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.     

Identification of the human liver UDP-glucuronosyltransferase involved in the metabolism of p-ethoxyphenylurea (dulcin)

Dulcin (DL), now banned, was once a widely used artificial sweetener. DL possesses an ureido group that is metabolized by direct glucuronidation in rabbit liver microsomes. Dulcin N-glucuronide (DNG) is the only type of ureido N-glucuronide known to date; ureido glucuronidation in humans has not been previously reported. Accordingly, the glucuronidation of DL was studied using human liver microsomes (HLM) and expressed human UDP-glucuronosyltransferase (UGT) enzymes. The average K _m and V _max values from nine HLM samples were 2.10 mM and 0.156 nmol/mg/min, respectively. Of the six human UGT isoforms screened for their ability to glucuronidate DL, only UGT1A1 and UGT1A9 showed activity. The apparent K _m values using UGT1A1 and UGT1A9 were 5.06 and 6.99 mM, and the apparent V _max values were 0.0461 and 0.106 nmol/min/mg, respectively. Phenolphthalein, a substrate for UGT1A9, inhibited DL glucuronidation in HLM competitively (K _i = 0.356 mM), but bilirubin, a substrate for UGT1A1, did not. These results suggest that UGT1A9 is a key enzyme catalyzing the glucuronidation of DL.     

Glycosidation of an endothelin ET(A) receptor antagonist and diclofenac in human liver microsomes: aglycone-dependent UDP-sugar selectivity.

Following the finding that UGT2B7 catalyzes the transfer of the glycosyl group from both UDP-glucuronic acid (UDP-GlcA) and UDP-glucose (UDP-Glc) to an endothelin ET(A) receptor antagonist, Compound A [(+)-(5S,6R,7R)-2-isopropylamino-7-[4-methoxy-2-((2R)-3-methoxy-2-methylpropyl)-5-(3,4-methylenedioxyphenyl)cyclopenteno[1,2-b] pyridine 6-carboxylic acid], to form an acyl glucuronide and a glucoside (Tang et al., 2003), two additional nucleotide sugars [UDP-galactose (UDP-gal) and UDP-N-acetyl glucosamine (UDP-GlcNAc)] were examined as cosubstrates in human liver microsomes. It was found that UDP-gal, but not UDP-GlcNAc, also served as a sugar donor primarily through catalysis by UGT2B7, although at a significantly reduced catalytic rate. These three UDP-sugars showed pH-dependent kinetics and appeared to compete with each other, with IC50 values parallel to their respective apparent K(m) values. In contrast, only UDP-GlcA served as the sugar donor for the conjugation of diclofenac, a known UGT2B7 substrate, with an apparent K(m) for UDP-GlcA of 96 +/- 17 microM. UDP-Glc and UDP-gal, two futile sugar donors for diclofenac, were found to competitively inhibit the glucuronidation of this aglycone. Different from the case with Compound A, UDP-Glc and UDP-gal displayed K(i) values of 2054 +/- 108 microM and 1277 +/- 149 microM, >10-fold greater than the K(m) for UDP-GlcA, indicating that these two nucleotide sugars were also capable of binding to the enzyme but with a lower affinity. The findings of this study indicate that the selectivity of UGT2B7 toward UDP-sugars is aglycone-dependent. With Compound A as the acceptor substrate, human UGT2B7 becomes more accommodative in the transfer of the glycosyl group from UDP-sugars beyond UDP-GlcA. The mechanism may involve enzyme conformational changes associated with Compound A binding to the enzyme.     

Contribution of UDP-glucuronosyltransferases 1A9 and 2B7 to the glucuronidation of indomethacin in the human liver

Objective We characterized the kinetics of indomethacin glucuronidation by recombinant UDP-glucuronosyltransferase (UGT) isozymes and human liver microsomes (HLM) and identified the human UGT isozymes involved. Methods Indomethacin glucuronidation was investigated using HLM and recombinant human UGT isozymes. Human UGTs involved in indomethacin glucuronidation were assessed in kinetic studies, chemical inhibition studies, and correlation studies. Results Among the UGT isozymes investigated, UGT1A1, 1A3, 1A9, and 2B7 showed glucuronidation activity for indomethacin, with UGT1A9 possessing the highest activity, followed by UGT2B7. Glucuronidation of indomethacin by recombinant UGT1A9 and 2B7 showed substrate inhibition kinetics with K _m values of 35 and 32 μM, respectively. The glucuronidation of indomethacin was significantly correlated with morphine 3OH-glucuronidation (r = 0.69, p < 0.05) and 3′-azido-3′-deoxythymidine glucuronidation (r = 0.82, p < 0.05), a reaction mainly catalyzed by UGT2B7. Propofol inhibited indomethacin glucuronidation in HLM with an IC₅₀ value of 248 μM, which is between the IC₅₀ value in recombinant UGT1A9 (106 μM) and UGT2B7 (> 400 μM). Conclusions These findings suggest that UGT2B7 plays a predominant role in indomethacin glucuronidation in the human liver and that UGT1A9 is partially involved.     

Species‐ and gender‐dependent differences in the glucuronidation of a flavonoid glucoside and its aglycone determined using expressed UGT enzymes and microsomes

Flavonoids occur naturally as glucosides and aglycones. Their common phenolic hydroxyl groups may trigger extensive UDP‐glucuronosyltransferase (UGT)‐ catalysed metabolism. Unlike aglycones, glucosides contain glucose moieties. However, the influence of these glucose moieties on glucuronidation of glucosides and aglycones remains unclear. In this study, the flavonoid glucoside tilianin and its aglycone acacetin were used as model compounds. The glucuronidation characteristics and enzyme kinetics of tilianin and acacetin were compared using human UGT isoforms, liver microsomes and intestinal microsomes obtained from different animal species. Tilianin and acacetin were metabolized into different glucuronides, with UGT1A8 produced as the main isoform. Assessment of enzyme kinetics in UGT1A8, human liver microsomes and human intestinal microsomes revealed that compared with tilianin, acacetin displayed lower K_m (0.6‐, 0.7‐ and 0.6‐fold, respectively), higher V_max (20‐, 60‐ and 230‐fold, respectively) and higher clearance (30‐, 80‐ and 300‐fold, respectively). Furthermore, glucuronidation of acacetin and tilianin showed significant species‐ and gender‐dependent differences. In conclusion, glucuronidation of flavonoid aglycones is faster than that of glucosides in the intestine and the liver. Understanding the metabolism and species‐ and gender‐dependent differences between glucosides and aglycones is crucial for the development of drugs from flavonoids. Copyright © 2015 John Wiley & Sons, Ltd.     

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.     

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.     

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.     

Mutual inhibition between carvedilol enantiomers during racemate glucuronidation mediated by human liver and intestinal microsomes

Carvedilol is administered orally as a racemic mixture of R(+)- and S(-)-enantiomers for treatment of angina pectoris, hypertension and chronic heart failure. We have reported that enzyme kinetic parameters for carvedilol glucuronidation by human liver microsomes (HLM) differed greatly depending on the substrate form, namely, racemic carvedilol and each enantiomer. These phenomena were thought to be caused by mutual inhibition between carvedilol enantiomers during racemate glucuronidation. The aim of this study was to clarify the mechanism of these phenomena in HLM and human intestinal microsomes (HIM) and its relevance to uridine 5'-diphosphate (UDP)-glucuronosyl transferase (UGT) 1A1, UGT2B4 and UGT2B7, which mainly metabolize carvedilol directly in phase II enzymes. HLM apparently preferred metabolizing (S)-carvedilol to (R)-carvedilol in the racemate, but true activities of HLM for both glucuronidation were approximately equal. By determination of the inhibitory effects of (S)-carvedilol on (R)-carvedilol glucuronidation and vice versa, it was shown that (R)-carvedilol glucuronidation was more easily inhibited than was (S)-carvedilol glucuronidation. UGT2B7 was responsible for (S)-carvedilol glucuronidation in HLM. Ratios of contribution to (R)-carvedilol glucuronidation were approximately equal among UGT1A1, UGT2B4 and UGT2B7. However, enzyme kinetic parameters were different between the two lots of HLM used in this study, depending on the contribution ratio of UGT2B4, in which (R)-glucuronidation was much more easily inhibited by (S)-carvedilol than was (S)-glucuronidation by (R)-carvedilol. Meanwhile, HIM preferred metabolizing (R)-carvedilol, and this tendency was not different between the kinds of substrate form.     

Assessment of UDP-glucuronosyltransferase catalyzed formation of Picroside II glucuronide in microsomes of different species and recombinant UGTs

1. This study compared the hepatic glucuronidation of Picroside II in different species and characterized the glucuronidation activities of human intestinal microsomes (HIMs) and recombinant human UDP-glucuronosyltransferases (UGTs) for Picroside II.

2. The rank order of hepatic microsomal glucuronidation activity of Picroside II was rat > mouse > human > dog. The intrinsic clearance of Picroside II hepatic glucuronidation in rat, mouse and dog was about 10.6-, 6.0- and 2.3-fold of that in human, respectively.

3. Among the 12 recombinant human UGTs, UGT1A7, UGT1A8, UGT1A9 and UGT1A10 catalyzed the glucuronidation. UGT1A10, which are expressed in extrahepatic tissues, showed the highest activity of Picroside II glucuronidation (K_m?=?45.1 μM, V_max?=?831.9 pmol/min/mg protein). UGT1A9 played a primary role in glucuronidation in human liver microsomes (HLM; K_m?=?81.3 μM, V_max?=?242.2 pmol/min/mg protein). In addition, both mycophenolic acid (substrate of UGT1A9) and emodin (substrate of UGT1A8 and UGT1A10) could inhibit the glucuronidation of Picroside II with the half maximal inhibitory concentration (IC₅₀) values of 173.6 and 76.2 μM, respectively.

4. Enzyme kinetics was also performed in HIMs. The K_m value of Picroside II glucuronidation was close to that in recombinant human UGT1A10 (K_m?=?58.6 μM, V_max?=?721.4 pmol/min/mg protein). The intrinsic clearance was 5.4-fold of HLMs. Intestinal UGT enzymes play an important role in Picroside II glucuronidation in human.

     

New Insights into SN‐38 Glucuronidation: Evidence for the Important Role of UDP Glucuronosyltransferase 1A9

Glucuronidation of SN‐38 serves as an important metabolic pathway in determining the toxic effects of irinotecan. The role of UDP‐glucuronosyltransferases (UGT) 1A9 in SN‐38 glucuronidation pathway is very confusing. This study re‐investigates the pathway through testing effects of bovine serum albumin (BSA) and the selective inhibitor on SN‐38 glucuronidation in pooled human liver microsomes (HLM) and recombinant UGT1A1/UGT1A9. For UGT1A1, SN‐38 glucuronidation was little affected by BSA. Whether in the presence of BSA or not, the reactions both obey Michaelis–Menten kinetics with closed Vmax/Km values. For UGT1A9 and HLM, BSA can significantly accelerate SN‐38 glucuronidation activities and similar effects are further observed on kinetic patterns. In the absence of BSA, reactions by HLM and UGT1A9 both display substrate inhibition kinetics. When BSA is included in the incubations, the reactions exhibit Michaelis–Menten kinetics. To get the true contribution of UGT1A9 in SN‐38 glucuronidation, a relative activity factor (RAF) approach was additionally used. It is suggested that UGT1A9 and 1A1 contribute equally to SN‐38 glucuronidation in HLM. Furthermore, in the presence of BSA, magnolol, a selective UGT1A9 inhibitor, displays moderate inhibition against HLM. Results together conclude that UGT1A9 serves as an additional important contributor to hepatic SN‐38 glucuronidation.     

Metabolism of the anthelmintic drug niclosamide by cytochrome P450 enzymes and UDP-glucuronosyltransferases: metabolite elucidation and main contributions from CYP1A2 and UGT1A1

1. Niclosamide is an old anthelmintic drug that shows potential in fighting against cancers. Here, we characterized the metabolism of niclosamide by cytochrome P450 enzymes (CYPs) and UDP-glucuronosyltransferases (UGTs) using human liver microsomes (HLM) and expressed enzymes.

2. NADPH-supplemented HLM (and liver microsomes from various animal species) generated one hydroxylated metabolite (M1) from niclosamide; and UDPGA-supplemented liver microsomes generated one mono-O-glucuronide (M2). The chemical structures of M1 (3-hydroxy niclosamide) and M2 (niclosamide-2-O-glucuronide) were determined through LC–MS/MS and/or NMR analyses.

3. Reaction phenotyping revealed that CYP1A2 was the main enzyme responsible for M1 formation. The important role of CYP1A2 in niclosamide metabolism was further confirmed by activity correlation analyses as well as inhibition experiments using specific inhibitors.

4. Although seven UGT enzymes were able to catalyze glucuronidation of niclosamide, UGT1A1 and 1A3 were the enzymes showed the highest metabolic activities. Activity correlation analyses demonstrated that UGT1A1 played a predominant role in hepatic glucuronidation of niclosamide, whereas the role of UGT1A3 was negligible.

5. In conclusion, niclosamide was subjected to efficient metabolic reactions hydroxylation and glucuronidation, wherein CYP1A2 and UGT1A1 were the main contributing enzymes, respectively.     

Quantitative prediction of in vivo inhibitory interactions involving glucuronidated drugs from in vitro data: the effect of fluconazole on zidovudine glucuronidation

AIMS: Using the fluconazole-zidovudine (AZT) interaction as a model, to determine whether inhibition of UDP-glucuronosyltransferase (UGT) catalysed drug metabolism in vivo could be predicted quantitatively from in vitro kinetic data generated in the presence and absence bovine serum albumin (BSA). METHODS: Kinetic constants for AZT glucuronidation were generated using human liver microsomes (HLM) and recombinant UGT2B7, the principal enzyme responsible for AZT glucuronidation, as the enzyme sources with and without fluconazole. K(i) values were used to estimate the decrease in AZT clearance in vivo. RESULTS: Addition of BSA (2%) to incubations decreased the K(m) values for AZT glucuronidation by 85-90% for the HLM (923 +/- 357 to 91 +/- 9 microm) and UGT2B7 (478-70 microm) catalysed reactions, with little effect on V(max). Fluconazole, which was shown to be a selective inhibitor of UGT2B7, competitively inhibited AZT glucuronidation by HLM and UGT2B7. Like the K(m), BSA caused an 87% reduction in the K(i) for fluconazole inhibition of AZT glucuronidation by HLM (1133 +/- 403 to 145 +/- 36 microm) and UGT2B7 (529 to 73 microm). K(i) values determined for fluconazole using HLM and UGT2B7 in the presence (but not absence) of BSA predicted an interaction in vivo. The predicted magnitude of the interaction ranged from 41% to 217% of the reported AUC increase in patients, depending on the value of the in vivo fluconazole concentration employed in calculations. CONCLUSIONS: K(i) values determined under certain experimental conditions may quantitatively predict inhibition of UGT catalysed drug glucuronidation in vivo.     

Identification of enzymes responsible for the N-oxidation of darexaban glucuronide, the pharmacologically active metabolite of darexaban, and the glucuronidation of darexaban N-oxides in human liver microsomes

Darexaban maleate is a novel oral direct factor Xa inhibitor. Darexaban glucuronide (YM-222714) was the major component in plasma after oral administration of darexaban to humans and is the pharmacologically active metabolite. Additionally, YM-222714 N-oxides were detected as minor metabolites in human plasma and urine. It is possible that YM-222714 N-oxides are formed by the N-oxidation of YM-222714 and/or the glucuronidation of darexaban N-oxides (YM-542845) in vivo. The former reaction is the pharmacological inactivation process. In this study, we identified the human enzymes responsible for YM-222714 N-oxidation and the uridine 5'-diphosphate (UDP)-glucuronosyltransferase (UGT) isoforms involved in YM-542845 glucuronidation in vitro. YM-222714 N-oxidation activity was detected in human liver microsomes (HLM), but not in human intestinal microsomes. In HLM, YM-222714 N-oxidation activities were significantly correlated with flavin-containing monooxygenase (FMO) marker enzyme activities (p<0.001) and inhibited by methimazole, a typical inhibitor of FMOs. Recombinant human FMO3 and FMO1 were capable of efficiently catalyzing YM-222714 N-oxidation, but not FMO5 or any recombinant human cytochrome P450 (CYP) isoforms. Considering the mRNA expression levels of FMO isoforms in human liver, these results strongly suggest that YM-222714 N-oxidation in HLM is mainly catalyzed by FMO3. In HLM, YM-542845 glucuronidation was strongly inhibited by typical substrates for UGT1A8, UGT1A9, and UGT1A10. Recombinant human UGT1A7, UGT1A8, UGT1A9, and UGT1A10 were capable of catalyzing YM-542845 glucuronidation, and UGT1A9 exhibited the highest intrinsic clearance. Considered together with the expression levels of UGT isoforms in human liver, these results strongly suggest that YM-542845 glucuronidation in HLM is mainly catalyzed by UGT1A9.     

Cis-resveratrol glucuronidation kinetics in human and recombinant UGT1A sources

1. It was hypothesized that cis-resveratrol glucuronidation contributes to a greater extent to in-vitro disposition of total resveratrol than previously assumed. To this end, the kinetic data for cis-resveratrol glucuronidation are reported.

2. Glucuronidation assays were conducted in human liver and intestinal microsomes and in uridine diphosphate-glucuronosyltransferases (UGTs) UGT1A1, UGT1A6, UGT1A9, and UGT1A10. Kinetic parameters were estimated for the major cis-resveratrol-3-O-glucuronide (cis-R3G). Substrate inhibition was observed with apparent V_max, K_m and K_i of 6.1?±?0.3/27.2?±?1.2 nmol min^?1 mg^?1, 415?±?48.1/989.9?±?92.8 and 789.6?±?76.3/1012?±?55.9?μM in human intestinal microsomes (HIMs) and UGT1A6, respectively (estimate?±?standard error (SE)). Biphasic kinetics were observed in human liver microsomes (HLMs), while sigmoidal kinetics were seen in UGT1A9 (V_max?=?11.92?±?0.2 nmol min^?1 mg^?1; K_m?=?360?μM; n?=?1.27?±?0.07). The 4′-O-glucuronide (cis-R4′G) exhibited atypical kinetics in HLM, HIM, UGT1A1, and UGT1A10. UGT1A9 catalysed cis-R4′G formation at high substrate concentrations (V_max?=?0.33?±?0.015 nmol min^?1 mg^?1; K_m?=?537.8?±?67.8?μM).

3. In conclusion, although the rates of formation of cis-R3G in HLM and UGT1A9 were higher than those for trans-R3G, the contribution to total resveratrol disposition could not be determined fully due to atypical kinetics observed.

     

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.     

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.     

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.     

In vitro characterization of the glucuronidation pathways of licochalcone A mediated by human UDP-glucuronosyltransferases

1. This study aimed to characterize the glucuronidation pathway of licochalcone A (LCA) in human liver microsomes (HLM).

2. HLM incubation systems were employed to catalyze the formation of LCA glucuronide. The glucuronidation activity of commercially recombinant UDP-glucuronosyltransferase (UGT) isoforms toward LCA was screened. Kinetic analysis was used to identify the UGT isoforms involved in the glucuronidation of LCA in HLM.

3. LCA could be metabolized to two monoglucuronides in HLM, including a major monoglucuronide, namely, 4-O-glucuronide, and a minor monoglucuronide, namely, 4′-O-glucuronide. Species-dependent differences were observed among the glucuronidation profiles of LCA in liver microsomes from different species. UGT1A1, UGT1A3, UGT1A7, UGT1A8, UGT1A9, UGT1A10 and UGT2B7 participated in the formation of 4-O-glucuronide, with UGT1A9 exhibiting the highest catalytic activity in this biotransformation. Only UGT1A1 and UGT1A3 were involved in the formation of 4′-O-glucuronide, exhibiting similar reaction rates. Kinetic analysis demonstrated that UGT1A9 was the major contributor to LCA-4-O-glucuronidation, while UGT1A1 played important roles in the formation of both LCA-4-O- and 4′-O-glucuronide.

4. UGT1A9 was the major contributor to the formation of LCA-4-O-glucuronide, while UGT1A1 played important roles in both LCA-4-O- and 4′-O-glucuronidation.