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.     

Regioselective glucuronidation of denopamine: marked species differences and identification of human udp-glucuronosyltransferase isoform.

Denopamine is one of the oral beta(1)-adrenoceptor-selective partial agonists. Denopamine glucuronide is the most abundant metabolite in human, rat, and dog urine when administered orally. Species differences in denopamine glucuronidation were investigated with liver microsomes obtained from humans and experimental animals. In rat and rabbit, only the phenolic glucuronide was detected, whereas in dog and monkey, not only the phenolic glucuronide but also the alcoholic glucuronide was found. In contrast, in humans, the alcoholic glucuronide was detected exclusively. The kinetics of denopamine glucuronidation in human liver microsomes showed a typical Michaelis-Menten plot. The K(m) and V(max) values accounted for 2.87 +/- 0.17 mM and 7.29 +/- 0.23 nmol/min/mg protein, respectively. With the assessment of denopamine glucuronide formation across a panel of recombinant UDP-glucuronosyltransferase (UGT) isoforms (UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B4, UGT2B7, UGT2B15, and UGT2B17), only UGT2B7 exhibited high denopamine glucuronosyltransferase activity. The K(m) value of denopamine glucuronidation in recombinant UGT2B7 microsomes was close to those in human liver and jejunum microsomes. The formation of denopamine glucuronidation by human liver, jejunum, and recombinant UGT2B7 microsomes was effectively inhibited by diclofenac, a known substrate for UGT2B7. The denopamine glucuronidation activities in seven human liver microsomes were significantly correlated with diclofenac glucuronidation activities (r(2) = 0.685, p < 0.05). These results demonstrate that the denopamine glucuronidation in human liver and intestine is mainly catalyzed by UGT2B7 and that glucuronidation of the alcoholic hydroxyl group, but not the phenolic hydroxyl group, occurs regioselectively in humans.     

Identification of human UDP-glucuronosyltransferase isoform(s) responsible for the glucuronidation of 2-(4-chlorophenyl)- 5-(2-furyl)-4-oxazoleacetic acid (TA-1801A)

We characterized the hepatic and intestinal UDP-glucuronosyltransferase (UGT) isoform(s) responsible for the glucuronidation of 2-(4-chlorophenyl)-5-(2-furyl)-4-oxazoleacetic acid (TA-1801A) in humans through several in vitro mechanistic studies. Assessment of a panel of recombinant UGT isoforms revealed the TA-1801A glucuronosyltransferase activity of UGT1A1, UGT1A3, UGT1A7, UGT1A9, and UGT2B7. Kinetic analyses of the TA-1801A glucuronidation by recombinant UGT1A1, UGT1A3, UGT1A9, and UGT2B7 showed that the K(m) value for UGT2B7 was apparently consistent with those in human liver and jejunum microsomes. The TA-1801A glucuronosyltransferase activity in human liver microsomes was inhibited by bilirubin (typical substrate for UGT1A1), propofol (typical substrate for UGT1A9), diclofenac (substrate for UGT1A9 and UGT2B7), and genistein (substrate for UGT1A1, UGT1A3, and UGT1A9). The inhibition by bilirubin, propofol, and diclofenac of the TA-1801A glucuronidation was less pronounced in jejunum microsomes than liver microsomes, suggesting that the contribution of UGT1A1, UGT1A9, and UGT2B7 to the TA-1801A glucuronidation is smaller in the intestine than the liver. In contrast, genistein strongly inhibited the TA-1801A glucuronosyltransferase activity in both human liver and jejunum microsomes. These results suggest that the glucuronidation of TA-1801A is mainly catalyzed by UGT1A1, UGT1A9, and UGT2B7 in the liver, and by UGT1A1, UGT1A3, and UGT2B7 in the intestine in humans.     

Characterization of in vitro glucuronidation clearance of a range of drugs in human kidney microsomes: comparison with liver and intestinal glucuronidation and impact of albumin

Previous studies have shown the importance of the addition of albumin for characterization of hepatic glucuronidation in vitro; however, no reports exist on the effects of albumin on renal or intestinal microsomal glucuronidation assays. This study characterized glucuronidation clearance (CL(int, UGT)) in human kidney, liver, and intestinal microsomes in the presence and absence of bovine serum albumin (BSA) for seven drugs with differential UDP-glucuronosyltransferase (UGT) 1A9 and UGT2B7 specificity, namely, diclofenac, ezetimibe, gemfibrozil, mycophenolic acid, naloxone, propofol, and telmisartan. The impact of renal CL(int, UGT) on accuracy of in vitro-in vivo extrapolation (IVIVE) of glucuronidation clearance was investigated. Inclusion of 1% BSA for acidic drugs and 2% for bases/neutral drugs in incubations was found to be suitable for characterization of CL(int, UGT) in different tissues. Although BSA increased CL(int, UGT) in all tissues, the extent was tissue- and drug-dependent. Scaled CL(int, UGT) in the presence of BSA ranged from 2.22 to 207, 0.439 to 24.4, and 0.292 to 23.8 ml · min(-1) · g tissue(-1) in liver, kidney, and intestinal microsomes. Renal CL(int, UGT) (per gram of tissue) was up to 2-fold higher in comparison with that for liver for UGT1A9 substrates; in contrast, CL(int, UGT) for UGT2B7 substrates represented approximately one-third of hepatic estimates. Scaled renal CL(int, UGT) (in the presence of BSA) was up to 30-fold higher than intestinal glucuronidation for the drugs investigated. Use of in vitro data obtained in the presence of BSA and inclusion of renal clearance improved the IVIVE of glucuronidation clearance, with 50% of drugs predicted within 2-fold of observed values. Characterization and consideration of kidney CL(int, UGT) is particularly important for UGT1A9 substrates.     

Altered AZT (3'-azido-3'-deoxythymidine) glucuronidation kinetics in liver microsomes as an explanation for underprediction of in vivo clearance: comparison to hepatocytes and effect of incubation environment.

Human liver microsomes are a reagent commonly used to predict human hepatic clearance of new chemical entities via phase 1 metabolism. Another common metabolic pathway, glucuronidation, can also be observed in human liver microsomes, although the scalability of this process has not been validated. In fact, several groups have demonstrated that clearance estimated from liver microsomes with UDP-glucuronic acid typically underpredicts the actual in vivo clearance more than 10-fold for compounds that are predominantly glucuronidated. In contrast, clearance predicted using human hepatocytes, for these same compounds, provides a more accurate assessment of in vivo clearance. We sought to characterize the kinetics of glucuronidation of the selective UGT2B7 substrate AZT (3'-azido-3'-deoxythymidine), a selective UGT2B7 substrate, in human liver microsomes (HLMs), recombinant UGT2B7, and human hepatocytes. Apparent Km values in these three preparations were 760, 490, and 87 microM with apparent Vmax values highest in hepatocytes. The IC50 for ibuprofen against AZT glucuronidation, when run at its Km concentration in HLMs and hepatocytes, was 975 and 170 microM respectively. Since incubation conditions have been shown to modulate glucuronidation rates, AZT glucuronidation was performed in various physiological and nonphysiological buffer systems, namely Tris, phosphate, sulfate, carbonate, acetate, human plasma, deproteinized human liver cytosol, and Williams E medium. The data showed that carbonate and Williams E medium, more physiologically relevant buffers, yielded the highest rates of AZT glucuronidation. Km observed in HLM/carbonate was 240 microM closer to that found in hepatocytes, suggesting that matrix differences might cause the kinetic differences observed between liver preparations. Caution should be exercised when extrapolating metabolic lability via glucuronidation or inhibition of UGT enzymes from human liver microsomes, since this system appears to underpredict the degree of lability or inhibition, respectively, due in part to an apparent decrease in substrate affinity.     

Carboxyl-glucuronidation of mitiglinide by human UDP-glucuronosyltransferases

Mitiglinide (MGN) is a new potassium channel antagonist for the treatment of type 2 diabetes mellitus. In the present study, a potential metabolic pathway of MGN, via carboxyl-linked glucuronic acid conjugation, was found. MGN carboxyl-glucuronide was isolated from a reaction mixture consisting of MGN and human liver microsomes fortified with UDP-glucuronic acid (UDPGA) and identified by a hydrolysis reaction with beta-glucuronidase and HPLC-MS/MS. Kinetic analysis indicated that MGN from four species had the highest affinity for the rabbit liver microsomal enzyme (K(m)=0.202 mM) and the lowest affinity for the dog liver microsomal enzyme (K(m)=1.164 mM). The metabolic activity (V(max)/K(m)) of MGN to the carboxyl-glucuronidation was in the following order: rabbit>dog>rat>human. With the assessment of MGN glucuronide formation across a panel of recombinant UDP-glucuronosyltransferase (UGT) isoforms (UGT1A3, UGT1A4, UGT1A6, UGT1A9, and UGT2B7), only UGT1A3 and UGT2B7 exhibited high MGN glucuronosyltransferase activity. The K(m) values of MGN glucuronidation in recombinant UGT1A3 and UGT2B7 microsomes were close to those in human liver microsomes. The formation of MGN glucuronidation by human liver microsomes was effectively inhibited by quercetin (substrate for UGT1A3) and diclofenac (substrate for UGT2B7), respectively. The MGN glucuronidation activities in 15 human liver microsomes were significantly correlated with quercetin (r(2)=0.806) and diclofenac glucuronidation activities (r(2)=0.704), respectively. These results demonstrate that UGT1A3 and UGT2B7 are catalytic enzymes in MGN carboxyl-glucuronidation in human liver.     

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

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

Drug-drug interaction potential of antitumor acridine agent C-1748: The substrate of UDP-glucuronosyltransferases 2B7, 2B17 and the inhibitor of 1A9 and 2B7

Background

The compound 9-(2′-hydroxyethylamino)-4-methyl-1-nitroacridine (C-1748), the promising antitumor agent developed in our laboratory was determined to undergo phase I metabolic pathways. The present studies aimed to know its biotransformation with phase II enzymes – UDP-glucuronosyltransferases (UGTs) and its potential to be engaged in drug-drug interactions arising from the modulation of UGT activity.

Predicting pharmacokinetics and drug interactions in patients from in vitro and in vivo models: the experience with 5,6-dimethylxanthenone-4-acetic acid (DMXAA), an anti-cancer drug eliminated mainly by conjugation

The novel anti-tumor agent 5,6-dimethylxanthenone-4-acetic acid (DMXAA) was developed in the Auckland Cancer Society Research Center. Its pharmacokinetic properties have been investigated using both in vitro and in vivo models, and the resulting data extrapolated to patients. The metabolism of DMXAA has been extensively studied mainly using hepatic microsomes, which indicated that UGT1A9 and UGT2B7-catalyzed glucuronidation on its acetic acid side chain and to a lesser extent CYP1A2-catalyzed hydroxylation of the 6-methyl group are the major metabolic pathways, resulting in DMXAA acyl glucuronide (DMXAA-G) and 6-hydroxymethyl-5-methylxanthenone-4-acetic acid. The predominant metabolite in human urine (up to 60% of total dose) was identified as DMXAA-G, which was chemically reactive, undergoing hydrolysis, intramolecular rearrangement, and covalent binding to plasma proteins. In vivo formation of DMXAA-protein adducts were also observed in cancer patients receiving DMXAA treatment. The comparison of the in vitro human hepatic microsomal metabolism and inhibition of DMXA by UGT and/or CYP substrates with animal species indicated species differences. Renal microsomes from all animal species examined had glucuronidation activity for DMXAA, but lower than the liver. In vitro-in vivo extrapolations based on human microsomal data indicated a 7-fold underestimation of plasma clearance in patients. In contrast, allometric scaling using in vivo data from the mouse, rat, and rabbit predicted a plasma clearance of 3.5 mL/min/kg, similar to that observed in patients (3.7 mL/min/kg). Based on in vitro metabolic inhibition studies, it appears possible to predict the effects on the plasma kinetic profile of DMXAA of drugs such as diclofenac, which are mainly metabolized by UGT2B7. However, it did not appear possible to predict the effect of thalidomide on the pharmacokinetics of DMXAA in patients based on in vitro inhibition and animal studies. These data indicate that preclincial pharmacokinetic studies using both in vitro and in vivo models play an important but different role in predicting pharmacokinetics and drug interactions in patients.     

The glucuronidation of morphine by dog liver microsomes: identification of morphine-6-O-glucuronide.

Canines are used extensively in the pharmaceutical industry for the preclinical screening of novel therapeutics, yet comparatively little is known about the phase 2 metabolism in this species. In humans, morphine is known to undergo extensive metabolism by glucuronidation, and the UDP-glucuronosyltransferase isoform, which catalyzes the formation of morphine-3-O-glucuronide and morphine-6-O-glucuronide is UGT2B7. This study was designed to investigate the glucuronidation of morphine using dog liver microsomes. Liver microsomes from beagle dogs catalyzed the glucuronidation of morphine-3(and 6)-O-glucuronide at rates 4 to 10 times that of rhesus monkey and human liver microsomes. The K(m) of morphine using beagle dog liver microsomes was approximately 270 microM, which is similar to that found for expressed human UGT2B7. The V(max) for morphine, using dog liver microsomes, was 27 nmol/min/mg of protein. Flunitrazepam inhibited the glucuronidation of morphine in dog liver microsomes, and the K(i) was 40 microM, which is similar to human UGT2B7 for other substrates. The effects of detergents were also investigated with dog liver microsomes, and Brij 35 and Brij 58 were found to be the best detergents to use for maximal activation of the dog liver morphine UGT. These studies suggest that dog has a UGT2B isoform similar to human UGT2B7.     

Identification of human UDP-glucuronosyltransferases involved in N-carbamoyl glucuronidation of lorcaserin

Lorcaserin, a selective serotonin 5-HT(2C) receptor agonist, is a weight management agent in clinical development. Lorcaserin N-carbamoyl glucuronidation governs the predominant excretory pathway of lorcaserin in humans. Human UDP-glucuronosyltransferases (UGTs) responsible for lorcaserin N-carbamoyl glucuronidation are identified herein. Lorcaserin N-carbamoyl glucuronide formation was characterized by the following approaches: metabolic screening using human tissues (liver, kidney, intestine, and lung) and recombinant enzymes, kinetic analyses, and inhibition studies. Whereas microsomes from all human tissues studied herein were found to be catalytically active for lorcaserin N-carbamoyl glucuronidation, liver microsomes were the most efficient. With recombinant UGT enzymes, lorcaserin N-carbamoyl glucuronidation was predominantly catalyzed by three UGT2Bs (UGT2B7, UGT2B15, and UGT2B17), whereas two UGT1As (UGT1A6 and UGT1A9) played a minor role. UGT2B15 was most efficient, with an apparent K(m) value of 51.6 ± 1.9 μM and V(max) value of 237.4 ± 2.8 pmol/mg protein/min. The rank order of catalytic efficiency of human UGT enzymes for lorcaserin N-carbamoyl glucuronidation was UGT2B15 > UGT2B7 > UGT2B17 > UGT1A9 > UGT1A6. Inhibition of lorcaserin N-carbamoyl glucuronidation activities of UGT2B7, UGT2B15, and UGT2B17 in human liver microsomes by mefenamic acid, bisphenol A, and eugenol further substantiated the involvement of these UGT2B isoforms. In conclusion, multiple human UGT enzymes catalyze N-carbamoyl glucuronidation of lorcaserin; therefore, it is unlikely that inhibition of any one of these UGT activities will lead to significant inhibition of the lorcaserin N-carbamoyl glucuronidation pathway. Thus, the potential for drug-drug interaction by concomitant administration of a drug(s) that is metabolized by any of these UGTs is remote.     

Characterization of hepatic and intestinal glucuronidation of magnolol: application of the relative activity factor approach to decipher the contributions of multiple UDP-glucuronosyltransferase isoforms

Magnolol is a food additive that is often found in mints and gums. Human exposure to this compound can reach a high dose; thus, characterization of magnolol disposition in humans is very important. Previous studies indicated that magnolol can undergo extensive glucuronidation in humans in vivo. In this study, in vitro assays were used to characterize the glucuronidation pathway in human liver and intestine. Assays with recombinant human UDP-glucuronosyltransferase enzymes (UGTs) revealed that multiple UGT isoforms were involved in magnolol glucuronidation, including UGT1A1, -1A3, -1A7, -1A8, -1A9, -1A10, and -2B7. Magnolol glucuronidation by human liver microsomes (HLM), human intestine microsomes (HIM), and most recombinant UGTs exhibited strong substrate inhibition kinetics. The degree of substrate inhibition was relatively low in the case of UGT1A10, whereas the reaction catalyzed by UGT1A9 followed biphasic kinetics. Chemical inhibition studies and the relative activity factor (RAF) approach were used to identify the individual UGTs that played important roles in magnolol glucuronidation in HLM and HIM. The results indicate that UGT2B7 is mainly responsible for the reaction in HLM, whereas UGT2B7 and UGT1A10 are significant contributors in HIM. In summary, the current study clarifies the glucuronidation pathway of magnolol and demonstrates that the RAF approach can be used as an efficient method for deciphering the roles of individual UGTs in a given glucuronidation pathway in the native tissue that is catalyzed by multiple isoforms with variable and atypical kinetics.     

Stereoselective conjugation of oxazepam by human UDP-glucuronosyltransferases (UGTs): S-oxazepam is glucuronidated by UGT2B15, while R-oxazepam is glucuronidated by UGT2B7 and UGT1A9.

(R,S)-Oxazepam is a 1,4-benzodiazepine anxiolytic drug that is metabolized primarily by hepatic glucuronidation. In previous studies, S-oxazepam (but not R-oxazepam) was shown to be polymorphically glucuronidated in humans. The aim of the present study was to identify UDP-glucuronosyltransferase (UGT) isoforms mediating R- and S-oxazepam glucuronidation in human liver, with the long term objective of elucidating the molecular genetic basis for this drug metabolism polymorphism. All available recombinant UGT isoforms were screened for R- and S-oxazepam glucuronidation activities. Enzyme kinetic parameters were then determined in representative human liver microsomes (HLMs) and in UGTs that showed significant activity. Of 12 different UGTs evaluated, only UGT2B15 showed significant S-oxazepam glucuronidation. Furthermore, the apparent K(m) for UGT2B15 (29-35 microM) was similar to values determined for HLMs (43-60 microM). In contrast, R-oxazepam was glucuronidated by UGT1A9 and UGT2B7. Although apparent K(m) values for HLMs (256-303 microM) were most similar to UGT2B7 (333 microM) rather than UGT1A9 (12 microM), intrinsic clearance values for UGT1A9 were 10 times higher than for UGT2B7. A common genetic variation results in aspartate (UGT2B15*1) or tyrosine (UGT2B15*2) at position 85 of the UGT2B15 protein. Microsomes from human embryonic kidney (HEK)-293 cells overexpressing UGT2B15*1 showed 5 times higher S-oxazepam glucuronidation activity than did UGT2B15*2 microsomes. Similar results were obtained for other substrates, including eugenol, naringenin, 4-methylumbelliferone, and androstane-3alpha-diol. In conclusion, S-oxazepam is stereoselectively glucuronidated by UGT2B15, whereas R-oxazepam is glucuronidated by multiple UGT isoforms. Allelic variation associated with the UGT2B15 gene may explain polymorphic S-oxazepam glucuronidation in humans.     

Inhibitory potential of nonsteroidal anti-inflammatory drugs on UDP-glucuronosyltransferase 2B7 in human liver microsomes

Objective A number of nonsteroidal anti-inflammatory drugs (NSAIDs) are subject to glucuronidation in humans, and UDP-glucuronosyltransferase (UGT) 2B7 is involved in the glucuronidation of many NSAIDs. The objective of this study was to identify a NSAID with potent inhibitory potential against UGT2B7 using liquid chromatography with tandem mass spectrometry (LC-MS/MS). Methods A rapid screening method for detecting the inhibitory potential of various drugs against UGT2B7 was established using a LC-MS/MS system. The effects of nine NSAIDs (acetaminophen, diclofenac, diflunisal, indomethacin, ketoprofen, mefenamic acid, naproxen, niflumic acid, and salicylic acid) against UGT2B7-catalyzed 3′-azido-3′-deoxythymidine glucuronidation (AZTG) were investigated in human liver microsomes (HLM) and recombinant human UGT2B7. Results Mefenamic acid inhibited AZTG most potently, with an IC₅₀ value of 0.3 μM, and its inhibition type was not competitive. The IC₅₀ values for diclofenac, diflunisal, indomethacin, ketoprofen, naproxen, and niflumic acid against AZTG were 6.8, 178, 51, 40, 23, and 83 μM, respectively, while those for acetaminophen and salicylic acid were >100 μM. The IC₅₀ values for NSAIDs against AZTG in recombinant human UGT2B7 were similar to those obtained in HLM. Conclusion The method established in this study is useful for identifying drugs with inhibitory potential against human UGT2B7. Among the nine NSAIDs investigated, mefenamic acid had the strongest inhibitory effect on UGT2B7-catalyzed AZTG in HLM. Thus, caution might be exercised when mefenamic acid is coadministered with drugs possessing UGT2B7 as a main elimination pathway.     

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.     

Udp-glucuronosyltransferase 2b7 is the major enzyme responsible for gemcabene glucuronidation in human liver microsomes.

The predominant metabolic pathway of gemcabene in humans is glucuronidation. The principal human UDP-glucuronosyltransferases (UGTs) involved in the glucuronidation of gemcabene were determined in this study. Glucuronidation of gemcabene was catalyzed by recombinant UGT1A3, recombinant UGT2B7, and recombinant UGT2B17, as well as by human liver microsomes (HLM). Gemcabene glucuronidation in recombinant UGTs and HLM followed non-Michaelis-Menten kinetics consistent with homotropic activation, but pharmacokinetics in humans were linear over the dose range tested (total plasma C(max), 0.06-0.88 mM). Gemcabene showed similar affinity (S(50)) for recombinant UGTs (0.92-1.45 mM) and HLM (1.37 mM). S-Flurbiprofen was identified as a more selective inhibitor of recombinant UGT2B7-catalyzed gemcabene glucuronidation (>23-fold lower IC(50)) when compared with recombinant UGT1A3- or recombinant UGT2B17-catalyzed gemcabene glucuronidation. The IC(50) for S-flurbiprofen inhibition of gemcabene glucuronidation was similar in HLM (60.6 microM) compared with recombinant UGT2B7 (27.4 microM), consistent with a major role for UGT2B7 in gemcabene glucuronidation in HLM. In addition, 5,6,7,3',4',5'-hexamethoxyflavone inhibited recombinant UGT1A3 and recombinant UGT2B17-catalyzed gemcabene glucuronidation (with 4-fold greater potency for recombinant UGT1A3) but did not inhibit gemcabene glucuronidation in HLM, suggesting that UGT1A3 and UGT2B17 do not contribute significantly to gemcabene glucuronidation. Reaction rates for gemcabene glucuronidation from a human liver bank correlated well (r(2)=0.722, P<0.0001; n=24) with rates of glucuronidation of the UGT2B7 probe substrate 3'-azido-3'-deoxythymidine. In conclusion, using the three independent experimental approaches typically used for cytochrome P450 reaction phenotyping, UGT2B7 is the major enzyme contributing to gemcabene glucuronidation in human liver microsomes.     

Acyl glucuronidation of fluoroquinolone antibiotics by the UDP-glucuronosyltransferase 1A subfamily in human liver microsomes.

Acyl glucuronidation is an important metabolic pathway for fluoroquinolone antibiotics. However, it is unclear which human UDP-glucuronosyltransferase (UGT) enzymes are involved in the glucuronidation of the fluoroquinolones. The in vitro formation of levofloxacin (LVFX), grepafloxacin (GPFX), moxifloxacin (MFLX), and sitafloxacin (STFX) glucuronides was investigated in human liver microsomes and cDNA-expressed recombinant human UGT enzymes. The apparent Km values for human liver microsomes ranged from 1.9 to 10.0 mM, and the intrinsic clearance values (calculated as Vmax/Km) had a rank order of MFLX > GPFX > STFX > > LVFX. In a bank of human liver microsomes (n = 14), the glucuronidation activities of LVFX, MFLX, and STFX correlated highly with UGT1A1-selective beta-estradiol 3-glucuronidation activity, whereas the glucuronidation activity of GPFX correlated highly with UGT1A9-selective propofol glucuronidation activity. Among 12 recombinant UGT enzymes, UGT1A1, 1A3, 1A7, and 1A9 catalyzed the glucuronidation of these fluoroquinolones. Results of enzyme kinetics studies using the recombinant UGT enzymes indicated that UGT1A1 most efficiently glucuronidates MFLX, and UGT1A9 most efficiently glucuronidates GPFX. In addition, the glucuronidation activities of MFLX and STFX in human liver microsomes were potently inhibited by bilirubin with IC50 values of 4.9 microM and 4.7 microM, respectively; in contrast, the glucuronidation activity of GPFX was inhibited by mefenamic acid with an IC50 value of 9.8 microM. These results demonstrate that UGT1A1, 1A3, and 1A9 enzymes are involved in the glucuronidation of LVFX, GPFX, MFLX, and STFX in human liver microsomes, and that MFLX and STFX are predominantly glucuronidated by UGT1A1, whereas GPFX is mainly glucuronidated by UGT1A9.     

The glucuronidation of Delta4-3-Keto C19- and C21-hydroxysteroids by human liver microsomal and recombinant UDP-glucuronosyltransferases (UGTs): 6alpha- and 21-hydroxyprogesterone are selective substrates for UGT2B7.

The stereo- and regioselective glucuronidation of 10 Delta(4)-3-keto monohydroxylated androgens and pregnanes was investigated to identify UDP-glucuronosyltransferase (UGT) enzyme-selective substrates. Kinetic studies were performed using human liver microsomes (HLMs) and a panel of 12 recombinant human UGTs as the enzyme sources. Five of the steroids, which were hydroxylated in the 6beta-, 7alpha-, 11beta- or 17alpha-positions, were not glucuronidated by HLMs. Of the remaining compounds, comparative kinetic and inhibition studies indicated that 6alpha- and 21-hydroxyprogesterone (OHP) were glucuronidated selectively by human liver microsomal UGT2B7. 6alpha-OHP glucuronidation by HLMs and UGT2B7 followed Michaelis-Menten kinetics, whereas 21-OHP glucuronidation by these enzyme sources exhibited positive cooperativity. UGT2B7 was also identified as the enzyme responsible for the high-affinity component of human liver microsomal 11alpha-OHP glucuronidation. In contrast, UGT2B15 and UGT2B17 were the major forms involved in human liver microsomal testosterone 17beta-glucuronidation and the high-affinity component of 16alpha-OHP glucuronidation. Activity of UGT1A subfamily enzymes toward the hepatically glucuronidated substrates was generally low, although UGT1A4 and UGT1A9 contribute to the low-affinity components of microsomal 16alpha- and 11alpha-OHP glucuronidation, respectively. Interestingly, UGT1A10, which is expressed only in the gastrointestinal tract, exhibited activity toward most of the glucuronidated substrates. The results indicate that 6alpha- and 21-OHP may be used as selective "probes" for human liver microsomal UGT2B7 activity and, taken together, provide insights into the regio- and stereoselectivity of hydroxysteroid glucuronidation by human UGTs.     

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

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

Preclinical Pharmacokinetics and In Vitro Metabolism of BMS-690514, a Potent Inhibitor of EGFR and VEGFR2

BMS-690514, a potent inhibitor of human epidermal growth factor receptor (HER) 1 (EGFR), 2, and 4, and vascular endothelial growth factor receptors (VEGFR) 1–3, is currently under investigation as an oral agent for the treatment of solid tumors. In vitro and in vivo studies were conducted to characterize the pharmacokinetics and metabolism. Through integration of in vitro and in vivo pharmacokinetic data and antitumor efficacy in nude mice, human pharmacokinetics and efficacious doses were projected for BMS-690514. The oral bioavailability of BMS-690514 was 78% in mice, ~ 100% in rats, 8% in monkeys, and 29% in dogs. The low oral bioavailability in monkeys could be attributed to high systemic clearance in that species, which was also consistent with predicted clearance using in vitro data from monkey liver microsomes. Permeability of BMS-690514 in Caco-2 cells was in the intermediate range with a moderate potential to be a P-gp substrate. Experiments using recombinant human CYP enzymes and human liver microsomes suggested that CYP2D6 and CYP3A4 are likely to play a key role in the metabolic clearance of BMS-690514; in addition, direct glucuronidation of BMS-690514 was also observed in human hepatocytes. BMS-690514 was able to cross the blood-brain barrier with a brain-to-plasma ratio of ~ 1. The preclinical ADME properties of BMS-690514 suggest good oral bioavailability in humans and metabolism by multiple pathways including oxidation and glucuronidation. Based on the efficacious AUC in nude mice and predicted human pharmacokinetics, the human efficacious QD dose is predicted to be in the range of 100-200 mg.