Spectral modification and catalytic inhibition of human cytochromes P450 1A1, 1A2, 1B1, 2A6, and 2A13 by four chemopreventive organoselenium compounds

Several organoselenium compounds including benzyl selenocyanate (BSC), 1,2-phenylenebis(methylene)selenocyanate (o-XSC), 1,3-phenylenebis(methylene)selenocyanate (m-XSC), and 1,4-phenylenebis(methylene)selenocyanate (p-XSC) have been shown to prevent cancers caused by polycyclic aromatic hydrocarbons (PAHs) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in experimental animals; these chemical carcinogens are activated by human P450 1 and 2A family enzymes, respectively, to carcinogenic metabolites. In this study, we examined whether these selenium compounds interact with and inhibit human P450 1 and 2A enzymes in vitro. Four organoselenium compounds induced reverse Type I binding spectra with P450 1A1, 1A2, and 1B1 and Type I binding spectra with P450 2A6 and 2A13. The spectral dissociation constants (K(s)) for the interaction of P450 1B1 with these chemicals were 3.6-5.7 μM; the values were lower than those with seen with P450 1A1 (19-30 μM) or 1A2 (6.3-13 μM). The K(s) values for Type I binding of P450 2A13 with m-XSC and BSC were both 0.20 μM; the values were very low compared to those for the interaction of P450 2A6 with m-XSC (5.7 μM) and BSC (2.0 μM). Four selenium compounds directly inhibited 7-ethoxyresorufin O-deethylation activities catalyzed by P450 1A1, 1A2, and 1B1 with IC(50) values <1.0 μM, except for the inhibition of P450 1A2 by BSC (1.3 μM). Coumarin 7-hydroxylation activities of P450 2A13 were more inhibited by four selenium compounds than those of P450 2A6, with IC(50) values of 0.22-1.4 μM for P450 2A13 and 2.4-6.2 μM for P450 2A6. Molecular docking studies of the interaction of four organoselenium compounds with human P450 enzymes suggest that these chemicals can be docked into the active sites of these human P450 enzymes and that the sites of the selenocyanate functional groups of these chemicals differ between the P450 1 and 2A family enzymes.     

Identification of human cytochrome P450 and flavin-containing monooxygenase enzymes involved in the metabolism of lorcaserin, a novel selective human 5-hydroxytryptamine 2C agonist

Lorcaserin, a selective serotonin 5-hydroxytryptamine 2C receptor agonist, is being developed for weight management. The oxidative metabolism of lorcaserin, mediated by recombinant human cytochrome P450 (P450) and flavin-containing monooxygenase (FMO) enzymes, was examined in vitro to identify the enzymes involved in the generation of its primary oxidative metabolites, N-hydroxylorcaserin, 7-hydroxylorcaserin, 5-hydroxylorcaserin, and 1-hydroxylorcaserin. Human CYP1A2, CYP2A6, CYP2B6, CYP2C19, CYP2D6, CYP3A4, and FMO1 are major enzymes involved in N-hydroxylorcaserin; CYP2D6 and CYP3A4 are enzymes involved in 7-hydroxylorcaserin; CYP1A1, CYP1A2, CYP2D6, and CYP3A4 are enzymes involved in 5-hydroxylorcaserin; and CYP3A4 is an enzyme involved in 1-hydroxylorcaserin formation. In 16 individual human liver microsomal preparations (HLM), formation of N-hydroxylorcaserin was correlated with CYP2B6, 7-hydroxylorcaserin was correlated with CYP2D6, 5-hydroxylorcaserin was correlated with CYP1A2 and CYP3A4, and 1-hydroxylorcaserin was correlated with CYP3A4 activity at 10.0 μM lorcaserin. No correlation was observed for N-hydroxylorcaserin with any P450 marker substrate activity at 1.0 μM lorcaserin. N-Hydroxylorcaserin formation was not inhibited by CYP1A2, CYP2A6, CYP2B6, CYP2C19, CYP2D6, and CYP3A4 inhibitors at the highest concentration tested. Furafylline, quinidine, and ketoconazole, selective inhibitors of CYP1A2, CYP2D6, and CYP3A4, respectively, inhibited 5-hydroxylorcaserin (IC(50) = 1.914 μM), 7-hydroxylorcaserin (IC(50) = 0.213 μM), and 1-hydroxylorcaserin formation (IC(50) = 0.281 μM), respectively. N-Hydroxylorcaserin showed low and high K(m) components in HLM and 7-hydroxylorcaserin showed lower K(m) than 5-hydroxylorcaserin and 1-hydroxylorcaserin in HLM. The highest intrinsic clearance was observed for N-hydroxylorcaserin, followed by 7-hydroxylorcaserin, 5-hydroxylorcaserin, and 1-hydroxylorcaserin in HLM. Multiple human P450 and FMO enzymes catalyze the formation of four primary oxidative metabolites of lorcaserin, suggesting that lorcaserin has a low probability of drug-drug interactions by concomitant medications.     

In vitro inhibitory effects of troglitazone and its metabolites on drug oxidation activities of human cytochrome P450 enzymes: comparison with pioglitazone and rosiglitazone

1. Investigated were the effects of a new oral antidiabetic drug, troglitazone, and its three metabolites and antidiabetic drug candidates pioglitazone and rosiglitazone on xenobiotic oxidations catalyzed by nine recombinant human cytochrome P450 (P450 or CYP) enzymes and by human liver microsomes. 2. Troglitazone (5 muM) significantly inhibited CYP2C8-dependent paclitaxel 6alpha- hydroxylation and CYP2C9-dependent S-warfarin 7-hydroxylation. On the other hand, pioglitazone and rosiglitazone (50 muM) only slightly inhibited these xenobiotic oxidation activities catalyzed by CYP2C enzymes. 3. The inhibitory potential of troglitazone (50% inhibition concentration, IC₅₀) was ~5 muM for drug oxidations catalyzed by CYP2C9 and CYP2C8 and C20 muM for activities catalyzed by CYP2C19 and CYP3A4 respectively. For the three metabolites of troglitazone tested, a quinone-type metabolite (M3) was the most potent inhibitor for CYP2C enzymes, followed by a sulphate conjugate (M1); effects of a glucuronide (M2) were very weak. The inhibitory effects of the parent drug were more potent than those of metabolites. Troglitazone and M3 inhibited P450 activities mainly through a competitive manner with K_i=0.2-1.7 muM and 1.4-8.8 muM respectively. 4. In three human liver microsomes, troglitazone and its metabolites also inhibited paclitaxel 6alpha-hydroxylation, S-warfarin 7-hydroxylation, S-mephenytoin 4'-hydroxylation, and testosterone 6beta-hydroxylation with similar IC₅₀, as observed for the recombinant P450 enzyme systems. 5. These results suggest that xenobiotic oxidations by P450 enzymes are more substantially affected by troglitazone and its metabolites than pioglitazone or rosiglitazone, and that drug interactions may be of much importance to understand the basis for the pharmacological and toxicological actions of this new oral antidiabetic drug.     

In vitro inhibitory effects of troglitazone and its metabolites on drug oxidation activities of human cytochrome P450 enzymes: comparison with pioglitazone and rosiglitazone

1. Investigated were the effects of a new oral antidiabetic drug, troglitazone, and its three metabolites and antidiabetic drug candidates pioglitazone and rosiglitazone on xenobiotic oxidations catalyzed by nine recombinant human cytochrome P450 (P450 or CYP) enzymes and by human liver microsomes. 2. Troglitazone (5 microM) significantly inhibited CYP2C8-dependent paclitaxel 6alpha-hydroxylation and CYP2C9-dependent S-warfarin 7-hydroxylation. On the other hand, pioglitazone and rosiglitazone (50 microM) only slightly inhibited these xenobiotic oxidation activities catalyzed by CYP2C enzymes. 3. The inhibitory potential of troglitazone (50% inhibition concentration, IC50) was approximately 5 microM for drug oxidations catalyzed by CYP2C9 and CYP2C8 and approximately 20 microM for activities catalyzed by CYP2C19 and CYP3A4 respectively. For the three metabolites of troglitazone tested, a quinone-type metabolite (M3) was the most potent inhibitor for CYP2C enzymes, followed by a sulphate conjugate (M1); effects of a glucuronide (M2) were very weak. The inhibitory effects of the parent drug were more potent than those of metabolites. Troglitazone and M3 inhibited P450 activities mainly through a competitive manner with Ki = 0.2-1.7 microM and 1.4-8.8 microM respectively. 4. In three human liver microsomes, troglitazone and its metabolites also inhibited paclitaxel 6alpha-hydroxylation, S-warfarin 7-hydroxylation, S-mephenytoin 4'-hydroxylation, and testosterone 6beta-hydroxylation with similar IC50, as observed for the recombinant P450 enzyme systems. 5. These results suggest that xenobiotic oxidations by P450 enzymes are more substantially affected by troglitazone and its metabolites than pioglitazone or rosiglitazone, and that drug interactions may be of much importance to understand the basis for the pharmacological and toxicological actions of this new oral antidiabetic drug.     

Bioactivation of 1,1-dichloroethylene to its epoxide by CYP2E1 and CYP2F enzymes.

1,1-Dichloroethylene (DCE) exposure to mice elicits lung toxicity that selectively targets bronchiolar Clara cells. The toxicity is mediated by DCE metabolites formed via cytochrome P450 metabolism. The primary metabolites formed are DCE epoxide, 2,2-dichloroacetaldehyde, and 2-chloroacetyl chloride. The major metabolite detected is 2-S-glutathionyl acetate [C], a putative conjugate of DCE epoxide with glutathione. In this investigation, studies were undertaken to test the hypothesis that CYP2E1 and CYP2F2 are involved in bioactivation of DCE to the epoxide in murine lung. We have developed a method using liquid chromatography/mass spectrometry (LC/MS) to evaluate the kinetics of the rates of production of conjugate [C] by recombinant CYP2E1 and CYP2F enzymes and lung microsomes. Concentration-dependent formation of conjugate [C] was found in incubations of DCE with recombinant CYP2E1 and CYP2F enzymes and lung microsomes from CD-1, wild-type (mixed 129/Sv and C57BL), and CYP2E1-null mice. Recombinant rat CYP2E1 exhibited greater affinity and catalytic efficiency for DCE metabolism than did recombinant human CYP2E1, mouse CYP2F2, goat CYP2F3 or rat CYP2F4. In the lung microsomal incubations, the rates of conjugate [C] production were higher in CD-1 mice than in either wild-type or CYP2E1-null mice; the level of [C] in CYP2E1-null mice was about 66% of that in wild-type mice. These results demonstrated that LC/MS analysis is a suitable method for detection and quantitation of conjugate [C], and that CYP2E1 and CYP2F2 catalyze the bioactivation of DCE to the epoxide in murine lung. The results also demonstrated that CYP2E1 is the high-affinity enzyme involved in DCE bioactivation.     

In vitro metabolism of the calmodulin antagonist DY-9760e (3-[2-[4-(3-chloro-2-methylphenyl)-1-piperazinyl]ethyl]-5,6-dimethoxy-1-(4-imidazolylmethyl)-1H-indazole dihydrochloride 3.5 hydrate) by human liver microsomes: involvement of cytochromes p450 in atypical kinetics and potential drug interactions.

Human cytochrome P450 (P450) isozyme(s) responsible for metabolism of the calmodulin antagonist 3-[2-[4-(3-chloro-2-methylphenyl)-1-piperazinyl]ethyl]-5,6-dimethoxy-1-(4-imidazolylmethyl)-1H-indazole dihydrochloride 3.5 hydrate (DY-9760e) and kinetic profiles for formation of its six primary metabolites [M3, M5, M6, M7, M8, and DY-9836 (3-[2-[4-(3-chloro-2-methylphenyl)piperazinyl]ethyl]-5,6-dimethoxyindazole)] were identified using human liver microsomes and recombinant P450 enzymes. In vitro experiments, including an immunoinhibition study, correlation analysis, and reactions with recombinant P450 enzymes, revealed that CYP3A4 is the primary P450 isozyme responsible for the formation of the DY-9760e metabolites, except for M5, which is metabolized by CYP2C9. Additionally, at clinically relevant concentrations, CYP2C8 and 2C19 make some contribution to the formation of M3 and M5, respectively. The formation rates of DY-9760e metabolites except for M8 by human liver microsomes are not consistent with a Michaelis-Menten kinetics model, but are better described by a substrate inhibition model. In contrast, the enzyme kinetics for all metabolites formed by recombinant CYP3A4 can be described by an autoactivation model or a mixed model of autoactivation and biphasic kinetics. Inhibition of human P450 enzymes by DY-9760e in human liver microsomes was also investigated. DY-9760e is a very potent competitive inhibitor of CYP2C8, 2C9 and 2D6 (Ki 0.25-1.7 microM), a mixed competitive and noncompetitive inhibitor of CYP2C19 (Ki 2.4 microM) and a moderate inhibitor of CYP1A2 and 3A4 (Ki 11.4-20.1 microM), suggesting a high possibility for human drug-drug interaction.     

In vitro interactions between a potential muscle relaxant E2101 and human cytochromes P450.

E2101 or N-methyl-[1-[1-(2-fluorophenethyl)piperidine-4-yl]-1H-indol-6-yl] acetamide, an antagonist of 5-hydroxytryptamine receptor subtypes 1A and 2, is currently under development for the potential treatment of skeletal muscle associated spasticity. Here we characterized the in vitro metabolism of E2101 using human liver enzymes including human liver microsomal preparations, human liver S9 fractions, and individual forms of recombinant cytochromes P450 (P450s). Our results showed that E2101 was metabolized by P450s to form monohydroxylated (M1 and M2), dihydroxylated (M3), and N-dealkylated metabolites (M4). The structures of these major microsomal metabolites were proposed based on LC/MS/MS analyses. All four metabolites, M1-M4, were formed by CYP3A4. Metabolites, M1, M2, and M4, were also formed by CYP2C19 and M2 and M3 by CYP2D6. The potential P450 inhibition and induction of E2101 were also evaluated. E2101 was determined to be a competitive inhibitor of CYP2C19 and CYP2D6 with K(i) of 15 and 48 microM, respectively, as determined by both Dixon plots and simultaneously nonlinear regression analyses. Induction of major P450 expression was not detected immunochemically after 72-h exposure to 10 or 50 microM E2101 in primary hepatocyte cultures obtained from three subjects. Taken together, E2101 is expected to metabolically interact with major human P450 enzymes including CYP2C19, CYP2D6, and CYP3A4, and a low risk of drug-drug interaction would be anticipated in clinical studies.     

Identification of the cytochrome P450 enzymes involved in the metabolism of domperidone

The objective was to identify the major cytochrome P450 enzyme(s) involved in the metabolism of domperidone. Experiments were performed using human liver microsomes (HLMs), recombinant human cytochrome P450 enzymes, cytochrome P450 chemical inhibitors and monoclonal antibodies directed against cytochrome P450 enzymes. Four metabolites were identified from incubations performed with HLMs and excellent correlations were observed between the formation of domperidone hydroxylated metabolites (M1, M3 and M4), N-desalkylated domperidone metabolite (M2) and enzymatic markers of CYP3A4/5 (r2 = 0.9427, 0.951, 0.9497 and 0.8304, respectively). Ketoconazole (1 microM) decreased the formation rate of M1, M2, M3 and M4 by 83, 78, 75 and 88%, respectively, whereas the effect of other inhibitors (quinidine, furafylline and sulfaphenazole) was minimal. Important decreases in the formation rate of M1 (68%), M2 (64%) and M3 (54%) were observed with anti-CYP3A4 antibodies. Formation of M1, M2 and M3 in HLMs exhibited Michaelis-Menten kinetics (Km: 166, 248 and 36 microM, respectively). Similar Km values were observed for M1, M2 and M3 when incubations were performed with recombinant human CYP3A4 (Km: 107, 273 and 34 microM, respectively). The data suggest that CYP3As are the major enzymes involved in the metabolism of domperidone.     

In vitro metabolism of tolcapone to reactive intermediates: relevance to tolcapone liver toxicity

Tolcapone is a catechol-O-methyltransferase (COMT) inhibitor used for control of motor fluctuations in Parkinson's disease (PD). Since its entry onto the market in 1998, tolcapone has been associated with numerous cases of hepatotoxicity, including three cases of fatal fulminant hepatic failure. The cause of this toxicity is not known; however, it does not occur with the use of the structurally similar drug entacapone. It is known that tolcapone is metabolized to amine (M1) and acetylamine (M2) metabolites in humans, but that the analogous metabolites were not detected in a limited human study of entacapone metabolism. We hypothesized that one or both of these tolcapone metabolites could be oxidized to reactive species and that these reactive metabolites might play a role in tolcapone-induced hepatocellular injury. To investigate this possibility, we examined the ability of M1 and M2 to undergo in vitro bioactivation by electrochemical and enzymatic methods. Electrochemical experiments revealed that M1 and M2 are more easily oxidized than the parent compound, in the order M1 > M2 > tolcapone. There was a general correlation between oxidation potential and the half-lives of the compounds in the presence of two oxidizing systems, horseradish peroxidase and myeloperoxidase. These enzymes catalyzed the oxidation of M1 and M2 to reactive species that could be trapped with glutathione (GSH) to form metabolite adducts (C1 and C2). Each metabolite was found to only form one GSH conjugate, and the structures were tentatively identified using LC-MS/MS. Following incubation of M1 and M2 with human liver microsomes in the presence of GSH, the same adducts were observed, and their structures were confirmed using LC-MS/MS and (1)H NMR. Experiments with chemical P450 inhibitors and cDNA-expressed P450 enzymes revealed that this oxidation is catalyzed by several P450s, and that P450 2E1 and 1A2 play the primary role in the formation of C1 while P450 1A2 is most important for the production of C2. Taken together, these data provide evidence that tolcapone-induced hepatotoxicity may be mediated through the oxidation of the known urinary metabolites M1 and M2 to reactive intermediates. These reactive species may form covalent adducts to hepatic proteins, resulting in damage to liver tissues, although this supposition was not investigated in this study.     

Novel metabolites of buprenorphine detected in human liver microsomes and human urine.

The in vitro metabolism of buprenorphine was investigated to explore new metabolic pathways and identify the cytochromes P450 (P450s) responsible for the formation of these metabolites. The resulting metabolites were identified by liquid chromatography-electrospray ionization-tandem mass spectrometry. In addition to norbuprenorphine, two hydroxylated buprenorphine (M1 and M2) and three hydroxylated norbuprenorphine (M3, M4, and M5) metabolites were produced by human liver microsomes (HLMs), with hydroxylation occurring at the tert-butyl group (M1 and M3) and at unspecified site(s) on the ring moieties (M2, M4, and M5). Time course and other data suggest that buprenorphine is N-dealkylated to form norbuprenorphine, followed by hydroxylation to form M3; buprenorphine is hydroxylated to form M1 and M2, followed by N-dealkylation to form M3 and M4 or M5. The involvement of selected P450s was investigated using cDNA-expressed P450s coupled with scaling models, chemical inhibition, monoclonal antibody (MAb) analysis, and correlation studies. The major enzymes involved in buprenorphine elimination and norbuprenorphine and M1 formation were P450s 3A4, 3A5, 3A7, and 2C8, whereas 3A4, 3A5, and 3A7 produced M3 and M5. Based on MAb analysis and chemical inhibition, the contribution of 2C8 was higher in HLMs with higher 2C8 activity, whereas 3A4/5 played a more important role in HLMs with higher 3A4/5 activity. Examination of human urine from subjects taking buprenorphine showed the presence of M1 and M3; most of M1 was conjugated, whereas 60 to 70% of M3 was unconjugated.     

In vitro characterization of 4′-(p-toluenesulfonylamide)-4-hydroxychalcone using human liver microsomes and recombinant cytochrome P450s

1.?4′-(p-Toluenesulfonylamide)-4-hydroxychalcone (TSAHC) is a synthetic sulfonylamino chalcone compound possessing anti-cancer properties. The aim of this study was to elucidate the metabolism of TSAHC in human liver microsomes (HLMs) and to characterize the cytochrome P450 (P450) enzymes that are involved in the metabolism of TSAHC.

2.?TSAHC was incubated with HLMs or recombinant P450 isoforms (rP450) in the presence of an nicotinamide adenine dinucleotide phosphate, reduced form (NADPH)-regenerating system. The metabolites were identified and analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). P450 isoforms, responsible for TSAHC metabolite formation, were characterized by chemical inhibition and correlation studies in HLMs and enzyme kinetic studies with a panel of rP450 isoforms.

3.?Two hydroxyl metabolites, that is M1 and M2, were produced from the human liver microsomal incubations (K_m and V_max values were 2.46?µM and 85.1?pmol/min/mg protein for M1 and 9.98?µM and 32.1?pmol/min/mg protein for M2, respectively). The specific P450 isoforms responsible for two hydroxy-TSAHC formations were identified using a combination of chemical inhibition, correlation analysis and metabolism by expressed recombinant P450 isoforms. The known P450 enzyme activities and the rate of TSAHC metabolite formation in the 15 HLMs showed that TSAHC metabolism is correlated with CYP2C and CYP3A activity. The P450 isoform-selective inhibition study in HLMs and the incubation study of cDNA-expressed enzymes also showed that two hydroxyl metabolites M1 and M2 biotransformed from TSAHC are mainly mediated by CYP2C and CYP3A, respectively. These findings suggest that CYP2C8, CYP2C9, CYP2C19, CYP3A4 and CYP3A5 isoforms are major enzymes contributing to TSAHC metabolism.     

Inhibition of human cytochrome P450 enzymes by 1,2-dithiole-3-thione, oltipraz and its derivatives, and sulforaphane

Recent studies have demonstrated that two chemoprotective agents, oltipraz (OPZ), a synthetic derivative of the natural compound 1, 2-dithiole-3-thione (D3T), and sulforaphane (SF), an isothiocyanate, are not only inducers of glutathione S-transferases but also inhibitors of some major cytochrome P450 enzymes (P450s) involved in xenobiotic metabolism. We examined P450 inhibition by the two compounds and compared two OPZ metabolites (OPZ M(3) and M(8)) and D3T using human P450s expressed in Escherichia coli membranes. OPZ was a more potent inhibitor than D3T or SF, in the following order of inhibition: P450 1A2 > 3A4 > 1A1 approximately 1B1 > 2E1. OPZ M(3) also inhibited P450s 1A2, 1A1, 1B1, and 3A4 but not more effectively than OPZ. OPZ M(8) was not inhibitory. OPZ behaved as a competitive inhibitor of P450 1A2, with a K(i) of 1.5 microM. Incubation of P450 1A2 with OPZ and NADPH led to a first-order loss of the P450 spectrum, and the loss was not blocked by glutathione. No such time-dependent loss of P450 was seen with P450 1A2 and D3T, P450 1A2 and OPZ M(3), P450 1A2 and SF, P450 3A4 and OPZ, P450 3A4 and D3T, P450 2E1 and OPZ, or P450 2E1 and D3T. The time- and concentration-dependent loss of P450 1A2 activity in the presence of OPZ was characterized with a K(i) of 9 microM and a k(inactivation) of 0.19 min(-)(1). The activation of 2-amino-3,5-dimethylimidazo[4, 5-f]quinoline (MeIQ) in an E. coli lac-based mutagenicity tester system containing functional human P450 1A2 was inhibited by OPZ (IC(50) < 1 microM) but not appreciably by 40 microM D3T. Our results indicate that OPZ is a competitive and mechanism-based inhibitor of P450 1A2, and the extent of this inhibition is significantly greater than that of other chemoprotective chemicals with P450 1A2 or other human P450s.     

Metabolism of capsaicin by cytochrome P450 produces novel dehydrogenated metabolites and decreases cytotoxicity to lung and liver cells

Capsaicin is a common dietary constituent and a popular homeopathic treatment for chronic pain. Exposure to capsaicin has been shown to cause various dose-dependent acute physiological responses including the sensation of burning and pain, respiratory depression, and death. In this study, the P450-dependent metabolism of capsaicin by recombinant P450 enzymes and hepatic and lung microsomes from various species, including humans, was determined. A combination of LC/MS, LC/MS/MS, and LC/NMR was used to identify several metabolites of capsaicin that were generated by aromatic (M5 and M7) and alkyl hydroxylation (M2 and M3), O-demethylation (M6), N- (M9) and alkyl dehydrogenation (M1 and M4), and an additional ring oxygenation of M9 (M8). Dehydrogenation of capsaicin was a novel metabolic pathway and produced unique macrocyclic, diene, and imide metabolites. Metabolism of capsaicin by microsomes was inhibited by the nonselective P450 inhibitor 1-aminobenzotriazole (1-ABT). Metabolism was catalyzed by CYP1A1, 1A2, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4. Addition of GSH (2 mM) to microsomal incubations stimulated the metabolism of capsaicin and trapped several reactive electrophilic intermediates as their GSH adducts. These results suggested that reactive intermediates, which inactivated certain P450 enzymes, were produced during catalytic turnover. Comparison of the rate and types of metabolites produced from capsaicin and its analogue, nonivamide, demonstrated similar pathways in the P450-dependent metabolism of these two capsaicinoids. However, production of the dehydrogenated (M4), macrocyclic (M1), and omega-1-hydroxylated (M3) metabolites was not observed for nonivamide. These differences may be reflective of the mechanism of formation of these metabolites of capsaicin. The role of metabolism in the cytotoxicity of capsaicin and nonivamide was also assessed in cultured lung and liver cells. Lung cells were markedly more sensitive to cytotoxicity by capsaicin and nonivamide. Cytotoxicity was enhanced 5 and 40% for both compounds by 1-ABT in BEAS-2B and HepG2, respectively. These data suggested that metabolism of capsaicinoids by P450 in cells represented a detoxification mechanism (in contrast to bioactivation).     

Inhibition of cytochrome p450 enzymes by quinones and anthraquinones

In silico docking studies and quantitative structure-activity relationship analysis of a number of in-house cytochrome P450 inhibitors have revealed important structural characteristics that are required for a molecule to function as a good inhibitor of P450 enzymes 1A1, 1A2, 2B1, and/or 2A6. These insights were incorporated into the design of pharmacophores used for a 2D search of the Chinese medicine database. Emodin, a natural anthraquinone isolated from Rheum emodi and known to be metabolized by cytochrome P450 enzymes, was one of the hits and was used as the lead compound. Emodin was found to inhibit P450s 1A1, 1A2, and 2B1 with IC(50) values of 12.25, 3.73, and 14.89 μM, respectively. On the basis of the emodin molecular structure, further similarity searches of the PubChem and ZINC chemical databases were conducted resulting in the identification of 12 emodin analogues for testing against P450s 1A1-, 1A2-, 2B1-, and 2A6-dependent activities. 1-Amino-4-chloro-2-methylanthracene-9,10-dione (compound 1) showed the best inhibition potency for P450 1A1 with an IC(50) value of 0.40 μM. 1-Amino-4-chloro-2-methylanthracene-9,10-dione (compound 1) and 1-amino-4-hydroxyanthracene-9,10-dione (compound 2) both inhibited P450 1A2 with the same IC(50) value of 0.53 μM. In addition, compound 1 acted as a mechanism-based inhibitor of cytochrome P450s 1A1 and 1A2 with K(I) and K(inactivation) values of 5.38 μM and 1.57 min(-1) for P450 1A1 and 0.50 μM and 0.08 min(-1) for P450 1A2. 2,6-Di-tert-butyl-5-hydroxynaphthalene-1,4-dione (compound 8) directly inhibited P450 2B1 with good selectivity and inhibition potency (IC(50) = 5.66 μM). Docking studies using the 3D structures of the enzymes were carried out on all of the compounds. The binding modes of these compounds revealed the structural characteristics responsible for their potency and selectivity. Compound 1, which is structurally similar to compound 2 with the presence of an amino group at position 1, showed a difference in the mechanism of inhibition toward P450s 1A1 and 1A2. The mechanism-based inhibition seen for compound 1 may be attributed to the presence of the methyl group at the 2-position, in close proximity to the amino group. Compound 2, which is otherwise similar, lacks that methyl moiety and did not show mechanism-based inhibition.     

Kava does not display metabolic toxicity in a homogeneous cellular assay

To determine whether kava (Kava kava, 'Awa, Yaqona, Piper methysticum Forst.), the popular herbal product associated recently with possible human hepatotoxicity, is bioactivated by cytochrome P450 enzymes to cytotoxic metabolites, three kava lactones (methysticin, yangonin, and desmethoxyyangonin) and an ethanolic extract of dried kava root were incubated over time in culture with MCL-5 cells, a human lymphoblastoid cell line that has been stably transfected with five human P450's (CYP 1A1, 1A2, 2A6, 2E1, and 3A4) and human epoxide hydrolase. Incubations were performed concurrently with a control cell line (cH2) that is derived from the same parental line as MCL-5, but transfected with two empty vectors. The kava compounds displayed varying degrees of toxicity (IC (50) values ranged from 50 to > 100 microM) to the MCL-5 and cH2 cell lines; however, both cell lines were equally sensitive to the test compounds. These results suggest that the parent compound for each of the four test compounds was primarily responsible for the observed cell toxicity and that CYP 1A1, 1A2, 2A6, 2E1, and 3A4 or epoxide hydroxylase did not appear to be involved. Thus, in vitro kava does not appear to be activated to toxic metabolites by enzymes known to be important in metabolic toxicity.     

Xenobiotic-metabolizing enzymes involved in activation and detoxification of carcinogenic polycyclic aromatic hydrocarbons

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental carcinogens and metabolized by a variety of xenobiotic-metabolizing enzymes such as cytochrome P450 (P450 or CYP), epoxide hydrolase, glutathione transferase, UDP-glucuronosyltransferase, sulfotransferase, NAD(P)H quinone oxidoreductase 1, and aldo-keto reductase. These enzymes mainly participate in the conversion of PAHs to more polar and water-soluble metabolites, and the resultant metabolites are readily excreted from the body. However, during the course of metabolism, a variety of unstable and reactive intermediates of PAHs are formed, and these metabolites attack DNA, causing cell toxicity and transformation. P450s and epoxide hydrolase convert PAHs to proximate carcinogenic metabolites, PAH-diols, and these products are further metabolized by P450s to ultimate carcinogenic metabolites, PAH diol-epoxides, or by aldo-keto reductase to reactive PAH o-quinones. PAHs are also activated by P450 and peroxidases to reactive radical cations that bind covalently to DNA. The oxygenated and reactive metabolites of PAHs are usually converted to more polar and detoxified products by phase II enzymes. Inter-individual differences exist in levels of expression and catalytic activities of a variety of enzymes that activate and/or detoxify PAHs in various organs of humans and these phenomena are thought to be critical in understanding the basis of individual differences in response to PAHs. Factors affecting such variations include induction and inhibition of enzymes by diverse chemicals and, more importantly, genetic polymorphisms of enzymes in humans.     

Evaluation of p450 inhibition and induction by artemisinin antimalarials in human liver microsomes and primary human hepatocytes

Artemisinin drugs have become the first-line antimalarials in areas of multidrug resistance. However, monotherapy with artemisinin drugs results in comparatively high recrudescence rates. Autoinduction of cytochrome P450 (P450)-mediated metabolism, resulting in reduced exposure, has been supposed to be the underlying mechanism. To better understand the autoinduction and metabolic drug-drug interactions (DDIs), we evaluated the P450s (particularly CYP2B6 and CYP3A4) inhibited or induced by two artemisinin drugs, Qing-hao-su (QHS) and dihydroartemisinin (DHA) using human liver microsome, recombinant P450 enzymes, and primary human hepatocytes. The results suggested that QHS was a weak reversible inhibitor of CYP2B6 (K(i) 4.6 μM), but not CYP3A4 (IC(50) ～ 50 μM) and did not show measurable time-dependent inhibition of either CYP2B6 or CYP3A4. DHA inhibited neither CYP2B6 nor CYP3A4 (IC(50) > 125 μM). In addition, it was found that QHS induced the activity of CYP3A4 (E(max) 3.5-fold and EC(50) 5.9 μM) and CYP2B6 (E(max) 1.9-fold and EC(50) 0.6 μM). Of the other P450s, UDP glucuronosyltransferases, and transporters studied, QHS and DHA had no significant effect except for minor induction of mRNA expression of CYP1A2 (E(max) 7.9-fold and EC(50) 5.2 μM) and CYP2A6 (E(max) 11.7-fold and EC(50) 4.0 μM) by QHS. Quantitative prediction of P450-mediated DDIs indicate autoinduction of QHS clearance with the AUC(i)/AUC ratio decreasing to 59%, as a result of a 1.9-fold increase in CYP3A4 and a 1.6-fold increase in CYP2B6 activity. These data suggest that QHS drugs are potential inducers of P450 enzymes, and the possible drug interactions (or lack thereof) with artemisinin drugs may be clinically relevant.     

Characterization of novel glutathione adducts of a non-nucleoside reverse transcriptase inhibitor, (S)-6-chloro-4-(cyclopropylethynyl)-4-(trifluoromethyl)-3, 4-dihydro-2(1H)-quinazolinone (DPC 961), in rats. Possible formation of an oxirene metabolic intermediate from a disubstituted alkyne

The postulated formation of oxirene-derived metabolites from rats treated with a disubstituted alkyne, (S)-6-chloro-4-(cyclopropylethynyl)-4-(trifluoromethyl)-3, 4-dihydro-2(1H)-quinazolinone (DPC 961), is described. The reactivity of this postulated oxirene intermediate led to the formation of novel glutathione adducts whose structures were confirmed by LC/MS and by two-dimensional NMR experiments. These metabolites were either excreted in rat bile or degraded to mercapturic acid conjugates and eliminated in urine. To demonstrate the oxidation of the triple bond, an analogue of DPC 961 was synthesized, whereby the two carbons of the alkyne moiety were replaced with (13)C stable isotope labels. Rats were orally administered [(13)C]DPC 961 and glutathione adducts isolated from bile. The presence of an oxygen atom on one of the (13)C labels of the alkyne was demonstrated unequivocally by NMR experiments. Administration of (14)C-labeled DPC 961 showed that biliary elimination was the major route of excretion with the 8-OH glucuronide conjugate (M1) accounting for greater than 90% of the eliminated radioactivity. On the basis of radiochemical profiling, the glutathione-derived metabolites were minor in comparison to the glucuronide conjugate. Studies with cDNA-expressed rat enzymes, polyclonal antibodies, and chemical inhibitors pointed to the involvement of P450 3A1 and P450 1A2 in the formation of the postulated oxirene intermediate. The proposed mechanism shown in Scheme 1 begins with P450-catalyzed formation of an oxirene, rearrangement to a reactive cyclobutenyl ketone, and a 1,4-Michael addition with endogenous glutathione to produce two isomeric adducts, GS-1 and GS-2. The glutathione adducts were subsequently catabolized via the mercapturic acid pathway to cysteinylglycine, cysteine, and N-acetylcysteine adducts. The transient existence of the alpha,beta-unsaturated cyclobutenyl ketone was demonstrated by incubating the glutathione adduct in the presence of N-acetylcysteine and monitoring the formation of N-acetylcysteine adducts by LC/MS. Epimerization of GS-1 to GS-2 was also observed when N-acetylcysteine was omitted from the incubation.     

Metabolism of 3-methylindole by porcine liver microsomes: responsible cytochrome P450 enzymes.

The role of different cytochrome P450 enzymes on the metabolism of 3-methylindole (3MI) was investigated using selective chemical inhibitors. Eight chemical inhibitors of P450 enzymes were screened for their inhibitory specificity towards 3MI metabolism in porcine microsomes: alpha-naphthoflavone (CYP1A1/2), 8-methoxypsoralen (CYP2A6), menthofuran (CYP2A6), diethyldithiocarbamate (CYP2A6), 4-methylpyrazole (CYP2E1), sulphaphenazole (CYP2C9), quinidine (CYP2D6), and troleandomycin (CYP3A4). The production of 3MI metabolites was only affected by the presence of inhibitors of CYP2A6 and CYP2E1 in the microsomal incubations. In a second experiment, a set of porcine microsomes (n = 30) was analyzed for CYP2A6 content by protein immunoblot analysis and for their coumarin 7-hydroxylation activity (CYP2A6 activity). Both CYP2A6 content and enzymatic activity were found to be highly and negatively correlated with 3MI fat content. The results of the present study indicate that the CYP2A6 porcine ortholog plays an important role in the metabolism of 3MI and that measurement of CYP2A6 levels and/or activity could be a useful marker for 3MI-induced boar taint.     

Structure-function relationships of inhibition of human cytochromes P450 1A1, 1A2, 1B1, 2C9, and 3A4 by 33 flavonoid derivatives

Structure-function relationships for the inhibition of human cytochrome P450s (P450s) 1A1, 1A2, 1B1, 2C9, and 3A4 by 33 flavonoid derivatives were studied. Thirty-two of the 33 flavonoids tested produced reverse type I binding spectra with P450 1B1, and the potencies of binding were correlated with the abilities to inhibit 7-ethoxyresorufin O-deethylation activity. The presence of a hydroxyl group in flavones, for example, 3-, 5-, and 7-monohydroxy- and 5,7-dihydroxyflavone, decreased the 50% inhibition concentration (IC50) of P450 1B1 from 0.6 μM to 0.09, 0.21, 0.25, and 0.27 μM, respectively, and 3,5,7-trihydroxyflavone (galangin) was the most potent, with an IC50 of 0.003 μM. The introduction of a 4'-methoxy- or 3',4'-dimethoxy group into 5,7-dihydroxyflavone yielded other active inhibitors of P450 1B1 with IC50 values of 0.014 and 0.019 μM, respectively. The above hydroxyl and/or methoxy groups in flavone molecules also increased the inhibition activity with P450 1A1 but not always toward P450 1A2, where 3-, 5-, or 7-hydroxyflavone and 4'-methoxy-5,7-dihydroxyflavone were less inhibitory than flavone itself. P450 2C9 was more inhibited by 7-hydroxy-, 5,7-dihydroxy-, and 3,5,7-trihydroxyflavones than by flavone but was weakly inhibited by 3- and 5-hydroxyflavone. Flavone and several other flavonoids produced type I binding spectra with P450 3A4, but such binding was not always related to the inhibitiory activities toward P450 3A4. These results indicate that there are different mechanisms of inhibition for P450s 1A1, 1A2, 1B1, 2C9, and 3A4 by various flavonoid derivatives and that the number and position of hydroxyl and/or methoxy groups highly influence the inhibitory actions of flavonoids toward these enzymes. Molecular docking studies suggest that there are different mechanisms involved in the interaction of various flavonoids with the active site of P450s, thus causing differences in inhibition of these P450 catalytic activities by flavonoids.