Glucuronidation In Liver Microsomes Research Articles

Atypical Kinetics and Albumin Effect of Glucuronidation of 5-n-Butyl-4-{4-[2-(1H-tetrazole-5- yl)-1H-pyrrol-1-yl]phenylmethyl}-2,4-dihydro-2-(2,6- dichlorophenyl)-3H-1,2,4-triazol-3-one, a Novel Nonpeptide Angiotensin Type 1 Receptor Antagonist, in Liver Microsomes and UDP-Glucuronosyl-transferase.

Ib is a new nonpeptide AT1 receptor antagonist, which plays an active role in cardiovascular protection. Ib monoglucuronide has been identified as its main metabolite. A detailed study of Ib glucuronidation is important for predicting potential DDI. Besides, the elucidation of the “BSA effect” in Ib glucuronidation would make obtained kinetic parameters more predictive in IVIVE. “BSA effect” means that there is a significant change in in vitro kinetic parameters when generated from incubations performed in the presence of bovine serum albumin (BSA). Five UGTs (UGT1A3, UGT2B4, UGT2B7, UGT1A9 and UGT1A8) were identified that produced abundant Ib monoglucuronide, especially UGT1A3. We investigated Ib glucuronidation in liver microsomes from different species (rat, dog, human) and in five identified major human UGTs. Ib glucuronidation in liver microsomes and recombinant human UGTs all showed substrate inhibition kinetics. DLM showed the strongest affinity and activity, HLM showed the lowest affinity, and RLM showed the weakest activity. The addition of BSA did not alter the enzyme kinetics, but significantly altered enzyme kinetic parameters resulting in a reduction in Km value and an increase in CLint value. However, high concentrations of BSA could significantly attenuate this positive effect on enzyme affinity and activity, and the effect of BSA on the Vmax of Ib glucuronidation was opposite in different enzyme sources. In conclusion, this study demonstrated the substrate inhibition kinetics of Ib glucuronidation in the liver metabolism and the effect of BSA on its kinetic parameters, in order to provide more accurate in vitro data for in vivo prediction.

Involvement of UDP-glucuronosyltransferases in higenamine glucuronidation and the gender and species differences in liver

ObjectivesHigenamine (HG), an active ingredient of Aconite root in Chinese herbal medicine, is mainly metabolized by UDP-glucuronosyltransferases (UGT). However, the systematic glucuronidation of HG in humans remains unclear. The purpose of this study was to investigate the glucuronidation of HG. Methods12 recombinant human UGT (rUGT) isozymes were used to characterize the HG glucuronidation. Liver microsomes from male and female mice, rats, guinea pigs, dogs, and humans were used to determine the species and gender differences using liquid chromatography-mass spectrometry. Key findingsOne monoglucuronide was detected in reactions catalyzed by rUGT1A6, rUGT1A8, rUGT1A9, also human and dog liver microsomes. UGT1A9 is the most important glucuronosyltransferase that metabolizes HG. Because carvacrol, a specific inhibitor of UGT1A9, can significantly decrease the glucuronidation of HG in Human liver microsomes and UGT1A9. HG metabolism by UGT1A9 described in Michaelis-Menten kinetics (Km=15.4 mM,Vmax=2.2 nmol/mg/min) and glucuronidation in liver microsomes were species dependent. Gender did not affect the kinetic parameters among species except in rats. ConclusionsUGT1A9 is a major isoenzyme responsible for the glucuronidation of HG in Human liver microsomes (HLMs). Dog may be an appropriate animal model to evaluate HG metabolism.

Identification of human UDP-glucuronosyltransferase isoforms involved in the isofraxidin glucuronidation and assessment of the species differences of the reaction

Isofraxidin, 7-Hydroxy-6.8-dimethoxy-2H-1-benzopyran-2-one, is a major active component of Acanthopanax senticosus, which has been used as Acanthopanax (Ciwujia) injection to treat cardiovascular and cerebrovascular diseases in China for more than thirty years. The purpose of this study was to identify the roles of human UDP-glucuronosyltransferases (UGTs) in isofraxidin glucuronidation in the liver and intestinal microsomes and to reveal the potential species differences by comparing the liver microsomal glucuronidation from different experimental animals. One metabolite was biosynthesized and characterized as isofraxidin-7-O-glucuronide by liquid chromatography tandem mass spectrometry (LC-MS/MS) and nuclear magnetic resonance (NMR). The intrinsic clearances in human liver and intestinal microsomes were 63.8 and 16.4μL/min/mg, respectively. Human liver microsomes displays higher potential for isofraxidin elimination than human intestinal microsomes. The reaction phenotyping analysis was conducted using cDNA-expressed human UGTs and chemical inhibitors. The results indicated that UGT1A1 and UGT1A9 were the main isoforms involved in the formation of isofraxidin-7-O-glucuronide. The isofraxidin glucuronidation in liver microsomes from human (HLM), rat (RLM), mouse (MLM), dog (DLM), monkey (CyLM), minipig (PLM), and guinea pig (GpLM) followed the Michealis-Menten model. The isofraxidin glucuronidation displays species differences in terms of catalytic activities. GpLM had the highest clearance with the CLint value of 152μL/min/mg. CyLM, RLM and MLM exhibit similar catalytic activities in isofraxidin glucuronidation with the intrinsic clearance values of 54.6, 58.0 and 50.2μL/min/mg, respectively, which are higher than those of PLM and DLM (23.9 and 37.7μL/min/mg, respectively). Rat exhibits the most similar intrinsic metabolic clearance (CLint) to human.

In vitro glucuronidation of Armillarisin A: UDP-glucuronosyltransferase 1A9 acts as a major contributor and significant species differences

1. This study is performed to investigate liver microsomal glucuronidation of Armillarisin A (A.A), an effective cholagogue drug, aiming at characterizing the involved UDP-glucuronosyltranferases (UGT) and revealing potential species differences.2. A.A glucuronidation in human liver microsomes (HLM) generates one metabolite (M2) glucuronidated at the phenol hydroxyl group, obeying Michaelis–Menten kinetic model. Multiple isoforms including UGT1A1, 1A7, 1A9 and 2B15 can catalyze A.A glucuronidation. Kinetic assays and chemical inhibition studies both demonstrate that UGT1A9 is responsible for A.A glucuronidation in HLM. A.A glucuronidation in Cynomolgus monkey microsomes (CyLM) also follows Michaelis–Menten model, but can additionally catalyze the traced glucuronosyl substitution at the alcohol hydroxyl group (M1). The reactions in liver microsomes from Sprague-Dawley rats (RLM), ICR/CD-1 mouse (MLM), Beagle dog (DLM) all display biphasic kinetics and only M2 is detected. HLM, RLM and CyLM exhibit very similar catalytic activities towards A.A glucuronidation, with the intrinsic clearance values of respective 38, 37 and 37 μL/min/mg, which are much higher than MLM and DLM.3. This in vitro study indicates that UGT1A9 acts as a major contributor to A.A glucuronidation in human liver, and the reaction displays large species differences.

Inhibition of Morphine Glucuronidation in the Liver Microsomes of Rats and Humans by Monoterpenoid Alcohols

Morphine is an important drug used to alleviate moderate to severe pain. This opiate is mainly metabolized by glucuronidation to a non-analgesic metabolite, morphine-3-glucuronide (M-3-G) and an active metabolite morphine-6-glucuronide (M-6-G). To understand the modulation of morphine glucuronidation activity by environmental factors, the effect of endogenous and food-derived compounds on morphine uridine 5'-diphosphate (UDP)-glucuronosyltransferase (UGT) in rat and human microsomes was evaluated examining the 50% inhibitory concentration (IC(50)). The liver microsomes from Sprague-Dawley rats (RLM) and humans (HLM, 150 donors, pooled microsomes) were used as enzyme sources. Of 27 compounds tested, monoterpenoid alcohols, such as borneol and iso-borneol, exhibited a strong inhibitory effect on morphine glucuronidation in rat liver microsomes (RLM), whereas we failed to detect any inhibitory effect of endogenous substances including amino acids and sugars. The substances which have the ability to inhibit the activity in RLM are also inhibitory toward morphine glucuronidation in HLM and UGT2B7 baculosomes. However, the difference was that while the strongest inhibitory effect was observed for iso-menthol in HLM, borneol was the strongest inhibitor of the activity mediated by RLM. Although zidovudine is a typical substrate of UGT2B7, the inhibition of morphine glucuronidation by zidovudine was far weaker than that of monoterpenoid alcohols. These results demonstrate that dietary and supplementary monoterpenoid alcohols modify the pharmacokinetics and pharmacodynamics of morphine through inhibition of UGT2B7.

Species and Gender Differences Affect the Metabolism of Emodin via Glucuronidation

The aim of the present study was to define the mechanisms responsible for poor bioavailability of emodin by determining its metabolism using in vitro and in situ disposition models of the intestine and liver. Liver microsomes of mice, rats, guinea pigs, dogs, and humans were used along with the rat intestinal perfusion model and the rat intestinal microsomes. In the rat intestine, excretion rates of emodin-3-O-glucuronide were significantly different (p < 0.05) in four regions of the intestine and were higher in males than in females (p < 0.01). Emodin glucuronidation in liver microsomes was species-dependent, and K (m) values varied 5.7-fold (3.2-18.2 microM) in males and 2.8-fold (4.6-13.0 microM) in females. The male intrinsic clearance (CL(int)) values differed by 5-fold (27.6-138.3 mL h(-1) mg(-1) protein), and female CL(int) values differed by 4.3-fold (24.3-103.5 mL h(-1) mg(-1) protein). Since CL(int) values of emodin glucuronidation were 10-fold higher than that of isoflavones, emodin was considered rapidly glucuronidated. In contrast to the large species-dependent effects on K (m) and CL(int) values, gender had a smaller effect on these kinetic parameters (2-fold, p < 0.05). Lastly, glucuronidation rates obtained using liver microsomes from various experimental animals of the same gender correlated well with those in human liver microsomes. In conclusion, Rapid metabolism by UDP-glucuronosyltransferase is the major reason why emodin has poor bioavailability. Species and gender affected emodin metabolism to a different degree, and experimental animals are expected to be useful in predicting emodin glucuronidation in humans.

Effect of rifampin and nelfinavir on the metabolism of methadone and buprenorphine in primary cultures of human hepatocytes.

We tested the hypothesis that primary cultures of human hepatocytes could predict potential drug interactions with methadone and buprenorphine. Hepatocytes (five donors) were preincubated with dimethyl sulfoxide (DMSO) (vehicle), rifampin, or nelfinavir before incubation with methadone or buprenorphine. Culture media (0-60 min) was analyzed by liquid chromatography-tandem mass spectrometry for R- and S-methadone and R- and S-2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) or for buprenorphine, norbuprenorphine, and their glucuronides [buprenorphine-3-glucuronide (B-3-G) and norbuprenorphine-3-glucuronide (N-3-G)]. R- and S-EDDP were detected in three of five, four of five, and five of five media from cells pretreated with DMSO, nelfinavir, and rifampin. R-EDDP increased 3.1- and 26.5-fold, and S-EDDP increased 2.5- and 21.3-fold after nelfinavir and rifampin. The rifampin effect was significant. B-3-G production was detected in media of all cells incubated with buprenorphine and accounted for most of the buprenorphine loss from culture media; it was not significantly affected by either pretreatment. Norbuprenorphine and N-3-G together were detected in three of five, four of five, and five of five donors pretreated with DMSO, nelfinavir and rifampin, and norbuprenorphine in one of five, one of five, and two of five donors. Although there was a trend for norbuprenorphine (2.8- and 4.9-fold) and N-3-G (1.7- and 1.9-fold) to increase after nelfinavir and rifampin, none of the changes were significant. To investigate low norbuprenorphine production, buprenorphine was incubated with human liver and small intestine microsomes fortified to support both N-dealkylation and glucuronidation; N-dealkylation predominated in small intestine and glucuronidation in liver microsomes. These studies support the hypothesis that methadone metabolism and its potential for drug interactions can be predicted with cultured human hepatocytes, but for buprenorphine the combined effects of hepatic and small intestinal metabolism are probably involved.

Glucuronidation of 2-(4-Chlorophenyl)-5-(2-Furyl)-4-Oxazoleacetic Acid (TA-1801A) in Humans: Species Differences in Liver and Intestinal Microsomes

The metabolism of ethyl 2-(4-chlorophenyl)-5-(2-furyl)-4-oxazoleacetate (TA-1801), a potent hypolipidemic agent, was studied in humans after oral administration and compared with that found in rats, rabbits, and dogs previously. Hydrolysis of the ethyl ester to produce metabolite M1 (TA-1801 active form; TA-1801A) is the first metabolic step and the subsequent biotransformation includes the glucuronidation to form the metabolite M4 and the oxidation to form the metabolites M2 and M3. The metabolism of TA-1801 in humans was qualitatively similar to that in the experimental animals studied, although species differences were seen in the amount of metabolites. M4, the glucuronide of TA-1801A was the most abundant metabolite in human urine (24.3% of the dose). In vitro studies using human liver and jejunum microsomes indicated that the TA-1801A glucuronosyltransferase activity in human jejunum microsomes was 2-fold higher than that in liver microsomes. With regard to the interspecies differences in the TA-1801A glucuronosyltransferase activities, the intrinsic clearance for the TA-1801A glucuronidation in liver microsomes was in the following order: rabbit>monkey>human=rat=dog. In jejunum microsomes, the intrinsic clearance for the TA-1801A glucuronidation was in the following order: human>monkey>rabbit>rat=dog. These results suggest that the species differences in the intestinal TA-1801A glucuronidation contribute to the species differences in the excretion rate of TA-1801A glucuronide into the urine.

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.

Dietary administration of an extract from rosemary leaves enhances the liver microsomal metabolism of endogenous estrogens and decreases their uterotropic action in CD-1 mice.

We evaluated the effects of a methanol extract from the leaves of the plant Rosmarinus officinalis L. (rosemary) on the metabolism and action of estradiol and estrone. Treatment of female CD-1 mice with 2% rosemary in AIN-76A diet for 3 weeks increased the liver microsomal 2-hydroxylation of estradiol and estrone by approximately 150%, increased their 6-hydroxylation by approximately 30% and inhibited the 16alpha-hydroxylation of estradiol by approximately 50%. Treatment of female CD-1 mice with 2% rosemary diet for 3 weeks also stimulated the liver microsomal glucuronidation of estradiol and estrone by 54-67% and 37-56%, respectively. In additional studies, feeding 2% rosemary diet to ovariectomized CD-1 mice for 3 weeks inhibited the uterotropic action of estradiol and estrone by 35-50% compared with animals fed a control diet. The results of this study showed that feeding female mice a 2% rosemary diet increased the liver microsomal oxidation and glucuronidation of estradiol and estrone and inhibited their uterotropic action.

Effects of tea polyphenols and flavonoids on liver microsomal glucuronidation of estradiol and estrone

Administration of 0.5 or 1% lyophilized green tea (5 or 10 mg tea solids per ml, respectively) as the sole source of drinking fluid to female Long–Evans rats for 18 days stimulated liver microsomal glucuronidation of estrone, estradiol and 4-nitrophenol by 30–37%, 15–27% and 26–60%, respectively. Oral administration of 0.5% lyophilized green tea to female CD-1 mice for 18 days stimulated liver microsomal glucuronidation of estrone, estradiol and 4-nitrophenol by 33–37%, 12–22% and 172–191%, respectively. The in vitro addition of a green tea polyphenol mixture, a black tea polyphenol mixture or (−)-epigallocatechin gallate inhibited rat liver microsomal glucuronidation of estrone and estradiol in a concentration-dependent manner and their ic 50 values for inhibition of estrogen metabolism were approximately 12.5, 50 and 10 μg/ml, respectively. Enzyme kinetic analysis indicates that the inhibition of estrone glucuronidation by 10 μM (−)-epigallocatechin gallate was competitive while inhibition by 50 μM (−)-epigallocatechin gallate was noncompetitive. Similarly, several flavonoids (naringenin, hesperetin, kaempferol, quercetin, rutin, flavone, α-naphthoflavone and β-naphthoflavone) also inhibited rat liver microsomal glucuronidation of estrone and estradiol to varying degrees. Naringenin and hesperetin displayed the strongest inhibitory effects ( ic 50 value of approximately 25 μM). These two hydroxylated flavonoids had a competitive mechanism of enzyme inhibition for estrone glucuronidation at a 10 μM inhibitor concentration and a predominantly noncompetitive mechanism of inhibition at a 50 μM inhibitor concentration.

Intestinal UDP-glucuronosyltransferase activities in rat and rabbit

1. Tissues other than the liver can contribute significantly to the drug-metabolizing capacity of an animal. In the current study, the glucuronidation of several aglycones in microsomes from the small intestinal mucosa of rat and rabbit has been investigated and compared with glucuronidation in liver microsomes. 2. UDP-glucuronosyltransferase activities in intestinal microsomes were generally higher in rabbit when compared with rat, ranging from 200 to 300% for 1-naphthol, 2- naphthol, 4-methylumbelliferone, 2-hydroxybiphenyl and 4-hydroxybiphenyl. 3. In contrast, hepatic activities were much higher in rat than in rabbit, ranging from 300 to 400% for 1-naphthol, 2-naphthol, 4-methylumbelliferone, 2-hydroxybiphenyl and testosterone; and from 150 to 250% for 4-nitrophenol and diclofenac. 4. In rabbit, activities in the small intestinal mucosa were comparable (70-100%) with hepatic activities for most aglycones. In rat, intestinal mucosa activities were much lower than in liver, with activities toward 1-naphthol, 2-naphthol, 4-nitrophenol, 4- methylumbelliferone, 2-hydroxybiphenyl and 4-hydroxybiphenyl in the small intestine representing 5-15% of hepatic activities. 5. With a higher intestine:liver activity ratio, intestinal UDP-glucuronosyltransferases could be anticipated to contribute more to overall drug glucuronidation in rabbit as compared with rat, thereby contributing more to reducing drug bioavailability.

Changes in liver drug glucuronidation during cholestasis are non predictable.

Liver microsomal glucuronidation of acetaminophen, chloramphenicol, salicylic acid, lorazepam, p-nitrophenol and morphine were measured in 8 days bile duct ligated rats. Compared to normals, cholestatic rats showed a decrease of 31% for p-nitrophenol glucuronidation; salicylic acid glucuronidation increased 281%; acetaminophen glucuronidation increased 38% while morphine, chloramphenicol and lorazepam values were similar to controls. We concluded that cholestasis produces non predictable changes on liver drug glucuronidation pathways.

P-Nitrophenol glucuronidation in bile duct ligated rats.

Liver microsomal glucuronidation of p-nitrophenol was measured in normal, 2 and 8 day bile duct ligated rats. At low aglycone concentration (0.24-0.8 mM) a Michaelis-Menten kinetic was registered in the three groups of animals but cholestatic rats showed a decrease of activity related to the time that cholestasis was maintained. At higher substrate concentration (1.2-2.0 mM), a very different behavior between the three groups was observed: normal rats showed a substrate inhibition phenomena; 2 day rats maintained plateau values and a remarkable activation effect of the activity was seen in the 8 day group. We concluded that cholestasis produced pronounced changes in p-nitrophenol glucuronidation pathway.

UDP-glucuronyltransferase in perfused rat liver and in microsomes—III. Effects of galactosamine and carbon tetrachloride on the glucuronidation of 1-naphthol and bilirubin

Galactosamine treatment (400 mg/kg, i.p., 4 hr) markedly decreased the level of UDP-glucuronic acid (UDPGA) and 1-naphthol glucuronidation in perfused liver. In contrast, bilirubin glucuronidation was not affected. In non-activated microsomes both 1-naphthol and bilirubin glucuronidation were dependent upon the concentration of UDPGA. In UDP- N-acetylglucosamine-activated microsomes, 1-naphthol glucuronidation remained dependent upon UDPGA whereas bilirubin glucuronidation tended to be independent of UDPGA. Carbon tetrachloride treatment (5 ml/kg, per os, 24 hr) strongly decreased 1-naphthol glucuronidation in the intact liver without altering the level of UDPGA. Bilirubin glucuronidation was affected similarly but to a lesser extent, In contrast. 1-naphthol glucuronidation in liver microsomes was increased under these conditions. In the presence of UDP- N-acetylglucosamine and UDP, however, enzyme activity in microsomes from CCl 4-treated rats was lower than in control microsomes. The results suggest a differential regulation of 1-naphthol and bilirubin glucuronidation and stress the importance of intracellular effectors for glucuronidation in the intact liver.

Drug hydroxylation and glucuronidation in liver microsomes of phenobarbital-treated rats.

Abstract1. The rise in p-nitroanisole O-demethylase activity in liver microsomes after phenobarbital treatment of rats slightly preceded, and was greater than, the increase in hepatic microsomal UDP-glucuronyltransferase activity.2. Predigestion of hepatic microsomes with trypsin was necessary in order to detect the increase in UDP-glucuronyltransferase activity. Digitonin was not able to reveal the increase induced by phenobarbital.3. Trypsin digestion of hepatic microsomes solubilized about 30% of the proteins in control rats and 45% in microsomes isolated from phenobarbital-treated rats. The amount of solubilized phospholipids was only about 6% in both cases.4. The proteins solubilized by trypsin contained some components of the mixed-function oxidase including all of the NADPH cytochrome c reductase and a part of the cytochrome P-450 (as cytochrome P-420).5. The trypsin digestion appears to peel off the superficial proteins of the microsomal vesicle and to uncover UDP-glucuronyltransferase from the de...

Nucleotide Activation of Liver Microsomal Glucuronidation

Nucleotide Activation of Liver Microsomal Glucuronidation