Stereoselective sulfoxidation of sulindac sulfide by flavin-containing monooxygenases. Comparison of human liver and kidney microsomes and mammalian enzymes

The stereoselective sulfoxidation of the pharmacologically active metabolite of sulindac, sulindac sulfide, was characterized in human liver, kidney, and cDNA-expressed enzymes. Kinetic parameter estimates (pH = 7.4) for sulindac sulfoxide formation in human liver microsomes (N = 4) for R- and S-sulindac sulfoxide were V(max) = 1.5 +/- 0.50 nmol/min/mg, K(m) = 15 +/- 5.1 microM; and V(max) = 1.1 +/- 0.36 nmol/min/mg, K(m) = 16 +/- 6.1 microM, respectively. Kidney microsomes (N = 3) produced parameter estimates (pH = 7.4) of V(max) = 0.9 +/- 0.29 nmol/min/mg, K(m) = 15 +/- 2.9 microM; V(max) = 0.5 +/- 0.21 nmol/min/mg, K(m) = 22 +/- 1.9 microM for R- and S-sulindac sulfoxide, respectively. In human liver and flavin-containing monooxygenase 3 (FMO3) the V(max) for R-sulindac sulfoxide increased 60-70% at pH = 8.5, but for S-sulindac sulfoxide was unchanged. In fourteen liver microsomal preparations, significant correlations occurred between R-sulindac sulfoxide formation and either immunoquantified FMO or nicotine N-oxidation (r = 0.88 and 0.83; P < 0.01). The R- and S-sulindac sulfoxide formation rate also correlated significantly (r = 0.85 and 0.75; P < 0.01) with immunoquantified FMO in thirteen kidney microsomal samples. Mild heat deactivation of microsomes reduced activity by 30-60%, and a loss in stereoselectivity was observed. Methimazole was a potent and nonstereoselective inhibitor of sulfoxidation in liver and kidney microsomes. n-Octylamine and membrane solubilization with lubrol were potent and selective inhibitors of S-sulindac sulfoxide formation. cDNA-expressed CYPs failed to appreciably sulfoxidate sulindac sulfide, and CYP inhibitors were ineffective in suppressing catalytic activity. Purified mini-pig liver FMO1, rabbit lung FMO2, and human cDNA-expressed FMO3 efficiently oxidized sulindac sulfide with a high degree of stereoselectivity towards the R-isomer, but FMO5 lacked catalytic activity. The biotransformation of the sulfide to the sulfoxide is catalyzed predominately by FMOs and may prove to be useful in characterizing FMO activity.     

The suncus (Suncus murinus) shows poor metabolic phenotype for trimethylamine N-oxygenation

In vitro and in vivo N-oxygenation of trimethylamine (TMA) in the suncus (Suncus murinus) was investigated. The N-oxygenation of TMA has been thought to be catalyzed by flavin-containing monooxygenase (FMO). In a previous study, we found that the levels of mRNAs for FMOs were extremely low in the suncus. Thus, we intended to evaluate the capacity of the suncus to N-oxygenate TMA compared to the rat. Eadie-Hofstee plots of the TMA N-oxygenation by suncus liver microsomes showed a biphasic pattern, suggesting that more than two enzymes were involved in this reaction. The low K(m) component in the suncus showed a twofold higher K(m) (55 vs. 31 microM) and a fourfold lower V(max) (0.61 vs 2.5 nmol/min/mg protein) values than those obtained using rat liver microsomes, resulting in a sevenfold lower V(max)/K(m) (11 vs 82 microl/min/mg protein) value. After an intraperitoneal administration of TMA (10 mg/kg body wt), the suncus excreted 39.6% of the dose in 24-h urine as TMA, whereas the rats excreted 6.3%. Metabolic ratio in the TMA N-oxygenation was 1.42 and 0.11 in the suncus and the rat, respectively. These results indicate that the suncus can be an animal model for a poor metabolizer phenotype in TMA metabolism.     

Sulphoxidation of ethyl methyl sulphide, 4-chlorophenyl methyl sulphide and diphenyl sulphide by purified pig liver flavin-containing monooxygenase

1. The biotransformation of ethyl methyl sulphide (EMS), 4-chlorophenyl methyl sulphide (CPMS) and diphenyl sulphide (DPS) to their corresponding sulphoxides by purified flavin-containing monooxygenase (FMO) is described. 2. Purified pig liver flavin-containing monooxygenase catalysed the sulphoxidation of EMS, CPMS and DPS to their corresponding sulphoxides and the reactions followed single enzyme Michelis-Menten kinetics. 3. The apparent K m and V max for the sulphoxidation of EMS were 1.38 ± 0.05 mM and 78.74 ± 3.9 nmoles?mg ? 1 protein?min ? 1, respectively. The apparent K m and V max for the sulphoxidation of CPMS were 0.185 ± 0.03 mM and 103 ± 5.0 nmoles?mg ? 1 protein?min ? 1, respectively. The apparent K m and V max for the sulphoxidation of DPS were 0.068 ± 0.002 mM and 49.26 ± 2.05 nmoles?mg ? 1 protein?min ? 1, respectively. 4. A significant reduction of the sulphoxidation of these simple sulphides was observed with addition of 1-naphthylthiourea in the incubation medium. On the other hand, incorporation of catalase and superoxide dismutase into the incubation media produced no appreciable inhibition of the observed sulphoxidation of the sulphides. 5. These results suggest that FMO is responsible, at least in part, for the sulphoxidation of nucleophilic sulphides as well as for the oxidation of sulphur atoms that reside within or adjacent to aromatic systems.     

In vitro evaluation of the disposition of A novel cysteine protease inhibitor.

K11777 (N-methyl-piperazine-Phe-homoPhe-vinylsulfone-phenyl) is a potent, irreversible cysteine protease inhibitor. Its therapeutic targets are cruzain, a cysteine protease of the protozoan parasite Trypanosoma cruzi, and cathepsins B and L, which are associated with cancer progression. We evaluated the metabolism of K11777 by human liver microsomes, isolated cytochrome P450 (CYP) enzymes, and flavin-containing monooxygenase 3 (FMO3) in vitro. K11777 was metabolized by human liver microsomes to three major metabolites: N-oxide K11777 (apparent K(m) = 14.0 +/- 4.5 microM and apparent V(max) = 3460 +/- 3190 pmol. mg(-1). min(-1), n = 4), beta-hydroxy-homoPhe K11777 (K(m) = 16.8 +/- 3.5 microM and V(max) = 1260 +/- 1090 pmol. mg(-1). min(-1), n = 4), and N-desmethyl K11777 (K(m) = 18.3 +/- 7.0 microM and V(max) = 2070 +/- 1830 pmol. mg(-1). min(-1), n = 4). All three K11777 metabolites were formed by isolated CYP3A and their formation by human liver microsomes was inhibited by the CYP3A inhibitor cyclosporine (50 microM, 54-62% inhibition) and antibodies against human CYP3A4/5 (100 microg of antibodies/100 microg microsomal protein, 55-68% inhibition). CYP2D6 metabolized K11777 to its N-desmethyl metabolite with an apparent K(m) (9.2 +/- 1.4 microM) lower than for CYP3A4 (25.0 +/- 4.0 microM) and human liver microsomes. The apparent K(m) for N-oxide K11777 formation by cDNA-expressed FMO3 was 109 +/- 11 microM. Based on the intrinsic formation clearances and the results of inhibition experiments (CYP2D6, 50 microM bufuralol; FMO3 mediated, 100 mM methionine) using human liver microsomes, it was estimated that CYP3A contributes to >80% of K11777 metabolite formation. K11777 was a potent (IC(50) = 0.06 microM) and efficacious (maximum inhibition 85%) NADPH-dependent inhibitor of human CYP3A4 mediated 6'beta-hydroxy lovastatin formation, suggesting that K11777 is not only a substrate but also a mechanism-based inhibitor of CYP3A4.     

Sulphoxidation of ethyl methyl sulphide, 4-chlorophenyl methyl sulphide and diphenyl sulphide by purified pig liver flavin-containing monooxygenase

1. The biotransformation of ethyl methyl sulphide (EMS), 4-chlorophenyl methyl sulphide (CPMS) and diphenyl sulphide (DPS) to their corresponding sulphoxides by purified flavin-containing monooxygenase (FMO) is described. 2. Purified pig liver flavin-containing monooxygenase catalysed the sulphoxidation of EMS, CPMS and DPS to their corresponding sulphoxides and the reactions followed single enzyme Michelis-Menten kinetics. 3. The apparent K(m) and V(max) for the sulphoxidation of EMS were 1.38+/-0.05 mM and 78.74+/-3.9 nmoles mg(-1) protein min(-1), respectively. The apparent K(m) and V(max) for the sulphoxidation of CPMS were 0.185+/-0.03 mM and 103+/-5.0 nmoles mg(-1) protein min(-1), respectively. The apparent K(m) and V(max) for the sulphoxidation of DPS were 0.068+/-0.002 mM and 49.26+/-2.05 nmoles mg(-1) protein min(-1), respectively. 4. A significant reduction of the sulphoxidation of these simple sulphides was observed with addition of 1-naphthylthiourea in the incubation medium. On the other hand, incorporation of catalase and superoxide dismutase into the incubation media produced no appreciable inhibition of the observed sulphoxidation of the sulphides. 5. These results suggest that FMO is responsible, at least in part, for the sulphoxidation of nucleophilic sulphides as well as for the oxidation of sulphur atoms that reside within or adjacent to aromatic systems.     

Glucosidation of hyodeoxycholic acid by UDP-glucuronosyltransferase 2B7

Previous studies have shown that several endogenous compounds, such as bilirubin and certain bile acids, are glucosidated in human liver. In this work, we have identified human UDP-glucuronosyltransferase 2B7 (UGT2B7) as the isoform that catalyzes the glucosidation of hyodeoxycholic acid (HDCA). The glucosidation by UGT2B7 was specific for HDCA and was not observed with the other bile acids examined, lithocholic acid, chenodeoxycholic acid, and ursodeoxycholic acid. The kinetics of HDCA glucuronidation and glucosidation by UGT2B7 were characterized. The K(m) values for glucuronidation and glucosidation of HDCA were 11.6 and 17.9 microM, respectively, with V(max) values of 4.15 nmol/min/mg protein for glucuronidation and 3.28 nmol/min/mg for glucosidation. At a fixed concentration of HDCA, the apparent K(m) for UDP-glucuronic acid was 89 microM with a V(max) of 3.53 nmol/min/mg. The corresponding parameters for UDP-glucose were 442 microM and 1.98 nmol/min/mg, respectively. UGT2B7 catalyzed the addition of the glucose and glucuronic acid moieties to an hydroxyl group on HDCA and also possessed some capacity to use UDP-xylose as sugar donor. The two polymorphic variants of UGT2B7, UGT2B7(*)1 and UGT2B7(*)2 could both glucosidate HDCA. This is the first report that identifies UGT2B7 as the enzyme responsible for the glucosidation of the bile acid, HDCA.     

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.     

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.     

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.     

Involvement of human blood arylesterases and liver microsomal carboxylesterases in nafamostat hydrolysis

Metabolism of nafamostat, a clinically used serine protease inhibitor, was investigated with human blood and liver enzyme sources. All the enzyme sources examined (whole blood, erythrocytes, plasma and liver microsomes) showed nafamostat hydrolytic activity. V(max) and K(m) values for the nafamostat hydrolysis in erythrocytes were 278 nmol/min/mL blood fraction and 628 microM; those in plasma were 160 nmol/min/mL blood fraction and 8890 microM, respectively. Human liver microsomes exhibited a V(max) value of 26.9 nmol/min/mg protein and a K(m) value of 1790 microM. Hydrolytic activity of the erythrocytes and plasma was inhibited by 5, 5'-dithiobis(2-nitrobenzoic acid), an arylesterase inhibitor, in a concentration-dependent manner. In contrast, little or no suppression of these activities was seen with phenylmethylsulfonyl fluoride (PMSF), diisopropyl fluorophosphate (DFP), bis(p-nitrophenyl)phosphate (BNPP), BW284C51 and ethopropazine. The liver microsomal activity was markedly inhibited by PMSF, DFP and BNPP, indicating that carboxylesterase was involved in the nafamostat hydrolysis. Human carboxylesterase 2 expressed in COS-1 cells was capable of hydrolyzing nafamostat at 10 and 100 microM, whereas recombinant carboxylesterase 1 showed significant activity only at a higher substrate concentration (100 microM). The nafamostat hydrolysis in 18 human liver microsomes correlated with aspirin hydrolytic activity specific for carboxylesterase 2 (r=0.815, p<0.01) but not with imidapril hydrolysis catalyzed by carboxylesterase 1 (r=0.156, p=0.54). These results suggest that human arylesterases and carboxylesterase 2 may be predominantly responsible for the metabolism of nafamostat in the blood and liver, respectively.     

Kinetic factors involved in the metabolism of benzene in mouse lung and liver

Benzene is an occupational and environmental toxicant. The main human health concern associated with benzene exposure is acute myelogenous leukemia. Benzene produces lung tumors in mice, while its effects on human lung are not clear. The adverse effects of benzene are dependent on its metabolism by the cytochrome P-450 enzyme system. The isozymes CYP2E1 and CYP2F2 play roles in the metabolism of benzene at low, environmentally relevant concentrations. Previous studies indicate that the mouse lung readily metabolizes benzene and that CYP2F2 plays a role in this biotransformation. The significance of CYP2E1 and CYP2F2 in benzene metabolism was determined by measuring their apparent kinetic parameters K(m) and V(max). Use of wild-type and CYP2E1 knockout mice and selective inhibitors allowed the determination of the individual importance of both CYP2E1 and CYP2F2 in mouse liver and lung. A simple Michaelis-Menten relationship involving Lineweaver-Burk plots for the microsomal metabolism of benzene shows the apparent kinetic factors are different between the wild-type (K(m): 30.4 microM, V(max): 25.3 pmol/mg protein/min) and knockout (K(m): 1.9 microM, V(max): 0.5 pmol/mg protein/min) mouse livers. Wild-type lung has a K(m) of 2.3 microM and V(max) of 0.9 pmol/mg protein/min. CYP2E1 knockout lung has similar affinity and metabolic activity with a K(m) of 3.7 microM and V(max) of 1.2 pmol/mg protein/min. These data suggest CYP2E1 is less important in the lung than liver, and that it has a lower affinity for benzene but higher rate of hydroxylated metabolite production than does CYP2F2, which plays the predominant role in metabolizing benzene in mouse lung.     

Validation of serotonin (5-hydroxtryptamine) as an in vitro substrate probe for human UDP-glucuronosyltransferase (UGT) 1A6.

Investigation of human UDP-glucuronosyltransferase (UGT) isoforms has been limited by a lack of specific substrate probes. In this study serotonin was evaluated for use as a probe substrate for human UGT1A6 using recombinant human UGTs and tissue microsomes. Of the 10 commercially available recombinant UGT isoforms, only UGT1A6 catalyzed serotonin glucuronidation. Serotonin-UGT activity at 40 mM serotonin concentration varied more than 40-fold among human livers (n = 54), ranging from 0.77 to 32.9 nmol/min/mg of protein with a median activity of 7.1 nmol/min/mg of protein. Serotonin-UGT activity kinetics of representative human liver microsomes (n = 7) and pooled human kidney, intestinal and lung microsomes and recombinant human UGT1A6 typically followed one enzyme Michaelis-Menten kinetics. Serotonin glucuronidation activity in these human liver microsomes had widely varying V(max) values ranging from 0.62 to 51.3 nmol/min/mg of protein but very similar apparent K(m) values ranging from 5.2 to 8.8 mM. Pooled human kidney, intestine, and lung microsomes had V(max) values (mean +/- standard error of the estimates) of 8.8 +/- 0.4, 0.22 +/- 0.00, and 0.03 +/- 0.00 nmol/min/mg of protein (respectively) and apparent K(m) values of 6.5 +/- 0.9, 12.4 +/- 2.0, and 4.9 +/- 3.3 mM (respectively). In comparison, recombinant UGT1A6 had a V(max) of 4.5 +/- 0.1 nmol/min/mg of protein and an apparent K(m) of 5.0 +/- 0.4 mM. A highly significant correlation was found between immunoreactive UGT1A6 protein content and serotonin-UGT activity measured at 4 mM serotonin concentration in human liver microsomes (R(s) = 0.769; P < 0.001) (n = 52). In conclusion, these results indicate that serotonin is a highly selective in vitro probe substrate for human UGT1A6.     

Metabolism of a disulfiram metabolite, S-methyl N,N-diethyldithiocarbamate, by flavin monooxygenase in human renal microsomes.

S-Methyl N,N-diethyldithiocarbamate (MeDDC), a metabolite of the alcohol deterrent disulfiram, is converted to MeDDC sulfine and then S-methyl N,N-diethylthiocarbamate sulfoxide, the proposed active metabolite in vivo. Several isoforms of CYP450 and to a lesser extent flavin monooxygenase (FMO) metabolize MeDDC in the liver. The human kidney contains FMO1 and several isoforms of CYP450, including members of the CYP3A, CYP4A, CYP2B, and CYP4F subfamilies. In this study the metabolism of MeDDC by the human kidney was examined, and the enzymes responsible for this metabolism were determined. MeDDC was incubated with human renal microsomes from five donors or with insect microsomes containing human FMO1, CYP4A11, CYP3A4, CYP3A5, or CYP2B6. MeDDC sulfine was formed at 5 microM MeDDC by renal microsomes at a rate of 210 +/- 50 pmol/min/mg of microsomal protein (mean +/- S.D., n = 5) and by FMO1 at 7.6 +/- 0.2 nmol/min/nmol (n = 3). Oxidation of 5 microM MeDDC was negligible by all CYP450 tested (< or =0.03 nmol/min/nmol). Inhibition of FMO by methimazole or heat diminished MeDDC sulfine formation 75 to 89% in renal microsomes. Inhibition of CYP450 in renal microsomes by N-benzylimidazole or antibody to the CYP450 NADPH reductase had no effect on MeDDC sulfine production. Benzydamine N-oxidation, a probe for FMO activity, correlated with MeDDC sulfine formation in renal microsomes (r = 0.951, p = 0.013). The K(M) values for MeDDC sulfine formation by renal microsomes and recombinant human FMO1 were 11 and 15 microM, respectively. These results demonstrate a role for the kidney and FMO1 in the metabolism of MeDDC in humans.     

Species comparison of vitamin K1 2,3-epoxide reductase activity in vitro: kinetics and warfarin inhibition

A comparative study of vitamin K(1) 2,3-epoxide reductase (VKOR) activity in vitro was conducted across species. The apparent kinetic constants K(m app), V(max), and Cl(int app) were determined in bovine, canine, equine, human, murine, ovine, porcine, and rat hepatic microsomes. In addition to these enzyme kinetic constants, the IC(50) of warfarin for VKOR was determined in human, murine, porcine, and rat hepatic microsomes. Interspecies differences were observed when comparing the K(m app) (range, 2.41-6.46 microM), V(max) (range, 19.5-85.7 nmol/mg/min), and Cl(int app) (range, 8.2-18.4 ml/mg/min) values. Comparison of the IC(50) values of warfarin, across the four species tested, revealed a significant species difference between murine microsomes (0.17 microM) and rat microsomes (0.07 microM). Overall, this study indicates that there are interspecies differences regarding the in vitro reduction of vitamin K(1) 2,3-epoxide by the warfarin-sensitive enzyme vitamin K(1) 2,3-epoxide reductase. Significant differences between the IC(50) values of murine and rat microsomes suggest differences in the susceptibility of these species to warfarin.     

Characterization of dextromethorphan O- and N-demethylation catalyzed by highly purified recombinant human CYP2D6.

The O-demethylation of dextromethorphan to dextrorphan in humans is catalyzed primarily by cytochrome P450 2D6 (CYP2D6). However, contrary to conventional wisdom, preparations of recombinant cytochrome P450 (P450) expressed from CYP2D6*1 cDNA also appear to produce significant amounts of 3-methoxymorphinan, the N-demethylated metabolite of dextromethorphan, when assayed in vitro. We hypothesized that both pathways were intrinsic to 2D6 and here further examine the kinetics of formation using a highly purified preparation of CYP2D6 in a reconstituted lipid system. Purified CYP2D6 protein with a measured molecular weight of 55772.0 (55769.6 Da predicted) was reconstituted into an active, lipid-vesicle environment with purified rat cytochrome P450 reductase before the addition of substrate and NADPH. Reaction kinetics were followed, and apparent Michaelis-Menten constants were determined for the appearance of each metabolite by high-pressure liquid chromatography, using both UV and fluorescence detection. In a 2-min assay, purified 2D6 catalyzed the formation of dextrorphan with an apparent K(m) value of 1.9 +/- 0.2 microM and a V(max) value of 8.5 +/- 0.2 nmol/nmol of P450/min and measured simultaneously the formation of 3-methoxymorphinan with an apparent K(m) value of 5000 +/- 700 microM and V(max) value of 176 +/- 12 nmol (nmol of P450)(-1) min(-1). These results indicate that at least two distinct binding orientations exist for dextromethorphan within the active site of CYP2D6.     

A highly sensitive fluorescent microplate method for the determination of UDP-glucuronosyl transferase activity in tissues and placental cell lines.

The fluorescent compound 4-methylumbelliferone (4MU) can be used to detect uridine diphosphate glucuronosyl transferase activity by observing the fall in fluorescence as the compound is converted to 4-methylumbelliferone glucuronide. A microplate assay has been developed that has improved sensitivity and is faster and cheaper than the historical extraction method. Activity is detectable with approximately 10% of the protein required in the extraction method. Absence of extraction and cleanup procedures and the ability to observe reaction rate directly are also of great advantage to the researcher. Michaelis-Menten kinetic data from one healthy female human liver is presented. The extraction method yielded a mean V(max) of 19.9 nmol/min/mg of protein and a mean K(m) of 652.5 microM on 1 day [n = 6, coefficients of variation (CV) 15 and 24%, respectively]. For the microplate method on 1 day, the mean V(max) was 36.21 +/- 1.3 nmol/min/mg of protein (CV = 3.7%), significantly (P <.0001) higher than for the extraction method. The mean K(m), 175. 4 +/- 24.2 microM (CV = 14.5%), was significantly lower (P <.0001) than observed in the extraction method. The assay was performed in replicates of six over 6 days; average intra- and interassay coefficients of variation were 9 and 22% for V(max) and 8 and 35% for K(m), respectively, for the microplate method. The microplate method has also detected activity in the placental trophoblast-derived cell lines JEG-3, JAr, and BeWo (5.5, 4.1, and 2. 6 nmol/min/mg of protein, respectively, at 200 microM 4MU concentration), indicating that placental cells may be capable of glucuronidating 4MU.     

Role of human liver cytochrome P4503A in the metabolism of etoricoxib, a novel cyclooxygenase-2 selective inhibitor.

Etoricoxib, a potent and selective cyclooxygenase-2 inhibitor, was shown to be metabolized via 6'-methylhydroxylation (M2 formation) when incubated with NADPH-fortified human liver microsomes. In agreement with in vivo data, 1'-N'-oxidation was a relatively minor pathway. Over the etoricoxib concentration range studied (1-1300 microM), the rate of hydroxylation conformed to saturable Michaelis-Menten kinetics (apparent K(m) = 186 +/- 84.3 microM; V(max) = 0.76 +/- 0.45 nmol/min/mg of protein; mean +/- S.D., n = 3 livers) and yielded a V(max)/K(m) ratio of 2.4 to 7.3 microl/min/mg. This in vitro V(max)/K(m) ratio was scaled, with respect to yield of liver microsomal protein and liver weight, to obtain estimates of M2 formation clearance (3.1-9.7 ml/min/kg of b.wt.) that agreed favorably with in vivo results (8.3 ml/min/kg of b.wt.) following i.v. administration of [(14)C]etoricoxib to healthy male subjects. Cytochrome P450 (P450) reaction phenotyping studies-using P450 form selective chemical inhibitors, immunoinhibitory antibodies, recombinant P450s, and correlation analysis with microsomes prepared from a bank of human livers-revealed that the 6'-methyl hydroxylation of etoricoxib was catalyzed largely (approximately 60%) by member(s) of the CYP3A subfamily. By comparison, CYP2C9 (approximately 10%), CYP2D6 (approximately 10%), CYP1A2 (approximately 10%), and possibly CYP2C19 played an ancillary role. Moreover, etoricoxib (0.1-100 microM) was found to be a relatively weak inhibitor (IC(50) > 100 microM) of multiple P450s (CYP1A2, CYP2D6, CYP3A, CYP2E1, CYP2C9, and CYP2C19) in human liver microsomes.     

UDP-glucuronosyltransferase 1A1 is the principal enzyme responsible for etoposide glucuronidation in human liver and intestinal microsomes: structural characterization of phenolic and alcoholic glucuronides of etoposide and estimation of enzyme kinetics.

Etoposide, an important anticancer agent, undergoes glucuronidation both in vitro and in vivo. In this study, three isomeric glucuronides of etoposide, including one phenolic (EPG) and two alcoholic glucuronides (EAG1 and EAG2), were biosynthesized in vitro with human liver microsomes (HLMs), and identified by liquid chromatography-electrospray ionization-mass spectrometry and confirmed by beta-glucuronidase cleavage. In vitro UDP-glucuronosyltransferase (UGT) reaction screening with 12 recombinant human UGTs demonstrated that etoposide glucuronidation is mainly catalyzed by UGT1A1. Although UGT1A8 and 1A3 also catalyzed the glucuronidation of etoposide, their activities were approximately 10 and 1% of UGT1A1. Enzyme kinetic study indicated that the predominant form of etoposide glucuronide in HLMs and human intestinal microsomes (HIMs) was EPG, whereas EAG1 and EAG2 were the minor metabolites, with approximately an 8 to 10% glucuronidation rate of EPG. For the formation of EPG, the V(max) of HLMs (110 pmol/min/mg protein) was very similar to that of recombinant UGT1A1 (124 pmol/min/mg protein), whereas the V(max) of HIMs (54.4 pmol/min/mg protein) was 2-fold lower than those of HIMs and UGT1A1. The K(m) values of HLMs (530 microM) and HIMs (608 microM) were 2-fold higher than that of UGT1A1 (285 microM). The V(max)/K(m) values for the formation of EPG were 0.21 and 0.09 microl/min/mg protein for HLMs and HIMs, respectively. The data indicated that UGT1A1 is principally responsible for the formation of etoposide glucuronides, mainly in the form of phenolic glucuronide, suggesting that etoposide can be used as a highly selective probe substrate for human UGT1A1 in vitro.