Human Liver Microsomal Samples Research Articles

Pharmacology and Metabolism of Renzapride

Renzapride (ATL-1251), a novel benzamide, is currently under clinical development for the treatment of irritable bowel syndrome (IBS). Previous in vitro and in vivo experimental studies have characterized renzapride as a full serotonin 5-HT(4) receptor agonist on the gut and a 5-HT(3) receptor antagonist. Clinical studies have confirmed the therapeutic efficacy, tolerability and safety of renzapride in patients with constipation-predominant IBS. This study set out to characterize the pharmacological profile of renzapride and its potential metabolic products at both 5-HT and other monoamine receptors in the gut. The affinity of renzapride, its (+) and (-) enantiomers, and its primary metabolite, renzapride N-oxide and its enantiomers, for serotonin receptors was assessed by means of in vitro radioligand binding inhibition studies. After membranes prepared from animal tissue or membranes of cell lines transfected with cloned human receptors had been incubated with radiolabelled ligand with high affinity for a specific receptor, renzapride was added to competitively inhibit this binding. Levels of bound radioligand were measured by filtration and counting of the bound radioactivity. In instances where >50% inhibition of radioligand binding had occurred, the inhibition constant (K(i)) was calculated. Metabolism of renzapride by liver microsomes was assessed by incubating 10 micromol/L renzapride with human liver microsome samples for 60 minutes at 37 degrees C. After the reaction was stopped, the samples were centrifuged and the supernatant analysed for metabolites by high-pressure liquid chromatography (HPLC). The potential inhibitory effects of renzapride on cytochrome P450 (CYP) enzymes were assessed by incubating renzapride at various concentrations over a 1-500 micromol/L concentration range with microsomes genetically engineered to express a single CYP. Renzapride was selective for serotonergic receptors and, in particular, had high affinity for human 5-HT(3) and guinea-pig 5-HT(4) receptors (K(i) 17 and 477 nm, respectively). Inhibitory properties at 5-HT(2B) receptors were also identified for renzapride, as well as some affinity for 5-HT(2A) and 5-HT(2C) receptors. Renzapride N-oxide and its enantiomers demonstrated much lower affinity for all 5-HT receptors compared with renzapride. Renzapride was metabolized by liver microsomes to a limited extent and there was no significant non-microsomal metabolism of renzapride. Renzapride did not inhibit the major CYP drug-metabolizing enzymes CYP2C9, CYP2D6, CYP1A2, CYP2A6, CYP2C19, CYP2E1 or CYP3A4 at concentrations consistent with use in a clinical setting. These results confirm and extend earlier studies in animal and human receptors that show renzapride is a potent and generally full 5-HT(4) receptor agonist and 5-HT(3) receptor antagonist. The results reported in the present study indicate that the metabolites of renzapride are minor and are unlikely to contribute to its therapeutic profile or lead to interaction of renzapride with other drugs that inhibit the major drug-metabolizing enzymes in the liver at therapeutic doses. These data contribute to the understanding of the pharmacological actions and metabolic fate of renzapride in vivo.

Glucuronidation of Nicotine and Cotinine by UGT2B10: Loss of Function by the UGT2B10 Codon 67 (Asp>Tyr) Polymorphism

Nicotine, the major addicting agent in tobacco and tobacco smoke, undergoes a complex metabolic pathway, with approximately 22% of nicotine urinary metabolites in the form of phase II N-glucuronidated compounds. Recent studies have shown that UGT2B10 is a major enzyme involved in the N-glucuronidation of several tobacco-specific nitrosamines. In the present study, microsomes of UGT2B10-overexpressing HEK293 cells exhibited high N-glucuronidation activity against both nicotine and cotinine with apparent KM's that were 37- and 3-fold lower than that observed for microsomes of UGT1A4-overexpressing cells against nicotine and cotinine, respectively. The KM of microsomes from wild-type (WT) UGT2B10-overexpressing cells for nicotine and cotinine was similar to that observed for human liver microsomes (HLM) against both substrates. The level of glucuronidated nicotine or cotinine in 112 HLM samples was correlated with UGT2B10 genotype; the levels of nicotine- and cotinine-glucuronide were 21% to 30% lower in specimens from subjects with the UGT2B10 (*1/*2) genotype compared with specimens from subjects with the WT UGT2B10 (*1/*1) genotype; a 5- and 16-fold lower level of nicotine- and cotinine-glucuronide formation, respectively, was observed in HLM from subjects with the UGT2B10 (*2/*2) genotype. In contrast to the relatively high activity observed for cells overexpressing WT UGT2B10 in vitro, little or no glucuronidation was observed for microsomes from cells overexpressing the UGT2B10*2 variant against either nicotine or cotinine. These data suggest that UGT2B10 is the major hepatic enzyme involved in nicotine/cotinine glucuronidation and that the UGT2B10*2 variant significantly reduces nicotine- and cotinine-N-glucuronidation formation and plays an important role in nicotine metabolism and elimination.

Identification of Human Liver Cytochrome P450 Enzymes Involved in Biotransformation of Vicriviroc, a CCR5 Receptor Antagonist

Vicriviroc (SCH 417690), a CCR5 receptor antagonist, is currently under investigation for the treatment of human immunodeficiency virus infection. The objective of this study was to identify human liver cytochrome P450 enzyme(s) responsible for the metabolism of vicriviroc. Human liver microsomes metabolized vicriviroc via N-oxidation (M2/M3), O-demethylation (M15), N,N-dealkylation (M16), N-dealkylation (M41), and oxidation to a carboxylic acid metabolite (M35b/M37a). Recombinant human CYP3A4 catalyzed the formation of all these metabolites, whereas CYP3A5 catalyzed the formation of M2/M3 and M41. CYP2C9 only catalyzed the formation of M15. There was a high correlation between the rates of formation of M2/M3, M15, and M41, which was determined using 10 human liver microsomal samples and testosterone 6beta-hydroxylation catalyzed by CYP3A4/5 (r > or = 0.91). Ketoconazole and azamulin (inhibitors of CYP3A4) were potent inhibitors of the formation of M2/M3, M15, M41, and M35b/M37a from human liver microsomes. A CYP3A4/5-specific monoclonal antibody (1 microg/microg of protein) inhibited the formation of all metabolites from human liver microsomes by 86 to 100%. The results of this study suggest that formation of the major vicriviroc metabolites in human liver microsomes is primarily mediated via CYP3A4. CYP2C9 and CYP3A5 most likely play a minor role in the biotransformation of this compound. These enzymology data will provide guidance to design clinical studies to address any potential drug-drug interactions.

Human carboxylesterases and their role in xenobiotic and endobiotic metabolism

Carboxylesterases (CEs) are traditionally regarded as xenobiotic metabolizing enzymes that hydrolyze esterified xenobiotics to alcohol and carboxylic acid products. However, there is a growing appreciation for the role of CEs in the processing of endobiotics, including cholesteryl esters and triacylglycerols. Human liver microsomes (HLMs) are often used in reaction phenotyping studies to discern interindividual variability in xenobiotic metabolism. The two major CE isoforms expressed in human liver are hCE1 and hCE2. These two isoforms are different gene products. We have begun studies to evaluate the CE phenotype'' of human liver samples, i.e. to determine both the levels of hCE1 and hCE2 protein and the hydrolytic activity of each. We have previously shown that there is little variation in hCE1 protein expression in HLM samples from 11 individuals [a 1.3-fold difference between the highest and lowest individuals; coefficient of variation (CV), 9%]. hCE2 protein expression in individual HLMs is only slightly more variable than hCE1 (2.3-fold difference between the highest and lowest individuals; CV, 36%). However, hCE1 protein is found in 46-fold higher amounts in HLMs than hCE2 protein (64.4 +/- 16.5 microg hCE1/mg microsomal protein compared to 1.4 +/- 0.2 microg hCE2/mg microsomal protein). The hydrolytic activity specifically attributable to hCE1 and hCE2 in individual HLMs was measured using bioresmethrin (a pyrethroid insecticide hydrolyzed specifically by hCE1, but not by hCE2) and procaine (an analgesic drug hydrolyzed by hCE2, but not by hCE1). The hydrolytic activity of individual HLMs toward bioresmethrin and procaine did not correlate with the protein content of hCE1 and hCE2. Thus, the mere abundance of CE proteins is not a good predictor of CE activity in HLMs. Identification of the factors that lead to altered CE activities in HLMs will be important to characterize since several pharmaceutical agents, environmental toxicants, and endobiotics are metabolized by these enzymes.

Human Cytochrome P450 Enzyme Specificity for the Bioactivation of Estragole and Related Alkenylbenzenes

Human cytochrome P450 enzymes involved in the bioactivation of estragole to its proximate carcinogen 1'-hydroxyestragole were identified and compared to the enzymes of importance for 1'-hydroxylation of the related alkenylbenzenes methyleugenol and safrole. Incubations with Supersomes revealed that all enzymes tested, except P450 2C8, are intrinsically able to 1'-hydroxylate estragole. Experiments with Gentest microsomes, expressing P450 enzymes to roughly average liver levels, indicated that P450 1A2, 2A6, 2C19, 2D6, and 2E1 might contribute to estragole 1'-hydroxylation in the human liver. Especially P450 1A2 is an important enzyme based on the correlation between P450 1A2 activity and estragole 1'-hydroxylation in human liver microsomal samples and inhibition of estragole 1'-hydroxylation by the P450 1A2 inhibitor alpha-naphthoflavone. Kinetic studies revealed that, at physiologically relevant concentrations of estragole, P450 1A2 and 2A6 are the most important enzymes for bioactivation in the human liver showing enzyme efficiencies (kcat/Km) of, respectively, 59 and 341 min-1 mM-1. Only at relatively high estragole concentrations, P450 2C19, 2D6, and 2E1 might contribute to some extent. Comparison to results from similar studies for safrole and methyleugenol revealed that competitive interactions between estragole and methyleugenol 1'-hydroxylation and between estragole and safrole 1'-hydroxylation are to be expected because of the involvement of, respectively, P450 1A2 and P450 2A6 in the bioactivation of these compounds. Furthermore, poor metabolizer phenotypes in P450 2A6 might diminish the chances on bioactivation of estragole and safrole, whereas lifestyle factors increasing P450 1A2 activities such as cigarette smoking and consumption of charbroiled food might increase those chances for estragole and methyleugenol.

Atmospheric pressure desorption/ionization on silicon ion trap mass spectrometry applied to the quantitation of midazolam in rat plasma and determination of midazolam 1′‐hydroxylation kinetics in human liver microsomes

The application of atmospheric pressure desorption/ionization on silicon (AP-DIOS) coupled with ion trap mass spectrometry (ITMS) was investigated for the quantification of midazolam in rat plasma, and determination of midazolam 1'-hydroxylation kinetics in pooled human liver microsomes. Results indicate good sensitivity with absolute detection limits for midazolam in rat plasma of approximately 300 femtograms. A linear dynamic range from approximately 10-5000 ng/mL was obtained in rat plasma with analysis times of 1 min per sample. Kinetic constants for midazolam 1'-hydroxylation in human liver microsomes yielded an apparent Km of 10.0 microM and Vmax of 6.4 nmol/min/mg. Studies investigating the inhibition of 1'-hydroxymidazolam formation by the cytochrome P450 3A4 model inhibitor ketoconazole yielded an IC50 of 0.03 microM. Quantitative precision for replicate analysis of rat plasma and human liver microsomal samples was variable with relative standard deviation (RSD) values ranging from a low of approximately 3% to over 50%, with the highest variability observed in data from human liver microsomal incubations. While preliminary studies investigating the application of AP-DIOS-ITMS suggested feasibility of this technique to typical pharmacokinetic applications, further work is required to understand the underlying causes for the high variability observed in these investigations.

Ethanol oxidation into acetaldehyde by 16 recombinant human cytochrome P450 isoforms: Role of CYP2C isoforms in human liver microsomes

The involvement of cytochromes P450 (CYPs) in the oxidation of ethanol into acetaldehyde was investigated by using 16 recombinant human CYP isoforms. Apparent K m and V m were determined for CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2B6, CYP2C8, CYP2C9*1, CYP2C9*2, CYP2C9*3, CYP2C18, CYP2C19, CYP2D6, CYP2E1, CYP2J2, CYP3A4 and CYP4A11. All of the tested CYPs, except CYP2A6 and CYP2C18, metabolized ethanol into significant amounts of acetaldehyde and displayed K m values around 10 mM. The significant correlation found between ethanol oxidation and CYP2E1, CYP3A4 and CYP1A2 catalytic activities in a panel of human liver microsomes confirmed the strong implication of these CYPs in ethanol metabolism. The contribution of CYP2C isoforms which are the most abundant in the liver after CYP3A4, was studied using selective inhibitors either with recombinant CYP2C isoforms or in human liver microsomes. Tienilic acid (100 μM) and ticlopidine (20 μM), mechanism-based inhibitors of CYP2C9 and CYP2C19, respectively, decreased ethanol oxidation by 8 ± 1.2% and 7.6 ± 1.6% in human liver microsomal samples while selective inhibitors of CYP2E1 (DEDTC 100 μM), CYP3A4 (TAO 50 μM) and CYP1A2 (furafylline 25 μM) decreased it by 11.9 ± 2.1%, 19.8 ± 1.9% and 16.3 ± 3.9%, respectively. As ethanol can be metabolized by most of CYPs, it helps to explain or predict alcohol–xenobiotics interactions which are of high importance in medical prescription.

Identification of the human liver UDP-glucuronosyltransferase involved in the metabolism of p-ethoxyphenylurea (dulcin)

Dulcin (DL), now banned, was once a widely used artificial sweetener. DL possesses an ureido group that is metabolized by direct glucuronidation in rabbit liver microsomes. Dulcin N-glucuronide (DNG) is the only type of ureido N-glucuronide known to date; ureido glucuronidation in humans has not been previously reported. Accordingly, the glucuronidation of DL was studied using human liver microsomes (HLM) and expressed human UDP-glucuronosyltransferase (UGT) enzymes. The average K (m) and V (max) values from nine HLM samples were 2.10 mM and 0.156 nmol/mg/min, respectively. Of the six human UGT isoforms screened for their ability to glucuronidate DL, only UGT1A1 and UGT1A9 showed activity. The apparent K (m) values using UGT1A1 and UGT1A9 were 5.06 and 6.99 mM, and the apparent V (max) values were 0.0461 and 0.106 nmol/min/mg, respectively. Phenolphthalein, a substrate for UGT1A9, inhibited DL glucuronidation in HLM competitively (K (i) = 0.356 mM), but bilirubin, a substrate for UGT1A1, did not. These results suggest that UGT1A9 is a key enzyme catalyzing the glucuronidation of DL.

Metabolism of Endosulfan-α by Human Liver Microsomes and Its Utility as a Simultaneous in Vitro Probe for CYP2B6 and CYP3A4

Endosulfan-alpha is metabolized to a single metabolite, endosulfan sulfate, in pooled human liver microsomes (Km = 9.8 microM, Vmax = 178.5 pmol/mg/min). With the use of recombinant cytochrome P450 (P450) isoforms, we identified CYP2B6 (Km = 16.2 microM, Vmax = 11.4 nmol/nmol P450/min) and CYP3A4 (Km = 14.4 microM, Vmax = 1.3 nmol/nmol P450/min) as the primary enzymes catalyzing the metabolism of endosulfan-alpha, although CYP2B6 had an 8-fold higher intrinsic clearance rate (CL(int) = 0.70 microl/min/pmol P450) than CYP3A4 (CL(int) = 0.09 microl/min/pmol P450). Using 16 individual human liver microsomes (HLMs), a strong correlation was observed with endosulfan sulfate formation and S-mephenytoin N-demethylase activity of CYP2B6 (r(2) = 0.79), whereas a moderate correlation with testosterone 6 beta-hydroxylase activity of CYP3A4 (r(2) = 0.54) was observed. Ticlopidine (5 microM), a potent CYP2B6 inhibitor, and ketoconazole (10 microM), a selective CYP3A4 inhibitor, together inhibited approximately 90% of endosulfan-alpha metabolism in HLMs. Using six HLM samples, the percentage total normalized rate (% TNR) was calculated to estimate the contribution of each P450 in the total metabolism of endosulfan-alpha. In five of the six HLMs used, the percentage inhibition with ticlopidine and ketoconazole in the same incubation correlated with the combined % TNRs for CYP2B6 and CYP3A4. This study shows that endosulfan-alpha is metabolized by HLMs to a single metabolite, endosulfan sulfate, and that it has potential use, in combination with inhibitors, as an in vitro probe for CYP2B6 and 3A4 catalytic activities.

Kinetic deuterium isotope effects for 7‐alkoxycoumarin O‐dealkylation reactions catalyzed by human cytochromes P450 and in liver microsomes

7-Ethoxy (OEt) coumarin has been used as a model substrate in many cytochrome P450 (P450) studies, including the use of kinetic isotope effects to probe facets of P450 kinetics. P450s 1A2 and 2E1 are known to be the major catalysts of 7-OEt coumarin O-deethylation in human liver microsomes. Human P450 1A2 also catalyzed 3-hydroxylation of 7-methoxy (OMe) coumarin at appreciable rates but P450 2E1 did not. Intramolecular kinetic isotope effects were used as estimates of the intrinsic kinetic deuterium isotope effects for both 7-OMe and 7-OEt coumarin dealkylation reactions. The apparent intrinsic isotope effect for P450 1A2 (9.4 for O-demethylation, 6.1 for O-deethylation) showed little attenuation in other competitive and noncompetitive experiments. With P450 2E1, the intrinsic isotope effect (9.6 for O-demethylation, 6.1 for O-deethylation) was attenuated in the noncompetitive intermolecular experiments. High noncompetitive intermolecular kinetic isotope effects were seen for 7-OEt coumarin O-deethylation in a baculovirus-based microsomal system and five samples of human liver microsomes (7.3-8.1 for O-deethylation), consistent with the view that P450 1A2 is the most efficient P450 catalyzing this reaction in human liver microsomes and indicating that the C-H bond-breaking step makes a major contribution to the rate of this P450 (1A2) reaction. Thus, the rate-limiting step appears to be the chemistry of the breaking of this bond by the activated iron-oxygen complex, as opposed to steps involved in the generation of the reactive complex. The conclusion about the rate-limiting step applies to all of the systems studied with this model P450 1A2 reaction including human liver microsomes, the most physiologically relevant.

IDENTIFICATION OF HUMAN LIVER CYTOCHROME P450 ENZYMES RESPONSIBLE FOR THE METABOLISM OF LONAFARNIB (SARASAR)

Lonafarnib (Sarasar), a farnesyl transferase inhibitor, is currently under development for the treatment of solid tumors. Incubation of lonafarnib with human liver microsomes resulted in the formation of four oxidative metabolites (M1, M2, M3, and M4). Minor to trace levels of these metabolites were detected in humans after multiple-dose administration of lonafarnib. Liquid chromatography-mass spectrometry analyses exhibited a mass to charge ratio (m/z) for the (M+H)(+) ion of M1, M2, M3, and M4 at 653, 635, 669, and 653 Th, respectively. These metabolites, respectively, resulted from changes of +O, -2H, +2O, and +O relative to lonafarnib. Recombinant human CYP3A4 and CYP3A5 exhibited catalytic activity with respect to the formation of M1, M2, and M3, whereas CYP2C8 exhibited catalytic activity with respect to the formation of M4. There was a high correlation between the formation of M1, determined in 10 human liver microsomal samples, and 6beta-hydroxylation of testosterone catalyzed by CYP3A4/5 (r = 0.93). The IC(50) values of ketoconazole for inhibition of M1 and M2 were 0.61 and 0.92 microM, respectively. The formation of M4 by human liver microsomes was inhibited 72% by 50 microM quercetin, suggesting that the formation of M4 was mediated via CYP2C8. A CYP3A4/5-specific inhibitory monoclonal antibody inhibited the formation of M1, M2, and M3 by 85, 75, and 100%, respectively. In conclusion, the formation of metabolites M1, M2, and M3 from lonafarnib was mediated via CYP3A4 and CYP3A5.

INTERACTIONS OF TWO MAJOR METABOLITES OF PRASUGREL, A THIENOPYRIDINE ANTIPLATELET AGENT, WITH THE CYTOCHROMES P450

The biotransformation of prasugrel to R-138727 (2-[1-2-cyclopropyl-1-(2-fluorophenyl)-2-oxoethyl]-4-mercapto-3-piperidinylidene]acetic acid) involves rapid deesterification to R-95913 (2-[2-oxo-6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl]-1-cyclopropyl-2-(2-fluorophenyl)ethanone) followed by cytochrome P450 (P450)-mediated formation of R-138727, the metabolite responsible for platelet aggregation. For identification of the P450s responsible for the formation of the active metabolite, the current studies were conducted with R-95913 as the substrate. Incubations required supplementation with reduced glutathione. Hyperbolic kinetics (K(m) 21-30 microM), consistent with a single enzyme predominating, were observed after incubations with human liver microsomes. Correlation analyses revealed a strong relationship between R-138727 formation and CYP3A-mediated midazolam 1'-hydroxylation (r(2) = 0.98; p < 0.001) in a bank of characterized human liver microsomal samples. The human lymphoblast-expressed enzymes capable of forming R-138727, in rank order of rates, were CYP3A4>CYP2B6>CYP2C19 approximately CYP2C9>CYP2D6. A monoclonal antibody to CYP2B6 and the CYP3A inhibitor ketoconazole substantially inhibited R-138727 formation, whereas inhibitors of CYP2C9 (sulfaphenazole) and CYP2C19 (omeprazole) did not. Scaling of in vitro intrinsic clearance values from expressed enzymes to the whole liver using a relative abundance approach indicated that either CYP3A4 alone or CYP3A4 and CYP2B6 are the major contributors to R-138727 formation. R-95913 and R-138727 were also examined for their ability to inhibit metabolism mediated by five P450s. R-138727 did not inhibit the P450s tested. In vitro, R-95913 inhibited CYP2C9, CYP2C19, CYP2D6, and CYP3A, with K(i) values ranging from 7.2 microM to 82 microM, but did not inhibit CYP1A2. These K(i) values exceed circulating concentrations in humans by 3.8- to 43-fold. Therefore, neither R-95913 nor R-138727 is expected to substantially inhibit the P450-mediated metabolism of coadministered drugs.

DETECTION OF A NOVEL REACTIVE METABOLITE OF DICLOFENAC: EVIDENCE FOR CYP2C9-MEDIATED BIOACTIVATION VIA ARENE OXIDES

A new glutathione adduct (M4) was tentatively identified, likely as 2'-hydroxy-3'-(glutathione-S-yl)-monoclofenac, using liquid chromatography-tandem mass spectrometry analysis of incubations of diclofenac with human liver microsomes. The same conjugate was not detected in incubations with either rat or monkey liver microsomes. Formation of M4 was mediated specifically by CYP2C9 in human liver microsomes, as evidenced by the following observations: 1) cDNA-expressed CYP2C9-catalyzing formation of M4; 2) inhibition of M4 formation by sulfaphenazole, a CYP2C9-selective inhibitor; and 3) strong correlation between the production of M4 and CYP2C9-mediated tolbutamide 4-hydroxylase activities in a panel of human liver microsome samples. Formation of M4 suggests the existence of a new reactive intermediate as diclofenac-2',3'-oxide. A tentative pathway states that diclofenac is oxidized to diclofenac-2',3'-oxide that reacts with glutathione (GSH) to form a thioether conjugate at the C-3' position, followed by a concomitant loss of chlorine to give rise to M4. Furthermore, a likely mechanism leading to the formation of diclofenac oxides is rationalized: CYP2C9-catalyzed oxidation at the C-3' position of the dichlorophenyl ring to form a cationic sigma-complex that subsequently results in diclofenac-3',4'-oxide and diclofenac-2',3'-oxide; the former oxide is converted to 4'-hydroxy-diclofenac as a major metabolite and can be trapped by GSH to produce 4'-hydroxy-3'-glutathione-S-yl diclofenac (M2), whereas the latter oxide forms 3'-hydroxy-diclofenac and can be trapped by GSH to produce M4. This mechanism is consistent with the structural modeling of the CYP2C9-diclofenac complex, which reveals that both the C-3' and C-4' of the dichlorophenyl ring are proximate to the heme group.

N-Glucuronidation of trans-3‘-Hydroxycotinine by Human Liver Microsomes

trans-3'-Hydroxycotinine is the major nicotine metabolite excreted in the urine of smokers and other tobacco or nicotine users. On average, about 30% of the trans-3'-hydroxycotinine in urine is present as a glucuronide conjugate. The O-glucuronide of trans-3'-hydroxycotinine has been isolated from smokers urine and appears to be the major glucuronide conjugate of trans-3'-hydroxycotinine in urine. However, nicotine and cotinine are both glucuronidated at the nitrogen atom of the pyridine ring. We report here that human liver microsomes catalyze both the N-glucuronidation and the O-glucuronidation of trans-3'-hydroxycotinine. The N-glucuronide was purified by HPLC, and its structure was confirmed by NMR. Both N- and O-glucuronidation of trans-3'-hydroxycotinine were detected in 13 of 15 human liver microsome samples. The ratio of N-glucuronidation to O-glucuronidation was between 0.4 and 2.7. One sample only catalyzed N-glucuronidation, and one sample did not catalyze either reaction. The rates of N-glucuronidation varied more than 6-fold from 6 to 38.9 pmol/min/mg protein; rates of O-glucuronidation ranged from 2.8 to 23.4 pmol/min/mg protein. The rate of trans-3'-hydroxycotinine N-glucuronidation by human liver microsomes correlated well with both the rate of nicotine and the rate of cotinine N-glucuronidation. trans-3'-Hydroxycotinine O-glucuronidation correlated with neither nicotine nor cotinine N-glucuronidation. These results suggest that the same enzyme(s) that catalyzes the N-glucuronidation of nicotine and cotinine may also catalyze the N-glucuronidation of trans-3'-hydroxycotinine in the human liver but that a separate enzyme catalyzes trans-3'-hydroxycotinine O-glucuronidation.

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

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

In vitro metabolism of genistein and tangeretin by human and murine cytochrome P450s.

Recombinant cytochrome P450 (CYP) 1A2, 3A4, 2C9 or 2D6 enzymes obtained from Escherichia coli and human liver microsomes samples were used to investigate the ability of human CYP enzymes to metabolize the two dietary flavonoids, genistein and tangeretin. Analysis of the metabolic profile from incubations with genistein and human liver microsomes revealed the production of five different metabolites, of which three were obtained in sufficient amounts to allow a more detailed elucidation of the structure. One of these metabolites was identified as orobol, the 3'-hydroxylated metabolite of genistein. The remaining two metabolites were also hydroxylated metabolites as evidenced by LC/MS. Orobol was the only metabolite formed after incubation with CYP1A2. The two major product peaks after incubation of tangeretin with human microsomes were identical with 4'-hydroxy-5,6,7,8-tetramethoxyflavone and 5,6-dihydroxy-4',7,8-trimethoxyflavone, previously identified in rat urine in our laboratory. By comparison with UV spectra and LC/MS fragmentation patterns of previously obtained standards, the remaining metabolites eluting after 14, 17 and 20 min. were found to be demethylated at the 4',7-, 4',6-positions or hydroxylated at the 3'- and demethylated at the 4'-positions, respectively. Metabolism of tangeretin by recombinant CYP1A2, 3A4, 2D6 and 2C9 resulted in metabolic profiles that qualitatively were identical to those observed in the human microsomes. Inclusion of the CYP1A2 inhibitor fluvoxamine in the incubation mixture with human liver microsomes resulted in potent inhibition of tangeretin and genistein metabolism. Other isozymes-selective CYP inhibitors had only minor effects on tangeretin or genistein metabolism. Overall the presented observations suggest major involvement of CYP1A2 in the hepatic metabolism of these two flavonoids.

High-throughput cytochrome p450 inhibition assays by ultrafast gradient liquid chromatography with tandem mass spectrometry using monolithic columns.

A generic method employing ultrafast liquid chromatography with tandem mass spectrometry (LC/MS/MS) was developed and employed for routine screening of drug candidates for inhibition of five major human cytochrome p450 (CYP) isozymes, CYP3A4, CYP2D6, CYP2C9, CYP2C19, and CYP1A2. The method utilized a monolithic silica rod column to allow fast flow rates to significantly reduce chromatographic run time. The major metabolites of six CYP-specific probe substrates for the five p450 isoforms were monitored and quantified to determine IC(50) values of five drug compounds against each p450 isozyme. Human liver microsomal incubation samples at each test compound concentration were combined and analyzed simultaneously by the LC/MS/MS method. Each pooled sample containing six substrates and an internal standard was separated and detected in only 24 seconds. The combination of ultrafast chromatography and sample pooling techniques has significantly increased sample throughput and shortened assay turnaround time, allowing a large number of compounds to be screened rapidly for potential p450 inhibitory activity, to aid in compound selection and optimization in drug discovery.

Role of Human Hepatic Cytochrome P-450S in Territrem a Metabolism

The ability of human liver microsomal preparations (HLM1, 2, 3, and 5), microsomes from human lymphoblasts expressing different cytochrome P-450 (CYP450) isoforms, and CYP3A4 cDNA-transfected V79 Chinese hamster cells to metabolize territrem A (TRA) was studied. The only metabolite generated by any of these preparations was 6 g -hydroxymethyl-6 g -demethylterritrem A (MA 1 ). MA 1 formation was observed with all four human liver microsomal samples. Of the eight microsomal preparations from human lymphoblasts expressing different cytochrome P-450 enzymes (1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, and 3A4) examined, only those expressing CYP2C9, CYP2D6, or CYP3A4 metabolized TRA, with that expressing CYP3A4 being the most active. No TRA metabolites were formed by control V79MZ cells, but MA 1 was formed by CYP3A4 cDNA-transfected V79 Chinese hamster cells. In order to investigate which CYP450 isoforms were involved in MA 1 formation in the human liver microsomal preparations, the effects of six isoform-specific chemical inhibitors (furafylline, sulfaphenazole, omeprazole, quinidine, ketaconazole, and diethyldithiocarbamate) and anti-3A4, anti-2C9, and anti-2D6 antibodies on TRA metabolism by HLM2 and HLM5 were examined. MA 1 formation was markedly inhibited by ketaconazole, with quinidine and sulfaphenazole having less of an effect. Anti-CYP3A4 antibody markedly inhibited MA 1 formation, while antibodies against CYP2C9 or CYP2D6 had little effect. The amount of MA 1 formed using different HLM preparations was related to the 6 g -testosterone hydroxylase activity and CYP3A4 protein content of the preparations. These results suggest that CYP3A4 is the major enzyme involved in TRA metabolism by human liver microsomes, with CYP2C9 and CYP2D6 playing a minor role.

Determination of the human cytochrome P450s involved in the metabolism of 2n -propylquinoline

1 2 n -Propylquinoline (2 n PQ) is a newly developed drug for visceral antileishmaniasis and its activity has been previously evaluated in mice following oral administration. The study was carried out to investigate the kinetic formation of 2 n PQ metabolites and to characterize the human liver CYP forms involved in its oxidative metabolism. 2. The inhibition of 2 n PQ metabolite formation by specific substrates or inhibitors of CYP forms and correlation studies were performed in human liver microsomes. 2 n PQ biotransformation was then studied in human lymphoblasts expressing specific CYPs and microsomal epoxide hydrolase. 3. Three major metabolites were produced by human liver microsomes and their structures were identified by ESI-LC/MS: dihydroxy-2 n -propylquinoline, 3'-hydroxy-2 n -propylquinoline and 1'-hydroxy-2 n -propylquinoline. An intermediary metabolite, epoxy-2 n -propylquinoline, formed by CYP was also biotransformed by microsomal epoxide hydrolase into dihydroxy-2 n -propylquinoline. 4. 2 n PQ oxidation follows Michaelis-Menten kinetics. In human liver microsomes, its metabolism was extremely inhibited by pilocarpine, coumarin and diethyldithiocarbamate. From a panel of 12 human liver microsome samples, the rate of 2 n PQ oxidation was highly correlated with the activities of CYP2A6 and CYP2E1. Human lymphoblasts expressing specific CYPs showed the involvement of CYP2A6, CYP2E1 and CYP2C19. 5. The results indicate that 2 n PQ metabolites are 3'- and 1'-hydroxylated by human liver microsomes and an epoxy-2 n -propylquinoline is biotransformed into a dihydroxy-2 n -propylquinoline by microsomal epoxide hydrolase.

Acyl glucuronidation and glucosidation of a new and selective endothelin ET(A) receptor antagonist in human liver microsomes.

Compound A [(+)-(5S,6R,7R)-2-isopropylamino-7-[4-methoxy-2-((2R)-3-methoxy-2-methylpropyl)-5-(3,4-methylenedioxyphenyl) cyclopenteno [1,2-b] pyridine 6-carboxylic acid] is a new and selective endothelin ET(A) receptor antagonist. It underwent significant acyl glucuronidation and acyl glucosidation in human liver microsomes supplemented with UDP-glucuronic acid (UDPGA) and UDP-glucose (UDPG). These two conjugations were observed in a panel of human liver microsomal samples (n = 16) that gave rise to varying activities but with no significant correlation with each other in the native and activator-treated microsomal preparations (r(2) <or= 0.4, p > 0.05). The lack of correlation may be explained by the involvement of multiple UDP-glucuronosyltransferases (UGTs; UGT1A1, 1A3, 1A9, 2B7 and 2B15) in the glucuronidation but essentially solely UGT2B7 in the glucosidation. Both reactions conformed to monophasic Michaelis-Menten kinetics in human liver microsomes. The glucuronidation reaction exhibited apparent K(m) values (mean +/- S.E.) for compound A and UDPGA of 8.4 +/- 0.6 and 605 +/- 35 microM, respectively, whereas the values for the glucosidation reaction were 10.2 +/- 1.5 and 670 +/- 120 microM, respectively. In both pooled human liver microsomes and expressed UGT2B7, UDPG and UDPGA competitively inhibited their counterpart conjugations with K(i) values close to their K(m) values, indicating a comparable affinity of the enzyme toward these two nucleotide sugars. We herein report a drug acyl glucoside formed in human liver microsomes at a considerable turnover rate and provide the evidence for a UGT isoform (UGT2B7) capable of transferring both glucuronic acid and glucose from UDPGA and UDPG to an aglycone.