Characterization of nicotine and cotinine N-glucuronidations in human liver microsomes.

The nicotine and cotinine N-glucuronidations in human liver microsomes were characterized. The Eadie-Hofstee plots of nicotine N-glucuronidation in human liver microsomes were clearly biphasic, indicating the involvement of multiple enzymes. The apparent K(m) and V(max) values were 33.1 +/- 28.1 micro M and 60.0 +/- 21.0 pmol/min/mg and 284.7 +/- 122.0 micro M and 124.0 +/- 44.0 pmol/min/mg for the high- and low-affinity components, respectively, in human liver microsomes (n = 4). However, the Eadie-Hofstee plots of cotinine N-glucuronidation in human liver microsomes were monophasic (apparent K(m) = 1.9 +/- 0.3 mM, V(max) = 655.6 +/- 312.3 pmol/min/mg). The nicotine and cotinine N-glucuronidations in the recombinant human UDP-glucuronosyltransferases (UGTs) (UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B7, and UGT2B15) expressed in baculovirus-infected insect cells or human B-lymphoblastoid cells that are commercially available were determined. However, no recombinant UGT isoforms showed detectable nicotine and cotinine N-glucuronides (the concentrations of nicotine and cotinine were 0.5 and 2 mM, respectively). Nicotine and cotinine N-glucuronidations in pooled human liver microsomes were competitively inhibited by bilirubin as a substrate for UGT1A1 (K(i) = 3.9 and 3.3 micro M), imipramine as a substrate for UGT1A4 (K(i) = 6.1 and 2.7 micro M), and propofol as a substrate for UGT1A9 (K(i) = 6.0 and 12.0 micro M). The nicotine N-glucuronidation (50 micro M nicotine) in 14 human liver microsomes was significantly (r = 0.950, P < 0.0001) correlated with the cotinine N-glucuronidation (0.2 mM cotinine), indicating that the same isoform(s) is involved in both glucuronidations. Furthermore, weak correlations between imipramine N-glucuronidation and nicotine N-glucuronidation (r = 0.425) or cotinine N-glucuronidation (r = 0.517) were observed. In conclusion, the involvement of UGT1A1 and UGT1A9 as well as UGT1A4 in nicotine and cotinine N-glucuronidations in human liver microsomes was suggested, although the contributions of each UGT isoform could not be determined conclusively.     

Microsomal N-glucuronidation of nicotine and cotinine: human hepatic interindividual, human intertissue, and interspecies hepatic variation.

Two of the abundant conjugates of human nicotine metabolism result from the N-glucuronidation of S-(-)-nicotine and S-(-)-cotinine, transformations we recently demonstrated in liver microsomes. We further studied these microsomal N-glucuronidation reactions with respect to human hepatic interindividual, human intertissue, and interspecies hepatic variation. The reactivities of microsomes from human liver (n = 12), various human tissues, and liver from eight species toward the N-glucuronidation of S-(-)-nicotine and S-(-)-cotinine, and also R-(+)-nicotine in human liver were examined. Assays with (14)C-labeled substrates involved radiometric high-performance liquid chromatography. For the human liver samples examined there were 13- to 17-fold variations in the catalytic activities observed toward S-(-)-nicotine, R-(+)-nicotine, and S-(-)-cotinine. Gender and smoking effects were studied, and after exclusion of an outlier a decrease in catalytic activity in females was observed. Significant correlations were observed between all three analytes, indicating that the same UDP-glucuronosyltransferase(s) enzyme is likely to be involved in these transformations. Catalytic activities were not observed for human gastrointestinal tract (colon, duodenum, ileum, jejunum, and stomach), kidney, or lung microsomes. For the seven animal species examined, activity was measurable only for monkey, guinea pig, and minipig, and only for S-(-)-nicotine N-glucuronidation and at rates 10- to 40-fold lower than humans. Activity was not measurable in the case of dog, mouse, rabbit, or rat, for the latter under five different treatment conditions for one of the strains. In conclusion, there are large hepatic interindividual variations in N-glucuronidation of S-(-)-nicotine and S-(-)-cotinine, in human extrahepatic metabolism seems limited, and none of the animal strains examined resembled human.     

Formation of the quaternary ammonium-linked glucuronide of nicotine in human liver microsomes: identification and stereoselectivity in the kinetics.

The formation of the N1-glucuronide metabolite of each nicotine enantiomer was studied in pooled human liver microsomes (n = 6). The metabolite formed from natural S(-)-nicotine was identified by comparison of the high-pressure liquid chromatography (HPLC) retention time and positive ion electrospray ionization-mass spectral characteristics with a synthetic reference standard. A radiometric HPLC method was used to quantify the metabolite. The specificity of the assay method was demonstrated by experiments in which beta-glucuronidase treatment of incubated assay samples resulted in elimination of the peak due to the N1-glucuronide metabolite. The glucuronides of S(-)- and R(+)-nicotine were formed by one-enzyme kinetics, with K(m) values of 0.11 and 0.23 mM and V(max) values of 132 and 70 pmol/min/mg of protein, respectively. There is marked stereoselectivity in the apparent intrinsic clearance values (V(max)/K(m)) in that the value for S(-)-nicotine is 4 times greater than for the R(+)-isomer (1.2 versus 0.31 microl/min/mg of protein).     

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

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

Imipramine N-glucuronidation in human liver microsomes: biphasic kinetics and characterization of UDP-glucuronosyltransferase isoforms.

A method for the direct determination of imipramine N-glucuronidation in human liver microsomes by high-performance liquid chromatography with UV detection was developed. Imipramine was incubated with human liver microsomes and UDP-glucuronic acid. The Eadie-Hofstee plots of imipramine N-glucuronidation in human liver microsomes were biphasic. For the high-affinity component, the K(m) was 97.2 +/- 39.4 microM and the V(max) was 0.29 +/- 0.03 nmol/min/mg of protein. For the low-affinity component, the K(m) was 0.70 +/- 0.29 mM and the V(max) was 0.90 +/- 0.28 nmol/min/mg of protein. The imipramine N-glucuronosyltransferase activities were not detectable in two samples of human jejunum microsomes. Among recombinant UDP-glucuronosyltransferases (UGTs) in baculovirus-infected insect cells (Supersomes or Bacurosomes) or human B-lymphoblastoid cells tested in the present study (UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B7, and UGT2B15), only UGT1A4 showed imipramine N-glucuronosyltransferase activity. The activity in UGT1A4 Supersomes was higher than that in recombinant UGT1A4 expressed in human B-lymphoblastoid cells at all imipramine concentration tested. The kinetics of imipramine N-glucuronidation in UGT1A4 Supersomes did not fit the Michaelis-Menten plot, showing a K(m) of >1 mM. In contrast, in UGT1A4 expressed in human B-lymphoblastoid cells, K(m) was 0.71 +/- 0.36 mM and the V(max) was 0.11 +/- 0.03 nmol/min/mg of protein. Interindividual differences in the imipramine N-glucuronidation in liver microsomes from 14 humans were at most 2.5-fold. The imipramine N-glucuronosyltransferase activities in 11 human liver microsomes were significantly (r = 0.817, P < 0.005) correlated with the glucuronosyltransferase activities of trifluoperazine, a typical substrate of UGT1A4. This is the first report of the biphasic kinetics of imipramine N-glucuronide in human liver microsomes.     

N-glucuronidation of nicotine and cotinine by human liver microsomes and heterologously expressed UDP-glucuronosyltransferases.

Nicotine is considered the major addictive agent in tobacco. Tobacco users extensively metabolize nicotine to cotinine. Both nicotine and cotinine undergo N-glucuronidation. Human liver microsomes have been shown to catalyze the formation of these N-glucuronides. However, which UDP-glucuronosyltransferases contribute to this catalysis has not been identified. To identify these enzymes, we initially measured the rates of glucuronidation by 15 human liver microsome samples. Fourteen of the samples glucuronidated both nicotine and cotinine at rates ranging from 146 to 673 pmol/min/mg protein and 140 to 908 pmol/min/mg protein, respectively. The rates of nicotine glucuronidation and cotinine glucuronidation by these 14 samples were correlated, r = 0.97 (p < 0.0001). The glucuronidation of nicotine and cotinine by heterologously expressed UGT1A3, UGT1A4, and UGT1A9 was also determined. All three enzymes catalyzed the glucuronidation of nicotine. However, the rate of catalysis by UGT1A4 Supersomes was more than 30-fold greater than that by either UGT1A3 Supersomes or UGT1A9 Supersomes. Interestingly, when expressed per UGT1A protein, measured by a UGT1A specific antibody, cell lysate from V79-expressed UGT1A9 catalyzed nicotine glucuronidation at a rate 17-fold greater than did UGT1A9 Supersomes. UGT1A4 Supersomes also catalyzed cotinine N-glucuronidation, but at one-tenth the rate of nicotine glucuronidation. Cotinine glucuronidation by either UGT1A3 or UGT1A9 was not detected. Both propofol, a UGT1A9 substrate, and imipramine, a UGT1A4 substrate, inhibited the glucuronidation of nicotine and cotinine by human liver microsomes. Taken together, these data support a role for both UGT1A9 and UGT1A4 in the catalysis of nicotine and cotinine N-glucuronidation.     

Characterization of afloqualone N-glucuronidation: species differences and identification of human UDP-glucuronosyltransferase isoform(s).

Afloqualone (AFQ) is one of the centrally acting muscle relaxants. AFQ N-glucuronide is the most abundant metabolite in human urine when administered orally, whereas it was not detected in the urine when administered to rats, dogs, and monkeys. Species differences in AFQ N-glucuronidation were investigated with liver microsomes obtained from humans and experimental animals. The kinetics of AFQ N-glucuronidation in human liver microsomes showed a typical Michaelis-Menten plot. The K(m) and V(max) values accounted for 2019 +/- 85.9 muM and 871.2 +/- 17.9 pmol/min/mg protein, respectively. The V(max) and intrinsic clearance (CL(int)) values of AFQ N-glucuronidation in human liver were approximately 4- to 10-fold and 2- to 4-fold higher than those in rat, dog, and monkey, respectively. Among 12 recombinant human UDP-glucuronosyltransferase (UGT) isoforms, both UGT1A4 and UGT1A3 exhibited high AFQ N-glucuronosyltransferase activities. The K(m) value of AFQ N-glucuronidation in recombinant UGT1A4 microsomes was very close to that in human liver microsomes. The formation of AFQ N-glucuronidation by human liver, jejunum, and recombinant UGT1A4 microsomes was effectively inhibited by trifluoperazine, a known specific substrate for UGT1A4. The AFQ N-glucuronidation activities in seven human liver microsomes were significantly correlated with trifluoperazine N-glucuronidation activities (r(2) = 0.798, p < 0.01). In contrast, the K(m) value of AFQ N-glucuronidation in recombinant UGT1A3 microsomes was relatively close to that in human jejunum microsomes. These results demonstrate that AFQ N-glucuronidation in human is mainly catalyzed by UGT1A4 in the liver and by UGT1A3, as well as UGT1A4 in the intestine.     

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.     

Epirubicin glucuronidation is catalyzed by human UDP-glucuronosyltransferase 2B7.

Epirubicin is one of the most active agents for breast cancer. The formation of epirubicin glucuronide by liver UDP-glucuronosyltransferase (UGT) is its main inactivating pathway. This study aimed to investigate epirubicin glucuronidation in human liver microsomes, to identify the specific UGT isoform for this reaction, and to correlate epirubicin glucuronidation with other UGT substrates. Microsomes from human livers were used. UGTs specifically expressed in cellular systems, as well as two UGT2B7 variants, were screened for epirubicin glucuronidation. Epirubicin, morphine, and SN-38 glucuronides were measured by high-pressure liquid chromatography. The mean +/- S.D. formation rate of epirubicin glucuronide in human liver microsomes (n = 47) was 138 +/- 37 pmol/min/mg (coefficient of variation, 24%). This phenotype was normally distributed. We screened commercially available UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A9, UGT2B7, and UGT2B15 for epirubicin glucuronidation. Only UGT2B7 converted epirubicin to its glucuronide. No differences in epirubicin glucuronidation were found in HK293 cells expressing the two UGT2B7 variants at position 268. Catalytic efficiency (V(max)/K(m)) of epirubicin glucuronidation was 1.4 microl/min/mg, a value higher than that observed for morphine, a substrate of UGT2B7. Formation of epirubicin glucuronide was significantly related to that of morphine-3-glucuronide (r = 0.76, p < 0.001) and morphine-6-glucuronide (r = 0.73, p < 0.001). No correlation was found with SN-38, a substrate of UGT1A1 (r = 0.04). UGT2B7 is the major human UGT catalyzing epirubicin glucuronidation, and UGT2B7 is the candidate gene for this phenotype. The reported tyrosine to histidine polymorphism in UGT2B7 does not alter the formation rate of epirubicin glucuronide, and undiscovered genetic polymorphisms in UGT2B7 might change the metabolic fate of this important anticancer agent.     

N-glucuronidation of perfluorooctanesulfonamide by human, rat, dog, and monkey liver microsomes and by expressed rat and human UDP-glucuronosyltransferases.

N-Alkylperfluorooctanesulfonamides have been used in a range of industrial and commercial applications. Perfluorooctanesulfonamide (FOSA) is a major metabolite of N-alkylperfluorooctanesulfonamides and has a long half-life in animals and in the environment and is biotransformed to FOSA N-glucuronide. The objective of this study was to identify and characterize the human and experimental animal liver UDP-glucuronosyltransferases (UGTs) that catalyze the N-glucuronidation of FOSA. The results showed that pooled human liver and rat liver microsomes had high N-glucuronidation activities. Expressed rat UGT1.1, UGT2B1, and UGT2B12 in HK293 cells catalyzed the N-glucuronidation of FOSA but at rates that were lower than those observed in rat liver microsomes. Of the 10 expressed human UGTs (1A1, 1A3, 1A4, 1A6, 1A9, 2B4, 2B7, 2B15, and 2B17) studied, only hUGT2B4 and hUGT2B7 catalyzed the N-glucuronidation of FOSA. The kinetics of N-glucuronidation of FOSA by rat liver microsomes and by hUGT2B4/7 was consistent with a single-enzyme Michaelis-Menten model, whereas human liver microsomes showed sigmoidal kinetics. These data show that rat liver UGT1.1, UGT2B1, and UGT2B12 catalyze the N-glucuronidation of FOSA, albeit at low rates, and that hUGT2B4 and hUGT2B7 catalyze the N-glucuronidation of FOSA.     

Comparative N-glucuronidation kinetics of ketotifen and amitriptyline by expressed human UDP-glucuronosyltransferases and liver microsomes.

Like other basic amphiphilic drugs, the (S)-enantiomer of the antiallergic drug ketotifen exhibited biphasic kinetics when it was converted to two isomeric quaternary ammonium-linked glucuronides in human liver microsomes. For (R)-ketotifen this applied when incubations were carried out in the absence of a detergent. Two UDP-glucuronosyltransferases (UGTs) present in human liver, UGT1A4 and UGT1A3, were previously shown to catalyze tertiary amine N-glucuronidation when expressed in HK293 cells. Therefore, the conjugation kinetics of (R)- and (S)-ketotifen were investigated with the two expressed proteins. When homogenates of HK293 cells expressing UGT1A4 were incubated without detergent, N-glucuronidation kinetics were monophasic with K(M) values of 59 +/- 5 microM for (R)- and 86 +/- 26 microM for (S)-ketotifen. In experiments with membranes containing expressed UGT1A3, somewhat higher K(M) values were obtained. These values correspond to the high rather than to the low K(M) components of ketotifen glucuronidation in liver microsomes, the latter exhibiting K(M) values around 2 and 1 microM, respectively, with (R)- and (S)-ketotifen. With amitriptyline as the substrate, N-glucuronidation kinetics in the absence of detergent were biphasic in human liver microsomes and monophasic with a high K(M) value in cell homogenates containing UGT1A4. The results suggest that UGT1A4 and UGT1A3 catalyze high-K(M) N-glucuronidation of tertiary amine drugs, whereas the low-K(M) reaction requires either an alternative enzyme or a special conformation of UGT1A4 or UGT1A3 that can be attained in liver microsomes, but not in HK293 cell membranes.     

Glucuronidation of DRF-6574, hydroxy metabolite of DRF-4367 (a novel COX-2 inhibitor) by pooled human liver, intestinal microsomes and recombinant human UDP-glucuronosyltransferases (UGT): role of UGT1A1, 1A3 and 1A8.

DRF-4367 is a novel COX-2 inhibitor, which showed good efficacy in several animal models of inflammation. In a comparative in vitro metabolism in various liver microsomes, DRF-4367 forms a hydroxy metabolite (DRF-6574) mediated by CYP2D6 and 2C19 isoenzymes. DRF-6574 readily undergoes Phase-II metabolism and forms glucuronide and sulfate conjugates both in vitro and in vivo. The objective of the present study was two folds: to study the glucuronidation of DRF-6574 in human liver and intestinal microsomes and to identify the recombinant human liver and intestinal UDP-glucuronosyltransferase (UGT) enzymes responsible for glucuronidation of DRF-6574. Of twelve recombinant UGTs tested, two hepatic UGTs viz., UGT1A1 and 1A3 and an extra hepatic UGT i.e., UGT1A8 showed the catalytic activity. The enzyme kinetics in pooled human liver, intestinal and recombinant UGT microsomes showed a typical Michaelis-Menten plot. The apparent Km and Vmax value for DRF-6574 was found to be 116 +/- 24 microM and 2.07 +/- 0.12 microg/min/mg protein and 142 +/- 17 microM and 3.83 +/- 0.15 microg/min/mg protein in pooled human liver and intestinal microsomes, respectively. The intrinsic clearance (Vmax/Km) value for DRF-6574 was estimated to be 0.043 and 0.065 ml/min/mg protein, respectively in pooled human liver and intestinal microsomes. Moreover we have determined the Km and Vmax and intrinsic clearance values for specific UGTs viz., UGT 1A1, 1A3 and 1A8. The apparent Km and Vmax values are 23 +/- 7.2 microM, 3.44 +/- 0.17 microg/min/mg protein for UGT1A1, 60 +/- 7.9 microM, 3.67 +/- 0.11 microg/min/mg protein for UGT1A3, 96 +/- 8.0 microM, 2.95 +/- 0.06 microg/min/mg protein for UGT1A8. The intrinsic clearance values (Vmax/Km) estimated were 0.367, 0.148, 0.074 ml/min/mg protein for UGT1A1, 1A3 and 1A8, respectively. The intrinsic clearance value in UGT1A8 was very close to that in human intestinal and liver microsomes. The formation of DRF-6574 glucuronide by human liver, intestinal and UGT1A1, 1A3 and 1A8 microsomes was effectively inhibited by phenylbutazone.     

Disposition kinetics of nicotine and cotinine enantiomers in rabbits and beagle dogs

The disposition kinetics of nicotine and cotinine enantiomers was determined in rabbits. The clearance of (R)-nicotine was similar to that of (S)-nicotine, but clearance of (R)-cotinine was twice that of (S)-cotinine. Fractional conversions of both enantiomers of nicotine to cotinine were approximately 50%. These results suggest that in rabbits the biotransformation pathways of cotinine, but not nicotine, are influenced by stereochemistry. The disposition kinetics of nicotine enantiomers in beagle dogs was also studied. In dogs, the clearance of (R)-nicotine was slightly greater than the clearance of (S)-nicotine. Methods for the synthesis of (R)-nicotine and (R)-cotinine of high enantiomeric purity and a gas chromatographic method for determination of nicotine enantiomeric purity are described.     

Quaternary ammonium-linked glucuronidation of tamoxifen by human liver microsomes and UDP-glucuronosyltransferase 1A4

Tamoxifen (TAM), a nonsteroidal antiestrogen, is the most widely used drug for chemotherapy of hormone-dependent breast cancer in women. In the present study, we found a new potential metabolic pathway of TAM via N-linked glucuronic acid conjugation for excretion in humans. TAM N(+)-glucuronide was isolated from a reaction mixture consisting of TAM and human liver microsomes fortified with UDP-glucuronic acid (UDPGA) and identified with a synthetic specimen by high-performance liquid chromatography-electrospray ionization-mass spectrometry. However, no TAM-glucuronidating activity was detected in microsomes from rat, mouse, monkey, dog, and guinea pig livers. A strong correlation (r(2) =0.92 ) was observed between N-glucuronidating activities toward TAM and trifluoperazine, a probe substrate for human UDP-glucuronosyltransferase (UGT) 1A4, in human liver microsomes from eight donors (five females, three males). However, no correlation ( (r(2) =0.02 )) was observed in the activities between 7-hydroxy-4-(trifluoromethyl)coumarin and TAM. Only UGT1A4 catalyzed the N-linked glucuronidation of TAM among recombinant UGTs (UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A9, UGT2B4, UGT2B7, UGT2B15, and UGT2B17) expressed in insect cells. Apparent K(m) values for TAM N-glucuronidation by human liver microsomes and recombinant UGT1A4 were 35.8 and 32.4 microM, respectively. These results strongly suggested that UGT1A4 could play a role in metabolism and excretion of TAM without Phase I metabolism in human liver. TAM N(+)-glucuronide still had binding affinity similar to TAM itself for human estrogen receptors, ERalpha and ERbeta, suggesting that TAM N(+)-glucuronide might contribute to the biological activity of TAM in vivo.     

Glucuronidation in the chimpanzee (Pan troglodytes): studies with acetaminophen, oestradiol and morphine

The chimpanzee has recently been characterized as a surrogate for oxidative drug metabolism in humans and as a pharmacokinetic model for the selection of drug candidates. In the current study, the glucuronidation of acetaminophen, morphine and oestradiol was evaluated in the chimpanzee to extend the characterization of this important animal model. Following oral administration of acetaminophen (600 mg) to chimpanzees (n=2), pharmacokinetics were comparable with previously reported human values, namely mean oral clearance 0.91 vs. 0.62+/-0.05 l h-1 kg-1, apparent volume of distribution 2.29 vs. 1.65+/-0.25 l kg-1, and half-life 1.86 vs. 1.89+/-7h, for chimpanzee vs. human, respectively. Urinary excretions (percentage of dose) of acetaminophen, acetaminophen glucuronide and acetaminophen sulfate were also similar between chimpanzees and humans, namely 2.3 vs. 5.0, 63.1 vs. 54.7, and 25.0 vs. 32.3%, respectively. Acetaminophen, oestradiol and morphine glucuronide formation kinetics were investigated using chimpanzee (n=2) and pooled human liver microsomes (n=10). V(max) (app) and K(m)(app) (or S(50)(app)) for acetaminophen glucuronide, morphine 3- and 6-glucuronide, and oestradiol 3- and 17-glucuronide formation were comparable in both species. Eadie-Hofstee plots of oestradiol 3-glucuronide formation in chimpanzee microsomes were characteristic of autoactivation kinetics. Western immunoblot analysis of chimpanzee liver microsomes revealed a single immunoreactive band when probed with anti-human UGT1A1, anti-human UGT1A6, and anti-human UGT2B7. Taken collectively, these data demonstrate similar glucuronidation characteristics in chimpanzees and humans.     

Characterization of raloxifene glucuronidation in vitro: contribution of intestinal metabolism to presystemic clearance.

Raloxifene, a selective estrogen receptor modulator used for the treatment of osteoporosis, undergoes extensive conjugation to the 6-beta- and 4'-beta-glucuronides in vivo. This paper investigated raloxifene glucuronidation by human liver and intestinal microsomes and identified the responsible UDP-glucuronosyltransferases (UGTs). UGT1A1 and 1A8 were found to catalyze the formation of both the 6-beta- and 4'-beta-glucuronides, whereas UGT1A10 formed only the 4'-beta-glucuronide. Expressed UGT1A8 catalyzed 6-beta-glucuronidation with an apparent K(m) of 7.9 microM and a V(max) of 0.61 nmol/min/mg of protein and 4'-beta-glucuronidation with an apparent K(m) of 59 microM and a V(max) of 2.0 nmol/min/mg. Kinetic parameters for raloxifene glucuronidation by expressed UGT1A1 could not be determined due to limited substrate solubility. Based on rates of raloxifene glucuronidation and known extrahepatic expression, UGT1A8 and 1A10 appear to be primary contributors to raloxifene glucuronidation in human jejunum microsomes. For human liver microsomes, the variability of 6-beta- and 4'-beta-glucuronide formation was 3- and 4-fold, respectively. Correlation analyses revealed that UGT1A1 was responsible for 6-beta- but not 4'-beta-glucuronidation in liver. Treatment of expressed UGTs with alamethicin resulted in minor increases in enzyme activity, whereas in human intestinal microsomes, maximal increases of 8-fold for the 6-glucuronide and 9-fold for the 4'-glucuronide were observed. Intrinsic clearance values in intestinal microsomes were 17 microl/min/mg for the 6-glucuronide and 95 microl/min/mg for the 4'-isomer. The corresponding values for liver microsomes were significantly lower, indicating that intestinal glucuronidation may be a significant contributor to the presystemic clearance of raloxifene in vivo.     

Trans-3'-hydroxycotinine O- and N-glucuronidations in human liver microsomes.

Trans-3'-hydroxycotinine is a major metabolite of nicotine in humans and is mainly excreted as O-glucuronide in smoker's urine. Incubation of human liver microsomes with UDP-glucuronic acid produces not only trans-3'-hydroxycotinine O-glucuronide but also N-glucuronide. The formation of N-glucuronide exceeds the formation of O-glucuronide in most human liver microsomes, although N-glucuronide has never been detected in human urine. Trans-3'-hydroxycotinine N-glucuronidation in human liver microsomes was significantly correlated with nicotine and cotinine N-glucuronidations, which are catalyzed mainly by UDP-glucuronosyltransferase (UGT)1A4 and was inhibited by imipramine and nicotine, which are substrates of UGT1A4. Recombinant UGT1A4 exhibited substantial trans-3'-hydroxycotinine N-glucuronosyltransferase activity. These results suggest that trans-3'-hydroxycotinine N-glucuronidation in human liver microsomes would be mainly catalyzed by UGT1A4. In the present study, trans-3'-hydroxycotinine O-glucuronidation in human liver microsomes was thoroughly characterized, since trans-3'-hydroxycotinine O-glucuronide is one of the major metabolites of nicotine. The kinetics were fitted to the Michaelis-Menten equation with a K(m) of 10.0 +/- 0.8 mM and a V(max) of 85.8 +/- 3.8 pmol/min/mg. Among 11 recombinant human UGT isoforms expressed in baculovirus-infected insect cells, UGT2B7 exhibited the highest trans-3'-hydroxycotinine O-glucuronosyltransferase activity (1.1 pmol/min/mg) followed by UGT1A9 (0.3 pmol/min/mg), UGT2B15 (0.2 pmol/min/mg), and UGT2B4 (0.2 pmol/min/mg) at a substrate concentration of 1 mM. Trans-3'-hydroxycotinine O-glucuronosyltransferase activity by recombinant UGT2B7 increased with an increase in the substrate concentration up to 16 mM (10.5 pmol/min/mg). The kinetics by recombinant UGT1A9 were fitted to the Michaelis-Menten equation with K(m) = 1.6 +/- 0.1 mM and V(max) = 0.69 +/- 0.02 pmol/min/mg of protein. Trans-3'-hydroxycotinine O-glucuronosyltransferase activities in 13 human liver microsomes ranged from 2.4 to 12.6 pmol/min/mg and were significantly correlated with valproic acid glucuronidation (r = 0.716, p < 0.01), which is catalyzed by UGT2B7, UGT1A6, and UGT1A9. Trans-3'-hydroxycotinine O-glucuronosyltransferase activity in human liver microsomes was inhibited by imipramine (a substrate of UGT1A4, IC(50) = 55 microM), androstanediol (a substrate of UGT2B15, IC(50) = 169 microM), and propofol (a substrate of UGT1A9, IC(50) = 296 microM). Interestingly, imipramine (IC(50) = 45 microM), androstanediol (IC(50) = 21 microM), and propofol (IC(50) = 41 microM) also inhibited trans-3'-hydroxycotinine O-glucuronosyltransferase activity by recombinant UGT2B7. These findings suggested that trans-3'-hydroxycotinine O-glucuronidation in human liver microsomes is catalyzed by mainly UGT2B7 and, to a minor extent, by UGT1A9.     

Effect of the beta-glucuronidase inhibitor saccharolactone on glucuronidation by human tissue microsomes and recombinant UDP-glucuronosyltransferases

Glucuronidation studies using microsomes and recombinant uridine diphosphoglucuronosyltransferases (UGTs) can be complicated by the presence of endogenous beta-glucuronidases, leading to underestimation of glucuronide formation rates. Saccharolactone is the most frequently used beta-glucuronidase inhibitor, although it is not clear whether this reagent should be added routinely to glucuronidation incubations. Here we have determined the effect of saccharolactone on eight different UGT probe activities using pooled human liver microsomes (pHLMs) and recombinant UGTs (rUGTs). Despite the use of buffered incubation solutions, it was necessary to adjust the pH of saccharolactone solutions to avoid effects (enhancement or inhibition) of lowered pH on UGT activity. Saccharolactone at concentrations ranging from 1 to 20 mM did not enhance any of the glucuronidation activities evaluated that could be considered consistent with inhibition of beta-glucuronidase. However, for most activities, higher saccharolactone concentrations resulted in a modest degree of inhibition. The greatest inhibitory effect was observed for glucuronidation of 5-hydroxytryptamine and estradiol by pHLMs, with a 35% decrease at 20 mM saccharolactone concentration. Endogenous beta-glucuronidase activities were also measured using various human tissue microsomes and rUGTs with estradiol-3-glucuronide and estradiol-17-glucuronide as substrates. Glucuronide hydrolysis was observed for pHLMs, lung microsomes and insect-cell expressed rUGTs, but not for kidney, intestinal or human embryonic kidney HEK293 microsomes. However, the extent of hydrolysis was relatively small, representing only 9-19% of the glucuronide formation rate measured in the same preparations. Consequently, these data do not support the routine inclusion of saccharolactone in glucuronidation incubations. If saccharolactone is used, concentrations should be titrated to achieve activity enhancement without inhibition.     

Synthesis and identification of the quaternary ammonium-linked glucuronide of 1-phenylimidazole in human liver microsomes and investigation of the human UDP-glucuronosyltransferases involved.

1-Phenylimidazole was investigated as a potential model substrate with respect to formation of a quaternary ammonium-linked glucuronide (N(+)-glucuronide) at an aromatic type tertiary amine. A reference sample of the potential N(+)-glucuronide metabolite of 1-phenylimidazole was obtained by organic synthesis. The structural identity of the metabolite formed by incubation of 1-phenylimidazole with human liver microsomes was proven to be the N(+)-glucuronide by exhibiting the same HPLC retention time and electrospray ionization mass spectrum as the reference sample. The screening of 1-phenylimidazole against a panel of nine expressed human UDP-glucuronosyltransferases indicated the involvement of UGT1A3 and UGT1A4 in the formation of the N(+)-glucuronide metabolite.