank of individual HLMs (n = 14). Based on the RAF approach, UGT1A1 and 2B7, respectively, contributed 55.40% and 32.20% of macelignan glucuronidation in pHLM. In conclusion, macelignan was efficiently metabolized via the glucuronidation pathway. It was also shown that UGT1A1 and 2B7 were probably the main contributors to the hepatic glucuronidation of macelignan. Copyright © 2014 John Wiley & Sons, Ltd.     

Stereoselective glucuronidation and hydroxylation of etodolac by UGT1A9 and CYP2C9 in man

1.?In vitro metabolic studies with etodolac were performed. S- and R-etodolac were converted to the acylglucuronide and hydroxylated metabolites by UDP-glucuronosyltransferase (UGT) and cytochrome P450 in microsomes. However, the stereoselectivities of UGT and P450 for the isomers were opposite. S-etodolac was glucuronidated preferentially than R-etodolac by UGT. In contrast, R-etodolac was hydroxylated preferentially than S-etodolac by P450.

2.?Of several human P450 enzymes, CYP2C9 had the greatest activity for hydroxylation of R-etodolac. Sulfaphenazole, an inhibitor of CYP2C9, and anti-CYP2C9 antibody inhibited the hydroxylation of R-etodolac in human liver microsomes. CYP2C9 therefore contributes to the stereoselective hydroxylation of R-etodolac.

3.?Of several human UGT enzymes, UGT1A9 had the greatest activity for glucuronidation of S-etodolac. Propofol and thyroxine, inhibitors of UGT1A9, inhibited the glucuronidation of S-etodolac in human liver microsomes. Therefore, UGT1A9 is mainly responsible for the stereoselective glucuronidation of S-etodolac.

4.?Because S-etodolac was metabolized more rapidly than R-etodolac in human cryopreserved hepatocytes, the stereoselectivities of UGT1A9 for etodolac substantially influenced the overall metabolism of S- and R-etodolac in man.     

Identification of UDP-glucuronosyltransferase isoforms responsible for leonurine glucuronidation in human liver and intestinal microsomes

Abstract

1.?Leonurine is a potent component of herbal medicine Herba leonuri. The detail information on leonurine metabolism in human has not been revealed so far.

2.?Two primary metabolites, leonurine O-glucuronide and demethylated leonurine, were observed and identified in pooled human liver microsomes (HLMs) and O-glucuronide is the predominant one.

3.?Among 12 recombinant human UDP-glucuronosyltransferases (UGTs), UGT1A1, UGT1A8, UGT1A9, and UGT1A10 showed catalyzing activity toward leonurine glucuronidation. The intrinsic clearance (CL_int) of UGT1A1 was approximately 15-to 20-fold higher than that of UGT1A8, UGT1A9, and UGT1A10, respectively. Both chemical inhibition study and correlation study demonstrated that leonurine glucuronidation activities in HLMs had significant relationship with UGT1A1 activities.

4.?Leonurine glucuronide was the major metabolite in human liver microsomes. UGT1A1 was principal enzyme that responsible for leonurine glucuronidation in human liver and intestine microsomes.     

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.     

Glucuronidation of piceatannol by human liver microsomes: major role of UGT1A1, UGT1A8 and UGT1A10

Objectives Piceatannol, a dietary polyphenol present in grapes and wine, is known for its promising anticancer and anti‐inflammatory activity. The aim of this study was to analyse the concentration‐dependent glucuronidation of piceatannol in vitro. Methods To determine the glucuronidation of piceatannol, experiments were conducted with human liver microsomes as well as using a panel of 12 recombinant UDP‐glucuronosyltransferase isoforms. Furthermore, the chemical structures of novel glucuronides were identified by liquid chromatography‐tandem mass spectrometry (LC‐MS/MS). Key findings Along with piceatannol it was possible to identify three metabolites whose structures were identified by LC‐MS/MS as piceatannol monoglucuronides (M1–M3). Formation of M1 and M3 exhibited a pattern of substrate inhibition, with apparent K_i and V_max/K_m values of 103 ± 26.6 µm and 3.8 ± 1.3 µl/mg protein per min, respectively, for M1 and 233 ± 61.4 µm and 19.8 ± 9.5 µl/mg protein per min, respectively, for M3. In contrast, formation of metabolite M2 followed classical Michaelis–Menten kinetics, with a K_m of 18.9 ± 8.1 µm and a V_max of 0.21 ± 0.02 nmol/mg protein per min. Incubation in the presence of human recombinant UDP‐glucuronosyltransferases (UGTs) demonstrated that M1 was formed nearly equally by UGT1A1 and UGT1A8. M2 was preferentially catalysed by UGT1A10 and to a lesser extent by UGT1A1 and UGT1A8. The formation of M3, however, was mainly catalysed by UGT1A1 and UGT1A8. Conclusions Our results elucidate the importance of piceatannol glucuronidation in the human liver, which must be taken into account in humans after dietary intake of piceatannol.     

Everolimus-inhibited multiple isoforms of UDP-glucuronosyltransferases (UGTs)

1.?Everolimus is an inhibitor of mammalian target of rapamycin (mTOR) and has been clinically utilized to prevent the rejection of organ transplants. This study aims to determine the inhibition of everolimus on the activity of phase-II drug-metabolizing enzymes UDP-glucuronosyltransferases (UGTs).

2.?The results showed that 100 μM of everolimus exerted more than 80% inhibition toward UGT1A1, UGT-1A3 and UGT-2B7. UGT1A3 and UGT2B7 were selected to elucidate the inhibition mechanism, and in silico docking showed that hydrogen bonds and hydrophobic interactions mainly contributed to the strong binding of everolimus toward the activity cavity of UGT1A3 and UGT2B7. Inhibition kinetic-type analysis using Lineweaver–Burk plot showed competitive inhibition toward all these UGT isoforms. The inhibition kinetic parameters (K_i) were calculated to be 2.3, 0.07 and 4.4 μM for the inhibition of everolimus toward UGT1A1, UGT-1A3 and UGT-2B7, respectively.

3.?In vitro–in vivo extrapolation (IVIVE) showed that [I]/K_i value was calculated to be 0.004, 0.14 and 0.002 for UGT1A1, UGT-1A3 and UGT-2B7, respectively. Therefore, high DDI potential existed between everolimus and clinical drugs mainly undergoing UGT1A3-catalyzed glucuronidation.     

In vitro metabolism of the anti-inflammatory clerodane diterpenoid polyandric acid A and its hydrolysis product by human liver microsomes and recombinant cytochrome P450 and UDP-glucuronosyltransferase enzymes

1.?The metabolism of the anti-inflammatory diterpenoid polyandric acid A (PAA), a constituent of the Australian Aboriginal medicinal plant Dodonaea polyandra, and its de-esterified alcohol metabolite, hydrolysed polyandric acid A (PAAH) was studied in vitro using human liver microsomes (HLM) and recombinant UDP-glucuronosyltransferase (UGT) and cytochrome P450 (CYP) enzymes.

2.?Hydrolysis of PAA to yield PAAH occurred upon incubation with HLM. Further incubations of PAAH with HLM in the presence of UGT and CYP cofactors resulted in significant depletion, with UGT-mediated depletion as the major pathway.

3.?Reaction phenotyping utilising selective enzyme inhibitors and recombinant human UGT and CYP enzymes revealed UGT2B7 and UGT1A1, and CYP2C9 and CYP3A4 as the major enzymes involved in the metabolism of PAAH.

4.?Analysis of incubations of PAAH with UDP-glucuronic acid-supplemented HLM and recombinant enzymes by UPLC/MS/MS identified three glucuronide metabolites. The metabolites were further characterised by β-glucuronidase and mild alkaline hydrolysis. The acyl glucuronide of PAAH was shown to be the major metabolite.

5.?This study demonstrates the in vitro metabolism of PAA and PAAH and represents the first systematic study of the metabolism of an active constituent of an Australian Aboriginal medicinal plant.