Identification of specific UGT1A9‐mediated glucuronidation of licoricidin in human liver microsomes

Licoricidin is a major prenylated isoflavone of Glycyrrhiza uralensis Fisch. (Leguminosae), and its pharmacological effects have been reported frequently. Typically, flavonoids having multiple hydroxyl groups are unambiguous substrates for glucuronyl conjugation by UDP‐glucuronosyltransferases (UGTs). The pharmacological effects of flavonoids are derived from the conjugation of glucuronide to yield the bioactive metabolite. Here, the metabolism of licoricidin in pooled human liver microsomes (HLMs) was investigated using high‐resolution quadrupole‐orbitrap mass spectrometry. One metabolite (M1) was identified in HLMs after incubation with licoricidin in the presence of uridine 5′‐diphosphoglucuronic acid (UDPGA) and NADPH. The structure of M1 was determined as a monoglucuronyl licoricidin, which was selectively produced by UGT1A9. Licoricidin showed a higher metabolic ratio and rapid metabolism with the recombinant human UGT1A9 than mycophenolic acid, a well‐known UGT1A9 substrate. In conclusion, the selective formation of 7‐glucuronyl licoricidin by UGT1A9 in HLMs could serve as a new selective substrate to determine the activity of UGT1A9 in vitro.     

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.     

Stereoselective glucuronidation of formoterol by human liver microsomes

AIMS: Formoterol is a beta2-adrenoceptor agonist marketed as a racemic mixture of the active (R; R)- and inactive (S; S)-enantiomers (rac-formoterol). The drug produces prolonged bronchodilation by inhalation but there is significant interpatient variability in duration of effect. Previous work has shown that in humans formoterol is metabolized by conjugation with glucuronic acid but little is known about the stereoselectivity of this reaction. The aim of the present study was to investigate the glucuronidation of formoterol enantiomers in vitro by human liver microsomes. METHODS: The kinetics of formation of formoterol glucuronides during incubation of racemate and of single formoterol enantiomers with human liver microsomes (n=9) was characterized by chiral h.p.l.c. assay. RESULTS: The kinetics of glucuronidation of the two formoterol enantiomers obeyed the Michaelis-Menten equation. Glucuronidation of formoterol was stereoselective and occurred more than two times faster for (S; S)-formoterol than for (R; R)-formoterol. In incubations with single formoterol enantiomers, the median (n=9) Km values for (R; R)-glucuronide and (S; S)-glucuronide were 827.6 and 840.4 microm, respectively, and the median V max values were 2625 and 4304 pmol min-1 mg-1, respectively. Corresponding values determined in incubations with rac-formoterol were 357.2 and 312.1 microm and 1435 and 2086 pmol min-1 mg-1 for (R; R)- and (S; S)-glucuronide, respectively. Interindividual variation was large with the ratio of V max/Km (S; S/R; R) ranging from 0.57 to 6.90 for incubations with rac-formoterol. CONCLUSIONS: Our study demonstrates that glucuronidation of formoterol by human liver microsomes is stereoselective and subject to high interindividual variability. These findings suggest that clearance of formoterol in humans is subject to variable stereoselectivity which could explain the variation in duration of bronchodilation produced by inhaled formoterol in patients with asthma.     

Raloxifene glucuronidation in human intestine, kidney, and liver microsomes and in human liver microsomes genotyped for the UGT1A1*28 polymorphism

Raloxifene, a selective estrogen receptor modulator, exhibits quite large interindividual variability in pharmacokinetics and pharmacodynamics. In women, raloxifene is metabolized extensively by different isoforms of UDP-glucuronosyltransferase (UGT) to its glucuronides. To gain an insight into intestine, kidney, liver, and lung glucuronidation of raloxifene, human microsomes of all tested organs were used. Raloxifene-6-β-glucuronide (M1) formation followed the Michaelis-Menten kinetics in intestinal, kidney, and liver microsomes; meanwhile, raloxifene-4'-β-glucuronide (M2) formation followed the substrate inhibition kinetics. Human lung microsomes did not show any glucuronidation activity. The tissue intrinsic clearances for kidney, intestine, and liver were 3.4, 28.1, and 39.6 ml · min(-1) · kg(-1), respectively. The aim of our in vitro study was to explain the mechanism behind the observed influence of UGT1A1*28 polymorphism on raloxifene pharmacokinetics in a small-sized in vivo study (Br J Clin Pharmacol 67:437-444, 2009). Incubation of raloxifene with human liver microsomes genotyped for UGT1A1*28 showed a significantly reduced metabolic clearance toward M1 in microsomes from donors with *28 allele. On the contrary, no significant genotype influence was observed on the formation of M2 because of the high variability in estimated apparent kinetic parameters, although a clear trend toward lower glucuronidation activities was observed when UGT1A1*28 polymorphism was present. The liver intrinsic clearances of both homozygotes differed significantly, whereas the clearance of heterozygotes did not differ from the wild-type and the mutated homozygotes. In conclusion, our results show the high importance of the liver and intestine in raloxifene glucuronidation. Moreover, the significant influence of UGT1A1*28 polymorphism on metabolism of raloxifene was confirmed.     

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.     

Identification of UDP-glucuronosyltransferase isoforms involved in hepatic and intestinal glucuronidation of phytochemical carvacrol

1. Carvacrol (2-methyl-5-(1-methylethyl)-phenol), one of the main components occurring in many essential oils of the family Labiatae, has been widely used in food, spice and pharmaceutical industries.

2. The carvacrol glucuronidation was characterized by human liver microsomes (HLMs), human intestinal microsomes (HIMs) and 12 recombinant UGT (rUGT) isoforms.

3. One metabolite was identified as a mono-glucuronide by liquid chromatography/mass spectrometry with HLMs, HIMs, rUGT1A3, rUGT1A6, rUGT1A7, rUGT1A9 and rUGT2B7.

4. The study with a chemical inhibition, rUGT, and kinetics study demonstrated that rUGT1A9 was the major isozyme responsible for glucuronidation in HLMs, and rUGT1A7 played a major role for glucuronidation in HIMs.

     

山姜素在人肝微粒体的葡萄糖醛酸化反应研究

目的研究体外肝微粒体孵育体系中山姜素的葡萄糖醛酸化代谢情况,鉴定参与山姜素葡萄糖醛酸化代谢的UGT亚型。方法用体外肝微粒体孵育体系,用HPLC-UV检测方法,检测山姜素的葡萄糖醛酸化代谢情况。将代谢产物进行纯化后,用质谱(MS)和核磁共振(NMR)法进一步鉴定其结构。用商业化重组表达的UGT单酶,鉴定代谢产物的结构和归属可能参与山姜素葡萄糖醛酸化代谢反应的葡萄糖醛酸转移酶(UGTs)亚型。结果山姜素葡萄糖醛酸代谢产生一个代谢产物,经结构鉴定为山姜素-氧-单葡萄糖醛酸化产物。人肝微粒体代谢山姜素的动力学行为,符合米方程且动力学参数:Vmax=(101.9±3.0)nmol·min-1·mg-1·pro,Km=(40.6±3.6)μmol·L-1。UGT1A1、UGT1A3、UGT1A9和UGT2B15均参与了山姜素的葡萄糖醛酸化反应。结论山姜素在人肝微粒体孵育体系中会被代谢成为一个单葡萄糖醛酸化产物,且归属了参与的UGT酶。     

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

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

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.     

Effect of aging on glucuronidation of valproic acid in human liver microsomes and the role of UDP-glucuronosyltransferase UGT1A4, UGT1A8, and UGT1A10

Valproic acid (VPA) is a widely used anticonvulsant that is also approved for mood disorders, bipolar depression, and migraine. In vivo, valproate is metabolized oxidatively by cytochromes P450 and beta-oxidation, as well as conjugatively via glucuronidation. The acyl glucuronide conjugate (valproate-glucuronide or VPAG) is the major urinary metabolite (30-50% of the dose). It has been hypothesized that glucuronidation of antiepileptic drugs is spared over age, despite a known decrease in liver mass. The formation rates of VPAG in a bank of elderly (65 years onward) human liver microsomes (HLMs) were measured by liquid chromatography/tandem mass spectrometry and compared with those in a younger (2-56 years) HLM bank. In vitro kinetic studies with recombinant UDP-glucuronosyltransferases (UGTs) were completed. A 5- to 8-fold variation for the formation of VPAG was observed within the microsomal bank obtained from elderly and younger donors. VPAG formation ranged from 6.0 to 53.4 nmol/min/mg protein at 1 mM substrate concentration (n=36). The average velocities at 0.25, 0.5, and 1 mM VPA were 7.0, 13.4, and 25.4 nmol/min/mg protein, respectively, in the elderly HLM bank. Rates of VPAG formation were not significantly different in the HLM bank obtained from younger subjects. Intrinsic clearances (V(max)/K(m)) for several cloned, expressed UGTs were determined. UGT1A4, UGT1A8, and UGT1A10 also were found to catalyze the formation of VPAG in vitro. This is the first reported activity of these UGTs toward VPA glucuronidation. UGT2B7 had the highest intrinsic clearance, whereas UGT1A1 demonstrated no activity. In conclusion, our investigation revealed no differences in VPAG formation in younger versus elderly HMLs and revealed three other UGTs that form VPAG in vitro.     

Limited influence of UGT1A1*28 and no effect of UGT2B7*2 polymorphisms on UGT1A1 or UGT2B7 activities and protein expression in human liver microsomes

AIMS: UGT1A1 and UGT2B7 are enzymes that commonly contribute to drug glucuronidation. Since genetic factors have been suggested to contribute to variability in activities and expression levels of these enzymes, a quantitative assessment of the influence of the major genotypes (UGT1A1*28 or UGT2B7*2) on enzyme activities was conducted. METHODS: Using a bank of microsomal samples from 59 human livers, the effect of UGT1A1*28 or UGT2B7*2 polymorphisms were investigated on rates of estradiol 3-glucuronidation (a marker of UGT1A1 enzyme activity) or zidovudine glucuronidation (a marker of UGT2B7 enzyme activity) and levels of immunoreactive protein for each enzyme. Glucuronidation rates for both enzymes were measured at K(m)/S(50) and 10 times K(m)/S(50) concentrations. RESULTS: UGT1A1 and UGT2B7 enzyme activities varied up to 16-fold and sixfold, respectively. Rates at K(m)/S(50) concentration closely correlated with rates at 10 times K(m)/S(50) concentration for both enzymes (but not at 1/10th K(m) for UGT2B7). Enzyme activities correlated with relative levels of immunoreactive protein for UGT1A1 and UGT2B7. Furthermore, rates of zidovudine glucuronidation correlated well with rates of glucuronidation of the UGT2B7 substrate gemcabene, but did not correlate with UGT1A1 enzyme activities. For the UGT1A1*28 polymorphism, consistent with levels of UGT1A1 immunoreactive protein, mean UGT1A1 activity was 2.5- and 3.2-fold lower for TA(6)/TA(7) (P < 0.05) and TA(7)/TA(7) (P < 0.001) genotypes in comparison with the TA(6)/TA(6) genotype. CONCLUSIONS: Relative to the observed 16-fold variability in UGT1A1 activity, these data indicate only a partial (approximately 40%) contribution of the UGT1A1*28 polymorphism to variability of interindividual differences in UGT1A1 enzyme activity. For the UGT2B7*2 polymorphism, genotype had no influence on immunoreactive UGT2B7 protein or the rate of 3'-azido-3'-deoxythymidine glucuronidation.     

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.

     

HPLC法测定大鼠UGT1A6的酶活性及体外动力学分析

目的:建立灵敏、稳定的高效液相色谱法,以对硝基酚为探针底物,评价体外大鼠肝微粒体中UGT1A6酶活性。方法:色谱柱为Agilent Zorbax SB-C18柱(250 mm×4.6 mm,5μm),流动相为20 mmol.L-1磷酸二氢钾缓冲液-甲醇(50∶50,V/V),流速0.8 mL.min-1,柱温30℃,检测波长280 nm。对硝基酚与大鼠肝微粒体在37℃共同孵育一段时间后,加入冰乙腈终止反应,于4℃下12000 r.min-1离心15 min后,吸取上清进行HPLC分析,以双倒数作图法计算酶动力学参数。结果:对硝基酚保留时间为8.95 min,线性范围为0.5～100μmol.L-1,回归方程为Y=5973.12X+571.58(r=0.9999),最低检测限(LOD)为0.1μmol.L-1(S/N≥3),最低定量限(LLOQ)为0.5μmol.L-1,回收率为104.5%～105.5%,日内、日间RSD均小于10%,温孵体系中其他内源性物质不干扰测定;计算酶动力学参数Km为24.63μmol.L-1,Vmax为18.87 nmol.min-1.mg.protein-1。结论:该方法灵敏度高、稳定性好,适合体外对硝基酚的测定,可用于大鼠体外UGT1A6酶活性的评价及酶动力学研究。     

Glucuronidation of 1-hydroxypyrene by human liver microsomes and human UDP-glucuronosyltransferases UGT1A6, UGT1A7, and UGT1A9: development of a high-sensitivity glucuronidation assay for human tissue.

Human UDP-glucuronosyltransferases (UGT, EC 2.4.1.17) involved in the biotransformation of pyrene were investigated by a sensitive fluorometric high-performance liquid chromatography (HPLC)method developed for determining activities toward 1-hydroxypyrene. The endpoint metabolite of pyrene, 1-pyrenylglucuronide, is a well-known urinary biomarker for the assessment of human exposure to polycyclic aromatic hydrocarbons. 1-Pyrenylglucuronide was synthesized using rat liver microsomes as biocatalyst. The yield was satisfactory, 22%. 1-Pyrenylglucuronide, identified by (1)H NMR and by electrospray mass spectrometry, was used for method validation and calibration. The HPLC assay was very sensitive with a quantitation limit of 3 pg (8 fmol) for 1-pyrenylglucuronide. The assay was precise, showing a relative standard deviation of 5% or less at 0.1 to 300 microM 1-hydroxypyrene. Only 2 microg of microsomal protein was required for the assay in human liver. The glucuronidation of 1-hydroxypyrene was catalyzed at high rates in microsomes from pooled or three individual liver samples, showing comparable apparent K(m) values. The formation of 1-pyrenylglucuronide was catalyzed by recombinant human UGT1A6, UGT1A7, and UGT1A9, the K(m) values being 45, 12, and 1 microM, respectively. The apparent K(m) values in human liver microsomes, ranging from 6.9 to 8.6 microM, agreed well with these results. The method provides a sensitive tool for measuring extremely low UGT activities and a specific means for assessing interindividual differences in 1-hydroxypyrene-metabolizing UGT activities in human liver and other tissues.     

Glucuronidation of flavonoids by recombinant UGT1A3 and UGT1A9

Flavonoids are highlighted for their potential roles in the prevention of oxidative stress-associated diseases. Their metabolisms in vivo, such as glucuronidation, are the key points to determine their health beneficial properties. In this paper, we tested the glucuronidation of nineteen flavonoids by both recombinant human UGT1A3 and UGT1A9. Eleven compounds could be catalyzed by both enzymes. In general, both enzymes showed moderate to high catalyzing activity to most flavonoid aglycones, while the catalyzing efficiency changed with structures. Each flavonoid produced more than one monoglucuronide with no diglucuronide detected by liquid chromatography-mass spectrometry (LC-MS). Enzymatic kinetic analysis indicated that the catalyzing efficiency (Vmax/Km) of UGT1A9 was higher than that of UGT1A3, suggesting its important role in flavonoid glucuronidation. Both human UGT1A3 and UGT1A9 preferred flavonoid aglycone to flavonoid glycoside, and their metabolism to arabinoside was stronger than to other glycosides. Of the flavonoids studied, it is the first time to report isorhamnetin, morin, silybin, kaempferol, daidzein, quercetin-3',4'-OCHO-, quercetin xylopyranoside and avicularin as substrates of UGT1A3. Apigenin, morin, daidzein, quercetin-3',4'-OCHO-, quercetin xylopyranoside and avicularin were the newly reported substrates of UGT1A9.     

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.     

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

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

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

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

大鼠微粒体代谢酶对黄芩素葡萄糖醛酸化代谢物影响的实验研究

目的 观察大鼠微粒体代谢酶对黄芩素(治疗呼吸道感染中药)代谢作用的影响.方法 用体外微粒体药物代谢酶孵育法;用HPLC法测定黄芩素及其葡萄糖醛酸化代谢物的含量,在不同孵育时间和不同浓度下,观察大鼠肝肠不同微粒体对黄芩素葡萄糖醛酸化代谢产物生成速率的影响.结果 5种不同微粒体药物代谢酶对黄芩素均有代谢作用,且随孵育时间延长,代谢作用也相应增加,呈较好的时间和剂量依赖关系.在5种微粒体中,十二指肠微粒体代谢作用最强;而肝代谢作用最弱.结论 黄芩素主要代谢部位是肠道.     

Quantitation of UGT1A1 in human liver microsomes using stable isotope-labelled peptides and mass spectrometry based proteomic approaches

1.?UDP-glucuronosyltransferases (UGTs) are a group of drug-metabolizing enzymes that catalyse the conjugation of endogeonous compounds and xenobiotics to yield hydrophilic glucuronides which subsequently undergo excretion. This report describes an approach for the identification and accurate quantitation of human UGT1A1 in complex biological matrices using liquid chromatography/mass spectrometry/mass spectrometry (LC-MS/MS) analysis of protein digests.

2.?A stable isotope-labelled (SIL) peptide of a unique peptide spanning residues 54–69 in exon 1 of the human UGT1A1 protein with the sequence RIYLSADPALVVIEHG was synthesized. The peptide sequence synthesized was in the reverse order of the human peptide with the stable isotope-labels in the amino acid arginine (¹³C₆¹⁵N₄) resulting in an increase in the mass of the SIL peptide of 10 amu, from 1753 to 1763. The SIL peptide was quantitated by injecting increasing concentrations of the peptide into the LC-MS to obtain a standard curve.

3.?The labelled peptide along with precursor ion monitoring was used to quantify the levels of UGT1A1 in commercial recombinant preparations (supersomes) and individual human liver microsomal samples and pooled human liver micrsomes obtained from BD Biosciences.

4.?Glucuronidation activity studies were performed, which demonstrated a positive correlation between enzyme activity levels and the UGT1A1 content in the liver microsomes obtained from individual human donors.