Role Of Human Cytochromes P450 Research Articles

In vitro metabolism of helenalin and its inhibitory effect on human cytochrome P450 activity.

Sesquiterpene lactone helenalin is used as an antiphlogistic in European and Chinese folk medicine. The pharmacological activities of helenalin have been extensively investigated, yet insufficient information exists about its metabolic properties. The objectives of the present study were (1) to investigate the in vitro NADPH-dependent metabolism of helenalin (5 and 100µM) using human and rat liver microsomes and liver cytosol, (2) to elucidate the role of human cytochrome P450 (CYP) enzymes in its oxidative metabolism, and (3) to study the inhibition of human CYPs by helenalin. Five oxidative metabolites were detected in NADPH-dependent human and rat liver microsomal incubations, while two reduced metabolites were detected only in NADPH-dependent human microsomal and cytosolic incubations. In human liver microsomes, the main oxidative metabolite was 14-hydroxyhelenalin, and in rat liver microsomes 9-hydroxyhelenalin. The overall oxidation of helenalin was several times more efficient in rat than in human liver microsomes. In humans, CYP3A4 and CYP3A5 followed by CYP2B6 were the main enzymes responsible for the hepatic metabolism of helenalin. The extrahepatic CYP2A13 oxidized helenalin most efficiently among CYP enzymes, possessing the Km value of 0.6µM. Helenalin inhibited CYP3A4 (IC50 = 18.7µM) and CYP3A5 (IC50 = 62.6µM), and acted as a mechanism-based inhibitor of CYP2A13 (IC50 = 1.1µM, KI = 6.7µM, and kinact = 0.58 ln(%)/min). It may be concluded that the metabolism of helenalin differs between rats and humans, in the latter its oxidation is catalyzed by hepatic CYP2B6, CYP3A4, CYP3A5, and CYP3A7, and extrahepatic CYP2A13.

Drug Interactions with Angiotensin Receptor Blockers: Role of Human Cytochromes P450.

Angiotensin receptor blockers (ARBs) are the most recent class of agents for the treatment of hypertension. However, ARBs may cause a low incidence of headache, upper respiratory infection, back pain, muscle cramps, fatigue, dizziness, and many other side effects. In some cases, such toxicity is associated with pharmacokinetic alterations. The cytochrome P450 (CYP) enzyme system plays an important role in a lot of clinically important pharmacokinetic drug interactions. To identify relevant studies on drug-drug and food-drug pharmacokinetic interactions with the ARBs, a literature search of Google Scholar was performed from January 1994 to June 2015, with the following keywords: 'losartan', 'valsartan,' 'candesartan,' 'irbesartan,' 'telmisartan,' 'eprosartan,' 'olmesartan,' and 'azilsartan', combined with the keyword 'pharmacokinetic interactions' and 'CYP'. Based on the literatures published, it has been demonstrated that pharmacokinetic interactions of losartan with other agents are mainly via CYP2C9- and CYP3A4-mediated, the role played by CYP enzyme system in the metabolism of valsartan, candesartan, irbesartan, and azilsartan appears modest, and cytochrome P450 system has no influence on the metabolism of telmisartan, eprosartan, olmesartan. Therefore, according to these pharmacokinetic findings, no dosage adjustment is recommended when eprosartan, telmisartan and olmesartan are combined with other pharmacological agents in patients with hypertension. This review summarize the available data on cytochrome P450 - related drug-drug interactions reported in the literature for the eight ARBs. Knowledge of the pharmacokinetic properties of the ARBs should allow the avoidance of the majority of drug interactions without compromising therapeutic benefits.

Abstract 5739: Characterization of the metabolism and DNA adduct formation of 2-amino-9H-pyrido[2,3-b]indole in human hepatocytes

Abstract 2-Amino-9H-pyrido[2-3-b]indole (AαC) is a heterocyclic aromatic amine (HAA) and considered to be a possible human carcinogen by the International Agency for Research on Cancer (IARC). Like other HAAs, AαC is metabolized in the liver and can undergo bioactivation to produce highly electrophilic derivatives that can form DNA adducts by covalent binding to DNA. Among the HAAs, AαC is of particular interest because it is present at high levels in tobacco smoke and ubiquitous in the environment. AαC was shown to form high levels of DNA adducts in primary cultured human hepatocytes. The adduct levels were higher than those formed by other HAAs such as 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) or 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and comparable to those derived from 4-aminobiphenyl (4-ABP), a known human carcinogen. An understanding of the metabolism of AαC and its potential to form DNA adducts would provide a better assessment of the human health risk posed by AαC. Indeed, the metabolism pathways of MeIQx, PhIP and AαC are different. These differences in metabolic processing of HAAs can lead to variable levels of DNA adducts formed in humans and impact the interindividual susceptibility towards these procarcinogens. Knowledge about the metabolism of AαC in humans is unknown. We have analyzed the role of human cytochromes P450, especially CYP1A2, in the metabolism of AαC and DNA adduct formation. The ring-oxidized 3-HO-AαC and 6-HO-AαC sulfate and glucuronide conjugates, AαC-N2-Gl, N2-acetyl-AαC and the glucuronide conjugates of 2-hydroxyamino-9H-pyrido[2,-3-b]indole (HONH-AαC) and the deoxyguanosine DNA adduct, dG-C8-AαC, were identified and quantified by liquid chromatography coupled with multistage mass spectrometry in human hepatocytes in primary culture. The levels of DNA adducts formed after a 24hr-treatment with AαC were (2 folds) lower when cells were pre-treated with furafylline, a specific CYP1A2 inhibitor; however, the levels of most of the metabolites formed were not significanly affected by furafylline. Our data suggests that CYPs other than CYP1A2 may be involved in AαC metabolism, while CYP1A2 is the main enzyme involved in the production of reactive metabolites leading to the formation of DNA adducts. The identification of CYPs and Phase II enzymes involved in AαC metabolism in human hepatocytes are under study to fully understand the health risk of AαC to humans. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5739. doi:1538-7445.AM2012-5739

In Vitro Profiling and Mass Balance of the Anti-Cancer Agent Laromustine [14C]-VNP40101M by Rat, Dog, Monkey and Human Liver Microsomes

Laromustine (VNP40101M; 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)-2-(methylamino) carbonylhydrazine) is a novel sulfonylhydrazine alkylating agent. For the first time In vitro profiling and mass balance of [14C] VNP40101M in rat, dog, monkey and human liver microsomes was investigated. Also, the role of human cytochrome P450 (CYP) and flavin-containing monooxygenase (FMO) enzymes in the conversion of [14C] VNP40101M by NADPH-fortified human liver microsomes was determined. In this study, [14C]-VNP40101M was converted to five radioactive components (C-1, C-2, C-3, C-4 and C-7) after 60 min of incubation with dog, monkey and human liver microsomes. With the exception of C-3, the same components were detected with rat liver microsomes. In the presence of NADPH, after 60 min of incubation, the loss of substrate for rat, dog, monkey and human was 63, 82, 76 and 64%, respectively and mass balance ranged from 91.0 – 99.3%. In the absence of NADPH, after 60 min of incubation with [14C] VNP40101M (100 µM), the loss of substrate for rat, dog, monkey and human liver microsomes was 59, 53, 61 and 59%, respectively and mass balance ranged from 100.6 – 116.4%. The profiles of metabolites were similar. The relative abundance of individual metabolites was not species dependent. The formation of C-7 was not observed in zero cofactor (no NADPH) or zero-protein samples, suggesting that its formation was enzymatic. The formation of C-1, C-2, C-3, and C-4 increased with respect to incubation time. Using a panel of CYP enzymes including rCYP1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6 and 3A4, it was shown that C-7 formation was catalyzed by CYP2B6 and CYP3A4/5. The results of this study suggest that (1) P450 plays a role in C-7 formation but plays little or no role in the conversion of [14C] VNP40101M to C-1 through C-4, and (2) the relative abundance of individual degradation/metabolite products were not species dependent. These findings provide a comprehensive understanding of the metabolism of this new agent.

Genotoxic activation of the environmental pollutant 3,6-dinitrobenzo[e]pyrene in Salmonella typhimurium umu strains expressing human cytochrome P450 and N-acetyltransferase

3,6-Dinitrobenzo[e]pyrene (DNBeP) is a potent mutagen identified in surface soil in two metropolitan areas of Japan. We investigated whether DNBeP can cause genotoxicity through any metabolic activation pathway in bacteria using the parental strain Salmonella enterica serovar Typhimurium (S. typhimurium) TA1535/pSK1002, nitroreductase (NR)-deficient strain NM1000, the O-acetyltransferase (O-AT)-deficient strain NM2000, bacterial O-AT-overexpressing strain NM2009, and bacterial NR- and O-AT-overexpressing strain NM3009 established in our laboratory. To further clarify the role of human cytochrome P450 (P450 or CYP) and N-acetyltransferase (NAT) enzymes in the bioactivation of DNBeP to genotoxic metabolites, we determined the genotoxicity of DNBeP using a variety of umu tester strains expressing human P450 and NAT enzymes. The dose-dependent induction of umuC by DNBeP was observed at concentrations between 0.01 and 1nM in the O-AT-expression strain, but not in the O-AT-deficient strain. In the CYP3A4-, CYP1A2-, CYP1A1-, and CYP1B1-expressing strains, DNBeP was found to be activated to reactive metabolites that cause the induction of umuC gene expression compared with the parent strain. The induction of DNBeP in the NAT2-expressing strain had a 10-fold lower concentration than that in the NAT1-expressing strain. Collectively, these results suggest that nitroreduction by human CYP1A2, CYP3A4, and CYP1A1 and O-acetylation by human NAT2 contributed to the genotoxic activation of DNBeP to its metabolites.

Hepatic Metabolism of MK-0457, a Potent Aurora Kinase Inhibitor: Interspecies Comparison and Role of Human Cytochrome P450 and Flavin-Containing Monooxygenase

MK-0457 (N-[4([4-(4-methylpiperazin-1-yl)-6-[(3-methyl-1H-pyrazol-5 -yl)amino]pyrimidin-2-yl]thio)phenyl]cyclopropanecarboxamide), an Aurora kinase inhibitor in development for the treatment of cancer, was evaluated for its in vitro metabolism in different species. This compound primarily underwent N-oxidation and N-demethylation in human, monkey, dog, and rat liver preparations. However, N-demethylation was less significant in dogs. The formation of minor metabolites varied with species, but all metabolites generated in human hepatocytes were observed in animals. Results of immunoinhibition, selective chemical inhibition, thermal inactivation, and metabolism by recombinant cytochromes P450 and flavin-containing monoogygenases (FMOs) strongly suggest that CYP3A4 and FMO3 comparably contributed to MK-0457 N-oxidation in human liver microsomes, where the reaction conformed to Michaelis-Menten kinetics. These studies indicate a major role of CYP2C8 in the N-demethylation reaction, whereas CYP3A4 only made a minor contribution. However, significant substrate inhibition was observed with MK-0457 N-demethylation at high substrate concentrations (>10 microM) in human liver microsomes relative to the anticipated therapeutic exposure. A multienzyme metabolic pathway such as this may mitigate the potential of drug interactions in clinical treatment with MK-0457.

Genotoxic Activation of the Environmental Pollutant 3-Nitrobenzanthrone by Human Cytochrome P450 Enzymes Expressed in Salmonella typhimurium umu Tester Strains

3-Nitrobenzanthrone (3-NBA) is a mutagenic and carcinogenic compound identified in diesel exhaust, airborne particulate matter, soil, and water. To address whether 3-NBA shows genotoxic effects through any metabolic activation pathway, we determined the genotoxicity of 3-NBA using the parental strain Salmonella enterica serovar Typhimurium (S. typhimurium) TA1535/pSK1002, nitroreductase (NR)-deficient strain NM1000, the O-acetyltransferase (O-AT)-deficient strain NM2000, bacterial O-AT-overexpressing strain NM2009, and bacterial NR- and O-AT-overexpressing strain NM3009 established in our laboratory. In order to further clarify the role of human cytochrome P450 (P450 or CYP) enzyme in the bioactivation of 3-NBA to genotoxic metabolites, umu tester strains S. typhimurium OY1022/1A1, OY1022/1A2, OY1022/1B1, and OY1022/3A4, which express four respective human cytochrome P450 enzymes, NADPH-cytochrome P450 reductases (NPR) and O-AT were established by introducing two plasmids into S. typhimurium TA1535NR/1,8-DNP (nitroreductase-deficient and resistant to 1,8-dinitropyrene, probably due to functional loss of O-AT), one carrying both P450 and reductase cDNA in a biocistronic construct under control of an IPTG-inducible double tac promoter and the other, pOA102, carrying O-AT and umuC'-'lacZ fusion genes. Induction of umuC gene expression could be monitored by measuring the cellular β-galactosidase activity produced by the umuC'-'lacZ fusion gene. Although the induction of umuC by 3-NBA was weak in TA1535/pSK1002, NM1000, and NM2000 strains, the induction was considerably potent in NM2009 and NM3009 strains. 3-NBA was also found to induce umuC gene expression in OY1022/1A1 and OY1022/3A4 strains and, to a lesser extent, OY1022/1A2 and OY1022/1B1 strains, at a much higher rate than the parental OY1022/pCW strain. Collectively, these results suggest nitroreduction and O-acetylation by bacterial NR and O-AT, respectively, and nitroreduction by human CYP1A1, 3A4, 1A2, and 1B1 and NPR could contribute to the genotoxic activation of 3-NBA to produce reactive metabolites.

Activation of aminophenylnorharman, aminomethylphenylnorharman and aminophenylharman to genotoxic metabolites by human N-acetyltransferases and cytochrome P450 enzymes expressed in Salmonella typhimurium umu tester strains

Norharman (9H-pyrido[3,4-b]indole) and harman (1-methyl-9H-pyrido[3,4-b]indole) contained in cigarette smoke and cooked foodstuffs, are non-mutagenic to Salmonella strains, but show co-mutagenicity with S9 mixture in the presence of aniline or o-toluidine. The resulting 9-(4'-aminophenyl)-9H-pyrido[3,4-b]indole (aminophenylnorharman, APNH), 9-(4'-amino-3'-methylphenyl)-9H-pyrido[3,4-b]indole (aminomethylphenylnorharman, AMPNH) and 9-(4'-aminophenyl)-1-methyl-9H-pyrido[3,4-b]indole (aminophenylharman, APH) are produced by coupling of norharman and aniline, norharman and o-toluidine, and harman and aniline in the presence of S9 mixture, respectively. To clarify the role of human cytochrome P450 (P450) and N-acetyltransferase (NAT) enzymes in the metabolic activation of APNH, AMPNH and APH, we determined the genotoxicity of these coupling chemicals using a variety of umu tester strains established in our laboratories. APNH, AMPNH and APH induced umuC gene expression more strongly in a bacterial O-acetyltransferase-overproducing strain than the parent strain. These chemicals were also found to induce umuC gene expression in NAT2-overexpressing strain at much higher rate than the NAT1-overexpressing strain. Among seven OY strains expressing human P450s and NADPH-P450 reductase used, the genotoxicity of APNH, AMPNH and APH was detected in OY1002/1A2 strain, OY1002/1A1 and OY1002/1A2 strains, and in OY1002/1A2 strain, respectively. From these results, it is concluded that APNH, AMPNH and APH are mainly bioactivated by P450 1A2 and NAT2, followed by NAT1 enzymes. P450 1A1 was also found to activate AMPNH at relatively slower rates.

Evidence for a new human CYP1A1 regulation pathway involving PPAR-α and 2 PPRE sites

Cytochrome P450 1A1 catalyzes the degradation of endobiotics (estradiol, fatty acids, and so on) and the bioactivation of numerous environmental procarcinogens, such as arylamines and polycyclic aromatic hydrocarbons, that are found in food. Several peroxisome proliferators and arachidonic acid derivatives enhance cytochrome P450 1A1 activity, but the mechanisms involved remain unknown. The aim of this work was to study the role of peroxisome proliferator-activated receptors in cytochrome P450 1A1 gene induction. The role of peroxisome proliferator-activated receptor transcription factors in cytochrome P450 1A1 induction was assessed by means of enzymatic activities, quantitative real-time polymerase chain reaction, gene reporter assays, mutagenesis, and electrophoretic mobility shift assay. We show that peroxisome proliferator-activated receptor-alpha agonists (WY-14643, bezafibrate, clofibrate, and phthalate) induce human cytochrome P450 1A1 gene expression, whereas 2,4-thiazolidinedione, a specific peroxisome proliferator-activated receptor-gamma agonist, represses it. The induction of cytochrome P450 1A1 transcripts by WY-14643 was associated with a marked increase of ethoxyresorufin O -deethylase activity (10-fold at 200 mumol/L). Transfection of peroxisome proliferator-activated receptor-alpha complementary DNA enhanced cytochrome P450 1A1 messenger RNA induction by WY-14643, although WY-14643 failed to activate xenobiotic responsive element sequences. Two peroxisome proliferator response element sites were located at positions -931/-919 and -531/-519 of the cytochrome P450 1A1 promoter. Their inactivation by directed mutagenesis suppressed the inductive effect of WY-14643 on cytochrome P450 1A1 promoter activation. Electrophoretic mobility shift assay and chromatin immunoprecipitation assay experiments showed that the 2 cytochrome P450 1A1 peroxisome proliferator response element sites bind the peroxisome proliferator-activated receptor-alpha/retinoid X receptor-alpha heterodimer. We describe here a new cytochrome P450 1A1 induction pathway involving peroxisome proliferator-activated receptor-alpha and 2 peroxisome proliferator response element sites, indicating that peroxisome proliferator-activated receptor-alpha ligands, which are common environmental compounds, may be involved in carcinogenesis.

CYP-specific bioactivation of four organophosphorothioate pesticides by human liver microsomes

The bioactivation of azinphos-methyl (AZIN), chlorpyrifos (CPF), diazinon (DIA), and parathion (PAR), four widely used organophosphorothioate (OPT) pesticides has been investigated in human liver microsomes (HLM). In addition, the role of human cytochrome P450 (CYPs) in OPT desulfuration at pesticide levels representative of human exposure have been defined by means of correlation and immunoinhibition studies. CYP-mediated oxon formation from the four OPTs is efficiently catalyzed by HLM, although showing a high variability (>40-fold) among samples. Two distinct phases were involved in the desulfuration of AZIN, DIA, and PAR, characterized by different affinity constants ( K mapp1 = 0.13–9 μM and K mapp2 = 5– 269 μM). Within the range of CPF concentrations tested, only the high-affinity component was evidenced ( K mapp1 = 0.27–0.94 μM). Oxon formation in phenotyped individual HLM showed a significant correlation with CYP1A2-, 3A4-, and 2B6-related activities, at different levels depending on the OPT concentration. Anti-human CYP1A2, 2B6, and 3A4 antibodies significantly inhibited oxon formation, showing the same OPT concentration dependence. Our data indicated that CYP1A2 is mainly involved in OPT desulfuration at low pesticide concentrations, while the role of CYP3A4 is more significant to the low-affinity component of OPT bioactivation. The contribution of CYP2B6 to total hepatic oxon formation was relevant in a wide range of pesticide concentrations, being a very efficient catalyst of both the high- and low-affinity phase. These results suggest CYP1A2 and 2B6 as possible metabolic biomarkers of susceptibility to OPT toxic effect at the actual human exposure levels.

ROLE OF HUMAN CYTOCHROME P450 (CYP) IN THE METABOLIC ACTIVATION OF NITROSAMINE DERIVATIVES: APPLICATION OF GENETICALLY ENGINEERED SALMONELLA EXPRESSING HUMAN CYP

The role of human cytochrome P450 (CYP) in the metabolic activation of tobacco-related N-nitrosamines was examined by Salmonella mutation test using a series of genetically engineered Salmonella typhimurium YG7108 strains each co-expressing a form of CYP (CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, and CYP3A5) together with human NADPH–cytochrome P450 reductase. Seven tobacco-related N-nitrosamines such as 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, N-nitrosodiethylamine, N-nitrosopyrrolidine, N-nitrosopiperidine, N-nitrosonornicotine, N-nitrosoanabasine, and N-nitrosoanatabine were used. The CYP2A6 was found to be responsible for the mutagenic activation of essentially all tobacco-related N-nitrosamines examined.On the basis of the evidence, genetic polymorphism of the CYP2A6 gene appeared to be one of the factors determining cancer susceptibility caused by smoking. Previously, we found the whole deletion of the CYP2A6 gene (CYP2A6*4C) as a type of genetic polymorphism in Japanese. We hypothesized that individuals possessing the gene homozygous for CYP2A6*4C were incapable of activating tobacco-related N-nitrosamines and showed lower susceptibility to lung cancer induced by tobacco smoke. Thus, the relationship between the CYP2A6*4C and the susceptibility to the lung cancer was evaluated. The frequency of the CYP2A6*4C was significantly lower in the lung cancer patients than healthy volunteers, suggesting that the subjects carrying the CYP2A6*4C alleles are resistant to carcinogenesis caused by N-nitrosamines because of the poor metabolic activation capacity.Taking these results into account, CYP2A6 is an enzyme enhancing lung cancer risk.

Role of human cytochrome P450 (CYP) in the metabolic activation of N-alkylnitrosamines: application of genetically engineered Salmonella typhimurium YG7108 expressing each form of CYP together with human NADPH-cytochrome P450 reductase

The role of human cytochrome P450 (CYP) in the metabolic activation of N-alkylnitrosamines was examined by Ames test using genetically engineered Salmonella typhimurium ( S. typhimurium)YG7108 cells expressing each form of human CYP together with human NADPH-cytochrome P450 reductase (OR). The relationship between the structure of N-alkylnitrosamines and CYP form(s) involved in the activation was evaluated. Eleven strains of S. typhimurium YG7108 cells expressing each form of CYP (CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4 or CYP3A5) were employed. Eight N-alkylnitrosamines including N-nitrosodimethylamine (NDMA), N-nitrosodiethylamine (NDEA), N-nitrosodipropylamine (NDPA), N-nitrosodibutylamine (NDBA), N-nitrosomethylethylamine (NMEA), N-nitrosomethylpropylamine (NMPA), N-nitrosomethylbutylamine (NMBA) and N-nitrosoethylbutylamine (NEBA) were examined. Minimal concentration (MC) value of a promutagen was defined as the concentration of a chemical giving a positive result. Mutagen-producing capacity of CYP, as indicated by induced revertants/nmol promutagen/pmol CYP, for an N-alkylnitrosamine was determined for all forms of CYP. These N-alkylnitrosamines were mainly activated by CYP2E1, CYP2A6 and CYP1A1. N-alkylnitrosamines with relatively short alkyl chains such as NDMA and NMEA were primarily activated by CYP2E1 as judged by mutagen-producing capacity. With the increase of the number of the carbon atoms of the alkyl chains, the contribution of CYP2A6 increased. CYP2A6 played major roles in the activation of NDEA, NDPA, NMPA, NMBA and NEBA. Interestingly, CYP1A1 became a molecular form of CYP playing a major role in the metabolic activation of NDBA.

Interspecies comparison and role of human cytochrome P450 and flavin-containing monooxygenase in hepatic metabolism of L-775,606, a potent 5-HT1D receptor agonist

1. Quantitative species differences and human liver enzymes involved in the metabolism of L-775,606, a potent and selective 5-HT1D receptor agonist developed for the acute treatment of migraine headache, have been investigated in vitro. 2. In human, monkey, dog and rat liver microsomes, formation of the hydroxylated M1 and the N-dealkylated M2 was mediated by enzyme(s) of high-affinity (apparent Km ~1-6 muM), and that of the two N-oxide isomers (M3) was catalysed by those of low affinity (apparent Km ~50-110 muM). In dog, M3 constituted a major pathway (~40%), whereas in all other species it was a minor metabolite (<5%). 3. In human liver microsomes, a marked inhibition ( 80%) of M1 and M2 formation was observed by SKF525-A, troleandomycin, ketoconazole and anti-CYP3A antibodies, whereas the inhibition was modest (~20-40%) with quercetin. Of seven cDNA-expressed human P450 tested, only CYP3A4 and CYP2C8 were capable of oxidizing L-775,606, resulting primarily in M1 and M2. However, CYP3A4 possessed much higher affinity ( 20-fold) and much higher intrinsic activity (>100-fold) than CYP2C8. 4. In contrast, N-oxidation was not inhibited by any inhibitors of P450 tested, but rather was reduced significantly by heat treatment and methimazole, and was increased substantially with an incubation pH > 7.4. Human flavin-containing monooxygenase form 3 (FMO3) catalysed exclusively the N-oxidation to M3, with apparent Km and optimum pH comparable with those observed in human liver microsomes. 5. These results demonstrated quantitative interspecies differences in the metabolism of L-775,606. In human, metabolism of L-775,606 to the principal metabolites, M1 and M2, was mediated primarily by CYP3A4 with minimal contribution from CYP2C8, whereas the minor N-oxidative pathway was catalysed mainly by FMO3.

Hepatic metabolism of diclofenac: role of human CYP in the minor oxidative pathways

The aim of this study was to re-examine the human hepatic metabolism of diclofenac, with special focus on the generation of minor hydroxylated metabolites implicated in the idiosyncratic hepatotoxicity of the drug. Different experimental approaches were used: human hepatocytes, human microsomes, and engineered cells expressing single human CYP (cytochromes P450). Human hepatocytes formed 3′-hydroxy-, 4′-hydroxy-, 5-hydroxy- 4′,5-dihydroxy-, and N,5-dihydroxydiclofenac, as well as several lactams. Formation of 4′- and 5-hydroxydiclofenac by human liver microsomes followed a Michaelis–Menten kinetics ( K m 9 ± 1 μM; V max 432 ± 15 pmol/min/mg and K m 43 ± 5 μM; and V max 15.4 ± 0.6 pmol/min/mg, respectively). Secondary metabolites were detected after incubation of 5-hydroxydiclofenac with human liver microsomes, yielding 4′,5-dihydroxydiclofenac ( K m 15 ± 1 μM; V max 96 ± 3 pmo1/min/mg) and small amounts of N,5-dihydroxydiclofenac (non-Michaelis–Menten kinetics). Based on microsome studies and the incubations with human hepatocytes and engineered cells, we estimated that in vivo CYP2C9 would be exclusively responsible for the 4′ hydroxylation of diclofenac (>99.5%) as well as 5-hydroxydiclofenac (>97%). CYP2C9 was exclusively responsible for the formation of 3′-hydroxydiclofenac. Multiple regression analysis evidenced that the rate of production of 5-hydroxydiclofenac in human microsomes followed the algorithm: 0.040 × S-mephenytoin 4′-hydroxylation + 0.083 × tolbutamide methylhydroxylation, (multiple correlation coefficient = 0.969). However, the incubation of diclofenac with cell lines expressing different human CYP suggested that 7 isoforms could be involved. Comparison of data obtained with CYP-expressing cells and human hepatocytes suggests that CYP2C8 > CYP2C19 ≅ CYP2C18 ≫ CYP2B6 are the isoforms implicated in the 5-hydroxylation of diclofenac in vivo.

Identification of the Human P‐450 Enzymes Responsible for the Sulfoxidation and Thiono‐Oxidation of Diethyldithiocarbamate Methyl Ester: Role of P‐450 Enzymes in Disulfiram Bioactivation

Diethyldithiocarbamate methyl ester (DDTC-Me) is a precursorto the formation of S-methyl-N,N-diethylthiolcarbamate sulfoxide, the active metabolite proposed to be responsible for the alcohol deterrent effects of disulfiram. The present study investigated the role of human cytochrome P-450 (CYP) enzymes in sulfoxidation and thiono-oxidation of DDTC-Me, intermediary steps in the activation of disulfiram. Several approaches were used in an attempt to delineate the particular P-450 enzyme(s) involved in the sulfoxidation and thiono-oxidation of DDTC-Me. These approaches included the use of cDNA-expressed human P-450 enzymes, correlation analysis with sample-to-sample variation in human P-450 enzymes in a bank of human liver microsomes, and chemical and antibody inhibition studies. Multiple human P-450 enzymes (CYP3A4, CYP1A2, CYP2A6, and CYP2D6) catalyzed the sulfoxidation of DDTC-Me, as determined with cDNA-expressed enzymes. Several lines of evidence suggest that the sulfoxidation of DDTC-Me by human liver microsomes is primarily catalyzed by CYP3A4/5, including (1) a high correlation between DDTC-Me sulfoxidation and testosterone 6beta-hydroxylation; (2) increased DDTC-Me sulfoxidation in the presence of alpha-naphthoflavone, an activator of CYP3A enzymes; (3) inhibition of this reaction by inhibitors of CYP3A4/5 enzymes, such as troleandomycin and ketoconazole; and (4) inhibition of DDTC-Me sulfoxidation by antibodies against CYP3A enzymes. On the other hand, several lines of evidence suggested that the thiono-oxidation of DDTC-Me by human liver microsomes is catalyzed in part by CYP1A2, CYP2B6, CYP2E1, and CYP3A4/5, including (1) these human P450 enzymes among others have the capacity to catalyze this reaction, as determined with cDNA-expressed enzymes; (2) a high correlation between DDTC-Me thiono-oxidation and testosterone 6beta-hydroxylation, weak inhibition by ketoconazole, troleandomycin, and anti-CYP3A antibodies suggested a minor role for CYP3A4; (3) a high correlation with immunoreactive CYP2B6 suggested involvement of this enzyme; (4) weak inhibition of DDTC-Me thiono-oxidation by furafylline and anti-CYP1A antibody suggested involvement of CYP1A2; and (5) inhibition of DDTC-Me thiono-oxidation by DDTC and anti-CYP2E antibodies suggested a role for CYP2E1. Collectively, these data suggested CYP3A4/5 enzymes are the major contributors to the sulfoxidation of DDTC-Me by human liver microsomes, and CYP1A2, CYP2B6, CYP2E1, and CYP3A4/5 contribute toward DDTC-Me thiono-oxidation by human liver microsomes. This study, in conjunction with others (Madan et al., Drug Metab. Dispos. 23:1153-1162, 1995), may help explain the variability in disulfiram's effectiveness as an alcohol deterrent.

Role of human cytochromes P450 in the metabolic activation of chemical carcinogens and toxins.

(1994). Role of Human Cytochromes P450 in the Metabolic Activation of Chemical Carcinogens and Toxins. Drug Metabolism Reviews: Vol. 26, No. 1-2, pp. 165-183.