Glucuronidation of anti-HIV drug candidate bevirimat: identification of human UDP-glucuronosyltransferases and species differences.

Bevirimat [BVM, PA-457, 3-O-(3',3'-dimethylsuccinyl)-betulinic acid], a new anti-human immunodeficiency virus drug candidate, is metabolized to two monoglucuronides [mono-BVMG (I) and mono-BVMG (II)] and one diglucuronide (di-BVMG) both in vivo and in vitro. UDP-glucuronosyltransferase (UGT) reaction screening, enzyme kinetics, and species differences for the glucuronidation of BVM in vitro were investigated with pooled human liver microsomes (HLMs) and human intestinal microsomes (HIMs), animal liver microsomes, and 12 recombinant human UGT isoforms. Glucuronidation of BVM with HLMs predominantly involved the formation of mono-BVMG (I) (V(max) = 61 pmol/min/mg protein, K(m) = 27 microM) and mono-BVMG (II) (V(max) = 48 pmol/min/mg protein, K(m) = 16 microM). Di-BVMG was also observed but was a minor metabolite. HIMs mainly revealed glucuronidation to form mono-BVMG (II) (V(max) = 90 pmol/min/mg of protein, K(m) = 8.3 microM). UGT1A3 predominantly formed mono-BVMG (I) (V(max) = 65 pmol/min/mg of protein, K(m) = 13 microM), whereas UGT1A4 is a less active isoform (V(max) = 1.8 pmol/min/mg of protein, K(m) = 5.6 microM). UGT2B7 was involved in the formation of both mono-BVMG (I) (V(max) = 6.1 pmol/min/mg of protein, K(m) = 6.0 microM) and mono-BVMG (II) (V(max) = 6.5 pmol/min/mg of protein, K(m) = 7.8 microM). Among the animal liver microsomes examined, all species (rat, mouse, dog, and marmoset) demonstrated conjugation to form both mono-BVMG (I) and mono-BVMG (II), with dog liver microsomes exhibiting a higher formation rate for mono-BVMG (I), whereas marmoset liver microsomes showed a higher formation rate for mono-BVMG (II). The data suggest a primary role of UGT1A3 for the glucuronidation of BVM.     

Glucuronidation of etoposide in human liver microsomes is specifically catalyzed by UDP-glucuronosyltransferase 1A1.

A metabolite formed by incubation of human liver microsomes, etoposide, and UDP-glucuronic acid was identified as etoposide glucuronide by liquid chromatography-tandem mass spectrometry analysis. According to the derivatization with trimethylsilylimidazole (Tri-Sil-Z), it was confirmed that the glucuronic acid is linked to an alcoholic hydroxyl group of etoposide and not to a phenolic group. Among nine recombinant human UGT isoforms (UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A8, UGT1A9. UGT1A10, UGT2B7, and UGT2B15), only UGT1A1 exhibited the catalytic activity of etoposide glucuronidation. The enzyme kinetics in pooled human liver microsomes and recombinant UGT1A1 microsomes showed a typical Michaelis-Menten plot. The kinetic parameters of etoposide glucuronidation were K(m) = 439.6 +/- 70.7 microM and V(max) = 255.6 +/- 19.2 pmol/min/mg of protein in human liver microsomes and K(m) = 503.2 +/- 110.2 microM and V(max) = was 266.5 +/- 28.6 pmol/min/mg of protein in recombinant UGT1A1. The etoposide glucuronidation in pooled human liver microsomes was inhibited by bilirubin (IC(50) = 31.7 microM) and estradiol (IC(50) = 34 microM) as typical substrates for UGT1A1. The inhibitory effects of 4-nitrophenol (IC(50) = 121.0 microM) as a typical substrate for UGT1A6 and UGT1A9, imipramine (IC(50) = 393.8 microM) as a typical substrate for UGT1A3 and UGT1A4, and morphine (IC(50) = 109.3 microM) as a typical substrate for UGT2B7 were relatively weak. The interindividual difference in etoposide glucuronidation in 13 human liver microsomes was 78.5-fold (1.4-109.9 pmol/min/mg of protein). The etoposide glucuronidation in 10 to 13 human liver microsomes was significantly correlated with beta-estradiol-3-glucuronidation (r = 0.841, p < 0.01), bilirubin glucuronidation (r = 0.935, p < 0.01), and the immunoquantified UGT1A1 protein content (r = 0.800, p < 0.01). These results demonstrate that etoposide glucuronidation in human liver microsomes is specifically catalyzed by UGT1A1.     

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

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. 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. 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 (IC(50)) values of 173.6 and 76.2 μM, respectively. 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.     

Involvement of human UGT2B7 and 2B15 in rofecoxib metabolism.

O-Glucuronidation of 5-hydroxyrofecoxib is the major biotransformation pathway of rofecoxib in human, rat, and dog. The glucuronide conjugate is also involved in the reversible metabolism of rofecoxib in rat and human. Atypical bimodal phenomena were observed in their plasma concentration-time curves with a large variability among different human subjects. It is unclear which family members of human UDP-glucuronosyltransferases (UGT) are involved in the formation of the glucuronide. O-Glucuronidation of 5-hydroxyrofecoxib by human liver microsomes and eight cDNA-expressed human UGT isoforms were investigated. Human liver microsomes formed 5-hydroxyrofecoxib glucuronide with apparent V(max) value of 1736 pmol/min/mg of protein and K(m) value of 44.2 microM. Eight individual cDNA-expressed human UGT isozymes (1A1, 1A3, 1A4, 1A6, 1A8, 1A9, 2B7, and 2B15) were evaluated for glucuronidation of 5-hydroxyrofecoxib. Among them UGT2B15 exhibited the highest metabolism rate with apparent V(max) value of 286 pmol/min/mg of protein and K(m) value of 16.1 microM, whereas UGT2B7 showed apparent V(max) value of 47.1 pmol/min/mg of protein and K(m) value of 41.6 microM. These results indicated that human UGT2B15 has the highest level of activity for catalyzing the glucuronidation of 5-hydroxyrofecoxib. Because polymorphisms have been identified in human UGT2B7, 2B15 genes and O-glucuronidation of 5-hydroxyrofecoxib plays a major role in biotransformation of rofecoxib, it is possible that human UGT2B7 and 2B15 polymorphisms for O-glucuronidation of 5-hydroxyrofecoxib are responsible for the high variability in bimodal patterns in human plasma concentration-time curves.     

Involvement of human hepatic UGT1A1, UGT2B4, and UGT2B7 in the glucuronidation of carvedilol.

Carvedilol ((+/-)-1-carbazol-4-yloxy)-3-[[2-(o-methoxyphenoxy)ethyl]amino]-2-propanol) is metabolized primarily into glucuronide conjugates. In the present study, we identified the human UDP-glucuronosyltransferase (UGT) isoforms involved in the glucuronidation of carvedilol by thin-layer chromatography using microsomes from human liver or insect cells expressing recombinant UGT isoforms. We observed two forms of carvedilol glucuronides, namely G1 and G2, in hepatic microsomes. The glucuronidation of carvedilol was catalyzed by at least three recombinant UGT isoforms: UGT1A1, UGT2B4, and UGT2B7. UGT2B4 formed both G1 and G2, whereas UGT1A1 and UGT2B7 were responsible for the formation of glucuronide G2 and G1, respectively. The enzyme kinetics for carvedilol glucuronidation by UGT1A1, UGT2B4, and UGT2B7 in addition to human liver microsomes were examined by Lineweaver-Burk analysis. The values of Km and Vmax for human liver microsomes were 26.6 microM and 106 pmol/min/mg protein for G1, and 46.0 microM and 44.5 pmol/min/mg protein for G2, respectively. The Km values for UGT1A1, UGT2B4, and UGT2B7 for G1 and G2 (22.1-55.1 microM) were comparable to those of the liver microsomes, whereas the Vmax values were in the range of 3.33 to 7.88 pmol/min/mg protein. The Km and Vmax/Km values for UGT2B4 and UGT2B7 for G1 were similar, whereas UGT2B4 had lower Km and higher Vmax/Km values for G2 compared with those of UGT1A1. These results suggest that G1 formation is catalyzed by UGT2B4 and UGT2B7, whereas G2 is formed by UGT2B4 and UGT1A1. These three hepatic UGT isoforms may have important roles in carvedilol metabolism.     

Sulfinpyrazone C-glucuronidation is catalyzed selectively by human UDP-glucuronosyltransferase 1A9.

The uricosuric agent sulfinpyrazone (SFZ) is metabolized via C-glucuronidation, an uncommon metabolic pathway, in humans. The present study aimed to characterize SFZ glucuronidation by human liver microsomes (HLMs) and identify the hepatic forms of UDP-glucuronosyltransferase responsible for this pathway. Incubations of SFZ with HLMs formed a single glucuronide that was resistant to beta-glucuronidase and acid hydrolysis, consistent with formation of a C-glucuronide. Mass spectral analysis confirmed the identity of the metabolite as SFZ glucuronide (sulfinpyrazone beta-D-glucuronide; SFZG). SFZ C-glucuronidation by HLMs exhibited Michaelis-Menten kinetics, with mean (+/- S.D.) Km and Vmax values of 51 +/- 21 microM and 2.6 +/- 0.6 pmol/min . mg, respectively. Fifteen recombinant human UDP-glucuronosyltransferases (UGTs), expressed in HEK293 cells, were screened for their capacity to catalyze SFZ C-glucuronidation. Of the hepatically expressed enzymes, only UGT1A9 formed SFZG. UGTs 1A7 and 1A10, which are expressed in the gastrointestinal tract, also metabolized SFZ, but rates of metabolism were low compared with UGT1A9. SFZ glucuronidation by UGT1A9 exhibited "weak" negative cooperative kinetics, which was modeled by the Hill equation (S50 16 microM). The data indicate that UGT1A9 is the enzyme responsible for hepatic SFZ C-glucuronidation and that SFZ may be used as a substrate "probe" for UGT1A9 activity in HLMs.     

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.     

Glucuronidation of antiallergic drug, Tranilast: identification of human UDP-glucuronosyltransferase isoforms and effect of its phase I metabolite.

Tranilast is an oral antiallergic agent widely used in Japan. Recently, in Western populations, hyperbilirubinemia induced by tranilast was suspected during clinical trials. Tranilast has been reported to be mainly metabolized to a glucuronide and a phase I metabolite, 4-demethyltranilast (N-3). In the present study, we investigated the in vitro metabolism of tranilast in human liver and jejunum microsomes and recombinant UDP-glucuronosyltransferases (UGTs). The glucuronidation of tranilast was clarified to be mainly catalyzed by UGT1A1 in human liver and intestine. The K(m) values of tranilast glucuronosyltransferase activity were 51.5, 50.6, and 38.0 microM in human liver microsomes, human jejunum microsomes, and recombinant UGT1A1, respectively. The V(max) values were 10.4, 42.9, and 19.7 pmol/min/mg protein in human liver microsomes, human jejunum microsomes, and recombinant UGT1A1, respectively. When the intrinsic clearance was calculated using the in vitro kinetic parameters, microsomal protein content, and weight of tissues, tranilast glucuronosyltransferase activity was 2.5-fold higher in liver than in intestine. Tranilast glucuronosyltransferase activity was strongly inhibited by bilirubin, a typical UGT1A1 substrate, and N-3, indicating that the phase I metabolite could affect the tranilast glucuronosyltransferase activity. In the case of N-3 formation, the K(m) and V(max) values were 37.1 microM and 27.6 pmol/min/mg protein in human liver microsomes. The bilirubin glucuronosyltransferase activity was strongly inhibited by both tranilast and N-3, suggesting that tranilast-induced hyperbilirubinemia would be responsible for the inhibition by tranilast and N-3 of the bilirubin glucuronosyltransferase activity, as would the UGT1A1 genotype.     

Characterization of N-glucuronidation of 4-(5-pyridin-4-yl-1H-[1,2,4]triazol-3-yl) pyridine-2-carbonitrile (FYX-051): a new xanthine oxidoreductase inhibitor.

In humans, orally administered 4-(5-pyridin-4-yl-1H-[1,2,4]triazol-3-yl) pyridine-2-carbonitrile (FYX-051) is excreted mainly as triazole N(1)- and N(2)-glucuronides in urine. It is important to determine the enzyme(s) that catalyze the metabolism of a new drug to estimate individual differences and/or drug-drug interactions. Therefore, the characterization and mechanism of these glucuronidations were investigated using human liver microsomes (HLMs), human intestinal microsomes (HIMs), and recombinant human UDP-glucuronosyltransferase (UGT) isoforms to determine the UGT isoform(s) responsible for FYX-051 N(1)- and N(2)-glucuronidation. FYX-051 was metabolized to its N(1)- and N(2)-glucuronide forms by HLMs, and their K(m) values were 64.1 and 72.7 microM, respectively; however, FYX-051 was scarcely metabolized to its glucuronides by HIMs. Furthermore, among the recombinant human UGT isoforms, UGT1A1, UGT1A7, and UGT1A9 catalyzed the N(1)- and N(2)-glucuronidation of FYX-051. To estimate their contribution to FYX-051 glucuronidation, inhibition analysis with pooled HLMs was performed. Mefenamic acid, a UGT1A9 inhibitor, decreased FYX-051 N(1)- and N(2)-glucuronosyltransferase activities, whereas bilirubin, a UGT1A1 inhibitor, did not affect these activities. Furthermore, in the experiment using microsomes from eight human livers, the N(1)- and N(2)-glucuronidation activity of FYX-051 was found to significantly correlate with the glucuronidation activity of propofol, a specific substrate of UGT1A9 (N(1): r(2) = 0.868, p < 0.01; N(2): r(2) = 0.775, p < 0.01). These results strongly suggested that the N(1)- and N(2)-glucuronidation of FYX-051 is catalyzed mainly by UGT1A9 in human livers.     

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.

     

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

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. The carvacrol glucuronidation was characterized by human liver microsomes (HLMs), human intestinal microsomes (HIMs) and 12 recombinant UGT (rUGT) isoforms. One metabolite was identified as a mono-glucuronide by liquid chromatography/mass spectrometry with HLMs, HIMs, rUGT1A3, rUGT1A6, rUGT1A7, rUGT1A9 and rUGT2B7. 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.     

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.     

Metabolism and pharmacokinetics of morinidazole in humans: identification of diastereoisomeric morpholine N+-glucuronides catalyzed by UDP glucuronosyltransferase 1A9

Morinidazole [R,S-1-(2-methyl-5-nitro-1H-imidazol-1-yl)-3-morpholinopropan-2-ol] is a new 5-nitroimidazole class antimicrobial agent. The present study aimed to determine the metabolism and pharmacokinetics of morinidazole in humans and to identify the enzymes responsible for the formation of the major metabolites. Plasma and urine samples were collected before and after an intravenous drip infusion of 500 mg of racemic morinidazole. Ultraperformance liquid chromatography/quadrupole time-of-flight mass spectrometry revealed 10 metabolites. Morinidazole glucuronidation, followed by renal excretion, was the major elimination pathway, accounting for 35% of the dose. The metabolic pathway displayed regioselectivities and stereoselectivities. Unexpectedly, the nitrogen atom of the morpholine ring, rather than the aliphatic hydroxyl group at the side chain, was glucuronidated to form S-morinidazole glucuronide (M8-1) and R-enantiomer glucuronide (M8-2). The plasma exposure of M8-2 was 6-fold higher than that of M8-1, accounting for 22.9 and 3.96% of the parent drug exposure, respectively. Investigation of morinidazole glucuronidation using human liver microsomes (HLMs) and 12 recombinant UDP glucuronosyltransferases (UGTs) indicated that this biotransformation was mainly catalyzed by UGT1A9. A kinetic study showed that N(+)-glucuronidation of racemic morinidazole in both HLMs and in UGT1A9 obeyed a typical Michaelis-Menten plot. The K(m) values for M8-1 and M8-2 formation by HLMs were similar (11.3 and 15.1 mM), but the V(max) values were significantly different (111 and 1660 pmol · min(-1) · mg protein(-1)). Overall, after an intravenous administration, morinidazole and its metabolites were eliminated in humans primarily via renal excretion. The major metabolites were two diastereoisomeric N(+)-glucuronides, and UGT1A9 played an important role in N(+)-glucuronidation.     

Conjugation of the enantiomers of ketotifen to four isomeric quaternary ammonium glucuronides in humans in vivo and in liver microsomes.

The antiallergic drug ketotifen is chiral due to a nonplanar seven-membered ring containing a keto group. Earlier studies have revealed glucuronidation at the tertiary amino group as a major metabolic pathway in humans. Chemical synthesis of glucuronides from racemic ketotifen now led to four isomers separable by HPLC of which two each could be ascribed to (R)-(+)- and (S)-(-)-ketotifen by synthesis from the enantiomers. According to (1)H NMR analysis of the (S)-ketotifen N-glucuronides, the conformation of the piperidylidene ring differs between the two isomers. Enzymatic hydrolysis with Escherichia coli beta-glucuronidase proceeded at a lower rate with the slower eluting (S)-ketotifen glucuronide than with the three other isomers. On incubation of the ketotifen enantiomers (0.5-200 microM) with human liver microsomes in the presence of UDP-glucuronic acid and Triton X-100, the N-glucuronides of (R)-ketotifen were produced with an apparent K(M) 15 microM and V(max) 470 pmol/min/mg protein. The two (S)-ketotifen glucuronides were formed by two-enzyme kinetics with K(M1) 1.3 microM and K(M2) 92 microM and V(max) values of 60 and 440 pmol/min/mg protein. After ingestion of 1 mg of racemic ketotifen, 10 healthy subjects excreted in urine 17 +/- 5% of the dose in the form of N-glucuronides. The (R)-ketotifen glucuronide isomers contributed one-sixth only, whereas the remainder consisted primarily of the (S)-ketotifen glucuronide isomer, which eluted last. Differential hydrolysis or membrane transport may be responsible for the discrepancy between N-glucuronide isomer ratios in vitro and in vivo.     

Characterization of rat and human UDP-glucuronosyltransferases responsible for the in vitro glucuronidation of diclofenac.

In the current study, the identification of the rat and human UDP-glucuronosyltransferase (UGT) isoforms responsible for the glucuronidation of diclofenac was determined. Recombinant human UGT1A9 catalyzed the glucuronidation of diclofenac at a moderate rate of 166-pmol/min/mg protein, while UGT1A6 and 2B15 catalyzed the glucuronidation of diclofenac at low rates (<20-pmol/min/mg protein). Conversely, human UGT2B7 displayed a high rate of diclofenac glucuronide formation (>500 pmol/min/mg protein). Recombinant rat UGT2B1 catalyzed the glucuronidation of diclofenac at a rate of 250-pmol/min/mg protein. Rat UGT2B1 and human UGT2B7 displayed a similar, low apparent Km value of <15 microM for both UGT isoforms and high Vmax values 0.3 and 2.8 nmol/min/mg, respectively. Using diclofenac as a substrate, enzyme kinetics in rat and human liver microsomes showed that the enzyme(s) involved in diclofenac glucuronidation had a low apparent Km value of <20 microM and a high Vmax value of 0.9 and 4.3 nmol/min/mg protein, respectively. Morphine is a known substrate for rat UGT2B1 and human UGT2B7 and both total morphine glucuronidation (3-O- and 6-O-glucuronides) and diclofenac glucuronidation reactions showed a strong correlation with one another in human liver microsome samples. In addition, diclofenac inhibited the glucuronidation of morphine in human liver microsomes. These data suggested that rat UGT2B1 and human UGT2B7 were the major UGT isoforms involved in the glucuronidation of diclofenac.     

Protein quantification of UDP-glucuronosyltransferases 1A1 and 2B7 in human liver microsomes by LC-MS/MS and correlation with glucuronidation activities

The aims of this study were to quantify absolute protein levels of uridine 5'-diphosphate-glucuronosyltransferases (UGTs) 1A1 and 2B7 in human liver microsomes (HLMs) and to investigate their correlation with marker activities. A quantification method for UGT1A1 and UGT2B7 in HLMs was developed. Unique tryptic peptides of UGT1A1 and UGT2B7 in tryptically digested HLMs were simultaneously quantified by liquid chromatography (LC) equipped with tandem mass spectrometry (MS) using corresponding stable isotope-labelled peptides as internal standards. Bovine serum albumin was used as a blank matrix for calibration curve samples. Our procedure had good digestion efficiency, sensitivity, calibration curve linearity, and reproducibility of digestion to quantification. In 16 individual HLMs, the protein levels of UGT1A1 and UGT2B7 ranged from 6.50 to 44.6 pmol/mg and 4.45 to 18.2 pmol/mg, respectively. Estradiol 3β-glucuronidation correlated strongly with the UGT1A1 level, indicating its high reliability as a reaction marker. Both morphine 3-O- and 6-O-glucuronidation significantly correlated with UGT2B7 level. However, the intercept of the linear regression clearly indicates that morphine glucuronidation was mediated by other UGT isoforms in addition to UGT2B7.     

Biosynthesis of imipramine glucuronide and characterization of imipramine glucuronidation catalyzed by recombinant UGT1A4

AIM: To study the profile of imipramine N+-glucuronidation using homogenates of recombinant uridine-5'-diphosphoglucuronosyltransferase 1A4 (UGT1A4) from baculovirus-infected sf9 cells. METHODS: Recombinant UGT1A4 was obtained from sf9 cells infected with recombinant baculovirus. Imipramine N(+)-glucuronide was biosynthesized by incubating imipramine with recombinant UGT1A4 and then purified with solid-phase cartridges. A reversed phase-high pressure liquid chromatography (RP-HPLC) assay method was used to directly measure the concentration of imipramine and its metabolite, imipramine N(+)-glucuronide, with p-nitrophenol as the internal standard. The validated method was used to characterize the activity of recombinant UGT1A4 and carry out kinetic studies on imipramine glucuronidation in vitro. RESULTS: The high concentration of imipramine inhibited glucuronide conjugation, so the formula V=V(max).S/(Km+S+S(2)/K(i)) was used to calculate the parameters, using MATLAB software. The values of apparent K(m), K(i), and V(max) for imipramine glucuronidation via UGT1A4 were 1.39+/-0.09 mmol/L, 6.24+/-0.45 mmol/L and 453.81+/-32.12 pmol/min per mg cell homogenate (n=3), respectively. CONCLUSION: As a specific substrate of UGT1A4, imipramine was used as a convenient method to characterize the activity of recombinant UGT1A4 by using HPLC. Furthermore, the profile of imipramine glucuronidation was evaluated by using recombinant UGT1A4 in vitro.     

Atorvastatin glucuronidation is minimally and nonselectively inhibited by the fibrates gemfibrozil, fenofibrate, and fenofibric acid.

Gemfibrozil coadministration generally results in plasma statin area under the curve (AUC) increases, ranging from moderate (2- to 3-fold) with simvastatin, lovastatin, and pravastatin to most significant with cerivastatin (5.6-fold). Inhibition of statin glucuronidation has been postulated as a potential mechanism of interaction (Drug Metab Dispos 30:1280-1287, 2002). This study was conducted to determine the in vitro inhibitory potential of fibrates toward atorvastatin glucuronidation. [(3)H]Atorvastatin, atorvastatin, and atorvastatin lactone were incubated with human liver microsomes or human recombinant UDP-glucuronosyltransferases (UGTs) and characterized using liquid chromatography (LC)/tandem mass spectrometry and LC/UV/beta-radioactivity monitor/mass spectrometry. [(3)H]Atorvastatin yields a minor ether glucuronide (G1) and a major acyl glucuronide (G2) with subsequent pH-dependent lactonization of G2 to yield atorvastatin lactone. Atorvastatin lactonization best fit substrate inhibition kinetics (K(m) = 12 microM, V(max) = 74 pmol/min/mg, K(i) = 75 microM). Atorvastatin lactone yields a single ether glucuronide (G3). G3 formation best fit Michaelis-Menten kinetics (K(m) = 2.6 microM, V(max) = 10.6 pmol/min/mg). Six UGT enzymes contribute to atorvastatin glucuronidation with G2 and G3 formation catalyzed by UGTs 1A1, 1A3, 1A4, 1A8, and 2B7, whereas G1 formation was catalyzed by UGTs 1A3, 1A4, and 1A9. Gemfibrozil, fenofibrate, and fenofibric acid inhibited atorvastatin lactonization with IC(50) values of 346, 320, and 291 microM, respectively. Based on unbound fibrate concentrations at the inlet to the liver, these data predict a small increase in atorvastatin AUC (approximately 1.2-fold) after gemfibrozil coadministration and no interaction with fenofibrate. This result is consistent with recent clinical reports indicating minimal atorvastatin AUC increases ( approximately 1.2- to 1.4-fold) with gemfibrozil.     

N-Glucuronidation of some 4-arylalkyl-1H-imidazoles by rat, dog, and human liver microsomes.

N-Glucuronidation in vitro of six 4-arylalkyl-1H-imidazoles (both enantiomers of medetomidine, detomidine, atipamezole, and two other closely related compounds) by rat, dog, and human liver microsomes and by four expressed human UDP-glucuronosyltransferase isoenzymes was studied. Human liver microsomes formed N-glucuronides of 4-arylalkyl-1H-imidazoles with high activity, with apparent V(max) values ranging from 0.59 to 1.89 nmol/min/mg of protein. In comparison, apparent V(max) values for two model compounds forming the N-glucuronides 4-aminobiphenyl and amitriptyline were 5.07 and 0.56 nmol/min/mg of protein, respectively. Atipamezole showed an exceptionally low apparent K(m) value of 4.0 microM and a high specificity constant (V(max)/K(m)) of 256 compared with 4-aminobiphenyl (K(m), 265 microM; V(max)/K(m), 19) and amitriptyline (K(m), 728 microM; V(max)/K(m), 0.8). N-Glucuronidation of medetomidine was highly enantioselective in human liver microsomes; levomedetomidine exhibited a 60-fold V(max)/K(m) value compared with dexmedetomidine. Furthermore, two isomeric imidazole N-glucuronides were formed from dexmedetomidine, but only one was formed from levomedetomidine. Dog liver microsomes formed N-glucuronides of 4-arylalkyl-1H-imidazoles at a low rate and affinity, with apparent V(max) values ranging from 0.29 to 0.73 nmol/min/mg of protein and apparent K(m) values from 279 to 1640 microM. Rat liver microsomes glucuronidated these compounds at a barely detectable rate. Four expressed human UDP-glucuronosyltransferase isoenzymes (UGT1A3, UGT1A4, UGT1A6, and UGT1A9) were studied for 4-arylalkyl-1H-imidazole-conjugating activity. Only UGT1A4 glucuronidated these compounds at an activity of about 5% of that measured for 4-aminobiphenyl. The observed activity of UGT1A4 does not explain the high efficiency of glucuronidation of 4-arylalkyl-1H-imidazoles in human liver microsomes.     

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.