Human Hepatic Cytochrome P450 Research Articles

Thalidomide Increases Human Hepatic Cytochrome P450 3A Enzymes by Direct Activation of the Pregnane X Receptor

Heterotropic cooperativity of human cytochrome P450 (P450) 3A4/3A5 by the teratogen thalidomide was recently demonstrated by H. Yamazaki et al. ( ( 2013 ) Chem. Res. Toxicol. 26 , 486 - 489 ) using the model substrate midazolam in various in vitro and in vivo models. Chimeric mice with humanized liver also displayed enhanced midazolam clearance upon pretreatment with orally administered thalidomide, presumably because of human P450 3A induction. In the current study, we further investigated the regulation of human hepatic drug metabolizing enzymes. Thalidomide enhanced levels of P450 3A4 and 2B6 mRNA, protein expression, and/or oxidation activity in human hepatocytes, indirectly suggesting the activation of upstream transcription factors involved in detoxication, e.g., the nuclear receptors pregnane X receptor (PXR) and constitutive androstane receptor (CAR). A key event after ligand binding is an alteration of nuclear receptor conformation and recruitment of coregulator proteins that alter chromatin accessibility of target genes. To investigate direct engagement and functional alteration of PXR and CAR by thalidomide, we utilized a peptide microarray with 154 coregulator-derived nuclear receptor-interaction motifs and coregulator and nuclear receptor boxes, which serves as a sensor for nuclear receptor conformation and activity status as a function of ligand. Thalidomide and its human proximate metabolite 5-hydroxythalidomide displayed significant modulation of coregulator interaction with PXR and CAR ligand-binding domains, similar to established agonists for these receptors. These results collectively suggest that thalidomide acts as a ligand for PXR and CAR and causes enzyme induction leading to increased P450 enzyme activity. The possibilities of drug interactions during thalidomide therapy in humans require further evaluation.

Effects of arecoline on hepatic cytochrome P450 activity and oxidative stress.

Betel-quid use is associated with the risk of liver cirrhosis and hepatocellular carcinoma. The aim of the present work was to evaluate the impact of arecoline on human hepatic cytochrome P450 (CYP) enzymes in vitro and rat hepatic CYP enzymes, as well as the hepatic oxidative stress and liver injury of rats in vivo. The in vitro results indicated that arecoline hydrobromide (AH) has no significant effect on the activities of CYP2B, 2C9, 3A4, 1A2, 2E1 and 2D6 in human liver microsome (HLM). However, oral administration of AH at 4 and 20 mg/kg/d for seven consecutive days significantly increased the activities of rat hepatic CYP2B, 2E1, 2D, 3A, 2C and 1A2. In addition, AH at 100 mg/kg/d significantly increased the levels of ALT, AST and MDA, decreased the levels of SOD, CAT, GSH-Px and GSH, in rat liver. The in vivo induction of AH on rat hepatic CYP isoforms suggested that the high risk of metabolic interaction should be existed when the substrate drugs of the six kinds of CYP isoforms was administered in betel-quid use human. Furthermore, the in vivo results also suggested that AH-induced hepatoxicity should be associated with the induction of AH on rat hepatic CYP2E1 and 2B.

Direct Transcriptional Regulation of Human Hepatic Cytochrome P450 3A4 (CYP3A4) by Peroxisome Proliferator–Activated Receptor Alpha (PPARα)

The nuclear receptor peroxisome proliferator-activated receptor (PPAR)α is known primarily as a regulator of fatty acid metabolism, energy balance, and inflammation, but evidence suggests a wider role in regulating the biotransformation of drugs and other lipophilic chemicals. We investigated whether PPARα directly regulates the transcription of cytochrome P450 3A4, the major human drug-metabolizing enzyme. Using chromatin immunoprecipitation in human primary hepatocytes as well as electrophoretic mobility shift and luciferase reporter-gene assays, we identified three functional PPARα-binding regions (PBR-I, -II, and -III) within ∼12 kb of the CYP3A4 upstream sequence. Furthermore, a humanized CYP3A4/3A7 mouse model showed in vivo induction of CYP3A4 mRNA and protein by [4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio]acetic acid (WY14,643) in liver but not in intestine, whereas hepatic occupancy of PBRs by PPARα was ligand independent. Using lentiviral gene knock-down and treatment with WY14,643 in primary human hepatocytes, PPARα was further shown to affect the expression of a distinct set of CYPs, including 1A1, 1A2, 2B6, 2C8, 3A4, and 7A1, but not 2C9, 2C19, 2D6, or 2E1. Interestingly, the common phospholipid 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine (16:0/18:1-PC), previously proposed to reflect nutritional status and shown to be a specific endogenous ligand of PPARα, induced CYP3A4 (up to 4-fold) and other biotransformation genes in hepatocytes with similar selectivity and potency as WY14,643. These data establish PPARα as a direct transcriptional regulator of hepatic CYP3A4. This finding warrants investigation of both known and newly developed PPARα-targeted drugs for their drug-drug interaction potential. Furthermore, our data suggest that nutritional status can influence drug biotransformation capacity via endogenous phospholipid signaling.

Identification of human cytochrome P450 enzymes involved in the metabolism of a novel к-opioid receptor agonist, nalfurafine hydrochloride

Nalfurafine hydrochloride (TRK-820) exhibits strong к-opioid agonistic activity and is a new antipruritic agent for uremic pruritus. This study was performed to identify the human hepatic cytochrome P450 isoforms involved in the metabolic conversion of nalfurafine to the decyclopropylmethylated form, de-CPM, using human liver microsomes and E. coli membrane fractions expressing human P450 isoforms. Samples were analysed by liquid chromatography with a radioactivity detector and liquid chromatography-tandem mass spectrometry. The metabolism of nalfurafine by human liver microsomes exhibited a biphasic kinetic profile. Experiments examining the metabolism by E. coli membrane fractions expressing human P450 isoforms indicated that CYP1A1, 2C8, 2C19 and 3A4 had the ability to produce de-CPM. In experiments with human liver microsomes that examined the inhibition of nalfurafine metabolism by anti-human P450 antibodies, anti-CYP3A4 antibody predominantly, and anti-CYP2C8 and 2C19 antibodies moderately, inhibited de-CPM formation. From these results, CYP3A4 appeared to be the major isoform involved in the metabolic decyclopropylmethylation of nalfurafine, while CYP2C8 and 2C19 most likely play a minor role in the formation of de-CPM.

The inhibition of major human hepatic cytochrome P450 enzymes by 18 pesticides: Comparison of the N-in-one and single substrate approaches

In the present study on human hepatic microsomes, the N-in-one assay with ten probe substrates for nine cytochrome-P450 enzymes (CYPs) was compared with the single substrate assays to investigate pesticides–CYP interactions. CYP inhibition was measured by liquid chromatography–tandem mass spectrometry (LC/MS–MS). As illustrated by the initial screening at 100μM concentration of 18 pesticides, CYPs are more sensitive to organophosphates (OPs) than to other pesticide groups. Chlorpyrifos and fenitrothion were most effective in inhibiting CYP1A1/2, and CYP2B6. Profenofos was also inhibitory towards multiple CYPs. Pyrethroids, e.g. deltamethrin, fenvalerate and lambda-cyhalothrin, potently inhibited CYP2D6. CYP3A4 activity was moderately inhibited by fenvalerate and potently by alpha-cypermethrin. The correlations between IC50 values obtained from the N-in-one and single substrate approaches were highly significant for CYP2Cs (r2=0.94), CYP3A4, omeprazole-sulfoxidation, (r2=0.89), followed by CYP1A2 and CYP2B6 (r2=0.82), and CYP2D6 (r2=0.80). In contrast no correlation was observed with CYP2E1 and CYP3A4 (midazolam-1′-hydroxylation). The N-in-one screening assay seems useful and reliable for most CYP activities when a comprehensive and quick evaluation of potential interactions with CYPs is needed. However, at the present moment, it does not enable discrimination on the basis of mechanism of inhibition. A strict comparison between single and N-in-one assays is a prerequisite for more extensive routine use.

Antifungal activities of novel non-azole molecules against S. cerevisiae and C. albicans

Because of the increasing number of immunocompromised patients and due to problems with antifungal treatment, especially with the most widely used antifungals, azoles, there is an urgent need for new, potent and safe antifungals with fewer cytochrome P450 (CYP)-mediated interactions with other drugs. In the present study, 54 novel non-azole molecules were selected with the help of molecular modelling and virtual molecule database screening to identify new fungistatic or fungicidic compounds with functional groups that would produce reactive intermediates killing the yeast cells. Database screening and selection of tested compounds were based on the construction of two pharmacophores and docking hits to the active site of the CYP51 homology model. Inhibition potency of the compounds was tested against Saccharomyces cerevisiae and/or Candida albicans. Two new structured compounds, 2-({4-[(2-cyanoethyl)(methyl) amino]benzylidene} amino)-5-(3,4-dimethoxyphenyl)-4-methylthiophene-3-carbonitrile and 2-[([1,1′-biphenyl]-4-ylmethylene)amino]-5-(3,4-dimethoxyphenyl)-4-methylthiophene-3-carbonitrile were discovered to have promising antifungal properties based on bioassays. Inhibition screen of human hepatic CYP enzymes revealed that these two compounds did not inhibit potently five human recombinant CYP enzymes. The results of this study indicate that the functional groups of the two compounds may produce reactive intermediates when located at the active site of CYP51.

Heterologous Expression of Human Cytochromes P450 2D6 and CYP3A4 in Escherichia coli and Their Functional Characterization

This study aimed to express two major drug-metabolizing human hepatic cytochromes P450 (CYPs), CYP2D6 and CYP3A4, together with NADPH-cytochrome P450 oxidoreductase (OxR) in Escherichia coli and to evaluate their catalytic activities. Full length cDNA clones of both isoforms in which the N-terminus was modified to incorporate bovine CYP17α sequence were inserted into a pCWori(+) vector. The modified CYP cDNAs were subsequently expressed individually, each together with OxR by means of separate, compatible plasmids with different antibiotic selection markers. The expressed proteins were evaluated by immunoblotting and reduced CO difference spectral scanning. Enzyme activities were examined using high performance liquid chromatography (HPLC) assays with probe substrates dextromethorphan and testosterone for CYP2D6 and CYP3A4, respectively. Results from immunoblotting demonstrated the presence of both CYP proteins in bacterial membranes and reduced CO difference spectra of the cell preparations exhibited the characteristic absorbance peak at 450nm. Co-expressed OxR also demonstrated an activity level comparable to literature values. Kinetic parameters, K(m) and V(max) values determined from the HPLC assays also agreed well with literature values. As a conclusion, the procedures described in this study provide a relatively convenient and reliable means of producing catalytically active CYP isoforms suitable for drug metabolism and interaction studies.

Characterization of inhibitory effects of perfluorooctane sulfonate on human hepatic cytochrome P450 isoenzymes: Focusing on CYP2A6

Perfluorooctane sulfonate (PFOS) is a chemically stable compound extensively used as oil and water repellent, surface active agents in our daily life. Accumulative research evidence gradually appears the toxicity of PFOS against mammals, but the whole figure remains to be elucidated. The present study was conducted to know the effects of PFOS on human hepatic drug metabolizing-type cytochrome P450 (CYP) isoenzymes such as CYP1A2 (7-ethoxyresorufin as a substrate), CYP2A6 (coumarin), CYP2B6 (7-ethoxy-4-trifluoromethylcoumarin), CYP2C8 (paclitaxel), CYP2C9 (diclofenac), CYP2C19 ( S-mephenytoin), CYP2D6 (bufuralol), CYP2E1 (chlorzoxazone) and CYP3A4 (testosterone) in human livers employing their typical substrates. Although all of the oxidation reactions tested were more or less inhibited by PFOS, diclofenac 4′-hydroxylation mediated mainly by CYP2C9 was most strongly inhibited ( K i value of 40 nM), followed by paclitaxel 6α-hydroxylation mediated mainly by CYP2C8 ( K i value of 4 μM). The substrate oxidation reactions catalyzed by CYP2A6, CYP2B6, CYP2C19 and CYP3A4 were moderately ( K i values of 35 to 45 μM), and those by CYP1A2, CYP2D6 and CYP2E1 were weakly inhibited by PFOS ( K i values of 190–300 μM). The inhibition by PFOS for coumarin 7-hydroxylation mainly catalyzed by human liver microsomal CYP2A6 as well as by the recombinant enzyme was found to be enhanced by the preincubation of PFOS with human liver microsomes and NADPH as compared to the case without preincubation. The inhibition of the human liver microsomal cumarin 7-hydroxylation was PFOS concentration-dependent, and exhibited pseudo-first-order kinetics with respect to preincubation time, yielding K inact and K I values of 0.06 min −1 and 23 μM, respectively. These results suggest that the metabolism of medicines which are substrates for CYP2C9 may be altered by PFOS in human bodies, and that PFOS is a mechanism-based inhibitor of CYP2A6.

In vitro determination of the effect of Andrographis paniculata extracts and andrographolide on human hepatic cytochrome P450 activities

We investigated the effects of Andrographis paniculata (AP) extracts and andrographolide on the catalytic activity of three human cDNA-expressed cytochrome P450 enzymes: CYP2C9, CYP2D6 and CYP3A4. In vitro probe-based high performance liquid chromatography assays were developed to determine CYP2C9-dependent tolbutamide methylhydroxylation, CYP2D6-dependent dextromethorphan O-demethylation and CYP3A4-dependent testosterone 6β-hydroxylation activities in the presence and absence of AP extracts and andrographolide. Our results indicate that AP ethanol and methanol extracts inhibited CYP activities more potently than aqueous and hexane extracts across the three isoforms. Potent inhibitory effects were observed on CYP3A4 and CYP2C9 activities (K (i) values below 20μg/ml). Andrographolide was found to exclusively but weakly inhibit CYP3A4 activity. In conclusion, data presented in this study suggest that AP extracts have the potential to inhibit CYP isoforms in vitro. There was, however, variation in the potency of inhibition depending on the extracts and the isoforms investigated.

Chemical inhibitors of cytochrome P450 isoforms in human liver microsomes: a re-evaluation of P450 isoform selectivity

The majority of marketed small-molecule drugs undergo metabolism by hepatic Cytochrome P450 (CYP) enzymes (Rendic 2002). Since these enzymes metabolize a structurally diverse number of drugs, metabolism-based drug-drug interactions (DDIs) can potentially occur when multiple drugs are coadministered to patients. Thus, a careful in vitro assessment of the contribution of various CYP isoforms to the total metabolism is important for predicting whether such DDIs might take place. One method of CYP phenotyping involves the use of potent and selective chemical inhibitors in human liver microsomal incubations in the presence of a test compound. The selectivity of such inhibitors plays a critical role in deciphering the involvement of specific CYP isoforms. Here, we review published data on the potency and selectivity of chemical inhibitors of the major human hepatic CYP isoforms. The most selective inhibitors available are furafylline (in co-incubation and pre-incubation conditions) for CYP1A2, 2-phenyl-2-(1-piperidinyl)propane (PPP) for CYP2B6, montelukast for CYP2C8, sulfaphenazole for CYP2C9, (-)-N-3-benzyl-phenobarbital for CYP2C19 and quinidine for CYP2D6. As for CYP2A6, tranylcypromine is the most widely used inhibitor, but on the basis of initial studies, either 3-(pyridin-3-yl)-1H-pyrazol-5-yl)methanamine (PPM) and 3-(2-methyl-1H-imidazol-1-yl)pyridine (MIP) can replace tranylcypromine as the most selective CYP2A6 inhibitor. For CYP3A4, ketoconazole is widely used in phenotyping studies, although azamulin is a far more selective CYP3A inhibitor. Most of the phenotyping studies do not include CYP2E1, mostly because of the limited number of new drug candidates that are metabolized by this enzyme. Among the inhibitors for this enzyme, 4-methylpyrazole appears to be selective.

Functional reconstitution of monomeric CYP3A4 with multiple cytochrome P450 reductase molecules in Nanodiscs

Traditional reconstitution of membrane cytochromes P450 monooxygenase system requires efficient solubilization of both P450 heme enzymes and redox partner NADPH dependent reductase, CPR, either in mixed micellar solution or by incorporation in liposomes. Here we describe a simple alternative approach to assembly of soluble complexes of monomeric human hepatic cytochrome P450 CYP3A4 with CPR by co-incorporation into nanoscale POPC bilayer Nanodiscs. Stable and fully functional complexes with different CPR:CYP3A4 stoichiometric ratios are formed within several minutes after addition of the full-length CPR to the solution of CYP3A4 preassembled into POPC Nanodiscs at 37°C. We find that the steady state rates of NADPH oxidation and testosterone hydroxylation strongly depend on CPR:CYP3A4 ratio and reach maximum at tenfold molar access of CPR. The binding of CPR to CYP3A4 in Nanodiscs is tight, such that complexes with different stoichiometry can be separated by size-exclusion chromatography. Reconstitution systems based on the co-incorporation of CPR into preformed Nanodiscs with different human cytochromes P450 are suitable for high-throughput screening of substrates and inhibitors and for drug–drug interaction studies.

Rapid screening of commercially available herbal products for the inhibition of major human hepatic cytochrome P450 enzymes using the N-in-one cocktail

Self-administration of complementary products concurrently with conventional medication is increasingly common. The potential for cytochrome P450 (CYP) inhibition requires investigation. The N-in-one assay with ten probe substrates for nine CYPs was used with human liver microsomes to investigate ten products. CYP inhibition was measured in a single liquid chromatography-tandem mass spectrometry (LC/MS-MS) analysis. Estimated IC50-values were determined for the extracts that produced significant inhibition (less than 100 μg ml−1).Inhibition of CYP2C19 by dong quai (IC50 = 13.7–14.3 μg ml−1 for the methanolic extract) and CYP2D6 by goldenseal (IC50 = 6.7 and 6.3 μg ml−1 for the aqueous and methanolic extracts, respectively), are of particular concern as the potential for adverse interactions is high. The inhibition of CYP2C8 by horsetail (IC50 = 93 μg ml−1 for the aqueous extract) requires further investigation, as the potential for concurrent use with products that require CYP2C8 for metabolism is significant. CYP3A4 inhibition varied depending on the probe reaction being monitored.The earlier reported findings of inhibition by black cohosh, goldenseal and gotu kola were confirmed. The present work has shown that the N-in-one cocktail is a rapid and reliable method that can be used as an initial screen to help prioritize products that require more detailed investigations and it can also be applied to monitor product variability.

Food-drug interactions: elucidating complex mixtures and individual constituents

Event Abstract Back to Event Food-drug interactions: elucidating complex mixtures and individual constituents Olavi Pelkonen1*, Pia Vuorela2, Moshe Finel3 and Jorma T. Ahokas4 1 University of Oulu, Department of Pharmacology and Toxicology, Finland 2 Åbo Akademi University, Finland 3 University of Helsinki, Molecular and biochemical pharmacology, Finland 4 RMIT-University, Australia Certain components in our food may affect the way patients respond to prescribed drugs, leading to either inefficacy of the treatment, or stimulating adverse drug effects. These two apparently opposite outcomes could both be due to interactions of certain food components with one or more of the enzymes that our body has for protection against chemicals from the environment (xenobiotics). These enzymes play important roles in regulating the concentration of most drugs and, therefore, their inhibition by food components, or up-regulating their expression level, could lead to unwanted and harmful consequences. Yet, it is currently very difficult to predict and thereby avoid such food-drug interactions due to a lack of sufficient and specific knowledge. The major goal of our ongoing project is to characterize the risks and reduce future cases of harmful food-drug interactions, particularly among the more sensitive sections of the population like the elderly and patients undergoing extensive drug treatments due to severe diseases. In order to reach this goal it is essential to develop tools and approaches to gain deeper knowledge into this topic. In line with this, we are working in four different but interacting topics: 1. We have studied the in vitro interactions of 10 most popular (in Australia) herbal preparations with human hepatic cytochrome P450 enzymes by employing an LC-MS method (Sevior et al, Xenobiotica 2010; 40(4): 245–254). A number of in vitro interactions were observed, with obvious implications for further more refined studies. 2. We have investigated the metabolism and potential interactions of thujone, a major neurotoxic alkaloid in Artemisia absinthum (absinthe drink), in a number of human liver preparations and identified the participating P450 enzymes. 3. We have studied metabolism and interactions of Angelica archangelica extracts as well as constituent furocoumarins, in order to elucidate possibilities to extrapolate from individual components to a complex mixture containing different amounts of those components. Angelica extract and many furocoumarins seem to be potent inhibitors of at least CYP1A2 and to a lesser extent, many other CYPs. After the above mentioned studies aimed at testing necessary tools and approaches and demonstrating the feasibility of such studies, we plan to proceed to targeted in vivo studies in human volunteers, to elucidate the possibilities to perform in vitro – in vivo extrapolations and to improve risk assessment. Support: the above mentioned studies are being supported by the Academy of Finland (Council for Health Sciences) and the Finnish Agency for Research and Innovation (TEKES). Keywords: Food-Drug Interactions, herbal medicines Conference: 8th Southeast European Congress on Xenobiotic Metabolism and Toxicity - XEMET 2010, Thessaloniki, Greece, 1 Oct - 5 Oct, 2010. Presentation Type: Invited speaker Topic: Food, drugs and environmental xenobiotics Citation: Pelkonen O, Vuorela P, Finel M and Ahokas JT (2010). Food-drug interactions: elucidating complex mixtures and individual constituents. Front. Pharmacol. Conference Abstract: 8th Southeast European Congress on Xenobiotic Metabolism and Toxicity - XEMET 2010. doi: 10.3389/conf.fphar.2010.60.00184 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 05 Mar 2011; Published Online: 04 Nov 2010. * Correspondence: Dr. Olavi Pelkonen, University of Oulu, Department of Pharmacology and Toxicology, Oulu, Finland, olavi.pelkonen@oulu.fi Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Olavi Pelkonen Pia Vuorela Moshe Finel Jorma T Ahokas Google Olavi Pelkonen Pia Vuorela Moshe Finel Jorma T Ahokas Google Scholar Olavi Pelkonen Pia Vuorela Moshe Finel Jorma T Ahokas PubMed Olavi Pelkonen Pia Vuorela Moshe Finel Jorma T Ahokas Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

In Vitro Study of the Inhibition and Induction of Human Cytochromes P450 by Crystalline Glucosamine Sulfate

The induction and inhibition of human hepatic cytochrome P450 (CYP) isoforms by crystalline glucosamine sulfate (CGS) was investigated in vitro. Inhibition of CYP1A2, CYP2E1, CYP2C19, CYP2C9, CYP2D6, and CYP3A4 by CGS was assessed using recombinant human enzymes incubated with CGS (up to 3 mM expressed as free base). Induction of CYP1A2, CYP2B6, CYP2C9, CYP2C19 and CYP3A4 by CGS (0.01, 0.3 and 3 mM) was evaluated in cryopreserved human hepatocytes, by determining CYP mRNA expression using quantitative RT-PCR. CGS produced no inhibition or induction of any the CYP enzymes tested at concentrations hundred folds higher than the steady state peak plasma concentrations (approximately 10 microM) observed in man after therapeutic doses of CGS of 1500 mg once a day. Therefore, no clinically relevant metabolic interactions are expected between CGS and co-administered drugs that are substrates of the CYP enzymes investigated.

CYP21-catalyzed production of the long-term urinary metandienone metabolite 17β-hydroxymethyl-17α-methyl-18-norandrosta-1,4,13-trien-3-one: a contribution to the fight against doping

Anabolic-androgenic steroids are some of the most frequently misused drugs in human sports. Recently, a previously unknown urinary metabolite of metandienone, 17beta-hydroxymethyl-17 alpha-methyl-18-norandrosta-1,4,13-trien-3-one (20OH-NorMD), was discovered via LC-MS/MS and GC-MS. This metabolite was reported to be detected in urine samples up to 19 days after administration of metandienone. However, so far it was not possible to obtain purified reference material of this metabolite and to confirm its structure via NMR. Eleven recombinant strains of the fission yeast Schizosaccharomyces pombe that express different human hepatic or steroidogenic cytochrome P450 enzymes were screened for production of this metabolite in a whole-cell biotransformation reaction. 17,17-Dimethyl-18-norandrosta-1,4,13-trien-3-one, chemically derived from metandienone, was used as substrate for the bioconversion, because it could be converted to the final product in a single hydroxylation step. The obtained results demonstrate that CYP21 and to a lesser extent also CYP3A4 expressing strains can catalyze this steroid hydroxylation. Subsequent 5 l-scale fermentation resulted in the production and purification of 10 mg of metabolite and its unequivocal structure determination via NMR. The synthesis of this urinary metandienone metabolite via S. pombe-based whole-cell biotransformation now allows its use as a reference substance in doping control assays.

Investigations on the human hepatic cytochrome P450 isozymes involved in the metabolism of 3,4-methylenedioxy-amphetamine (MDA) and benzodioxolyl-butanamine (BDB) enantiomers

3,4-Methylenedioxy-amphetamine (MDA) and benzodioxolyl-butanamine (BDB) are chiral designer drugs distributed on the illicit drug market and they are also N-dealkyl metabolites of 3,4-methylenedioxymethamphetamine (MDMA, Ecstasy, Adam), 3,4-methylenedioxyethylamphetamine (MDEA, Eve), and N-methyl-benzodioxolyl-butanamine (MBDB, Eden), respectively. MDA and BDB are mainly metabolized via demethylenation to the corresponding catecholamines. The aim of the present work was to elucidate the contribution of the relevant human P450s in the demethylenation of the MDA and BDB enantiomers. They were incubated using heterologously expressed human P450s and the corresponding metabolites dihydroxyamphetamine and 1,2-dihydroxy-4-[2-amino-butyl]benzene were determined. Highest contributions to the demethylenation as calculated from the enzyme kinetic data were obtained for CYP2D6 (MDA and BDB) and additionally CYP3A4 in the case of BDB at substrate concentrations corresponding to plasma concentrations of recreational users. A preferred transformation of the S-enantiomer could be observed for the CYP2D6- and CYP3A4-catalyzed reactions.

Interaction of antitumor platinum complexes with human liver microsomal cytochromes P450

Interaction of nine human hepatic cytochromes P450 (CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4) with six platinum complexes was studied using pooled human microsomes. The compounds used were cisplatin, oxaliplatin, carboplatin, transplatin, and trans-[PtCl2(NH3) (Am)], where Am=2-methylbutylamine or sec-butylamine. No significant inhibition of all CYP activities by carboplatin was observed. With cisplatin and oxaliplatin, a minor inhibition of CYP2C9 enzyme (75% of control at 400 miromol/l of these complexes) was seen; cisplatin also inhibited slightly the CYP2B6 activity (85% of control). With respect to plasma levels of cisplatin obtained in clinical applications, these effects are probably not important. In contrast, clinically ineffective transplatin, inhibited the CYP2B6 as well as CYP2C9 activities significantly (to 50-35% of control at 100 micromol/l); also, an inhibition of CYP2E1 activity was found here (to 70% at 100 micromol/l). Two other derivatives of transplatin (new antitumor agents with trans geometry), inhibited CYP activities more strongly reaching nearly a complete inhibition of the respective CYP activities at concentration of 200 micromol/l. Half maximal inhibitory concentration values were found in the range of tens of micromol/l indicating that there is a possibility of potential interactions of these compounds with drugs metabolized by CYP3A4, CYP2E1, CYP2D6, CYP2C19, CYP2B6, CYP2A6, and CYP1A2. Interestingly, clinically non-significant inhibition was found with the CYP2C9 and CYP2C8 indicating low probability of interactions with, for example, warfarin. The results document that the new antitumor agents based on the transplatin should be more thoroughly tested for interactions with liver microsomal drug-metabolizing cytochromes P450.

Intramolecular hydrogen bonding as a determinant of the inhibitory potency of N-unsubstituted imidazole derivatives towards mammalian hemoproteins

Enzymes involved in the mammalian microsomal metabolism of drugs are, in numerous cases, inhibited by compounds bearing an imidazolyl scaffold. However, the inhibition potency is highly dependent upon the accessibility of the imidazolyl nitrogen lone pair. In order to highlight some structural parameters of inhibitors that control this phenomenon, a series of compounds containing a nitrogen unsubstituted imidazolyl moiety with varying degrees of nitrogen lone pair accessibility was tested on human and rat hepatic cytochromes P450 and microperoxidase 8, an enzymatically active peptide derived from cytochrome c. In each case, we have shown that the accessibility of the imidazole lone pair determined the extent of inhibition. Nitrogen accessibility was tuned not only by varying the steric hindrance in the vicinity of the imidazolyl ring but also by modifying its surrounding hydrogen bonding network. Compounds in which there exists intramolecular hydrogen bonding between the imidazole moiety and an H-bond acceptor, such as an appropriately positioned amide carbonyl group, demonstrated enhanced inhibitory effects. Conversely, imidazole moieties which are in proximity to H-bond donors, such as an amide NH group, displayed reduced potency. This trend was observed in cyclo-peptide derivatives in which the intramolecular H-bond network was adjusted through the modification of the stereochemistry of a dehydrohistidine residue. It was observed that (Z)-isomers weakly bind heme, whereas (E)-isomers demonstrated higher degrees of metal binding. Therefore, enzymatic inhibition of heme-containing proteins by compounds bearing a dehydrohistidine motif seems to be closely related to its stereochemistry and hydrogen binding propensity. At neutral pH, these differences in binding affinities can be confidently attributed to the ambident H-bond properties of imidazole nitrogen atoms. This structure-activity relationship may be of use for the design of novel imidazolyl compounds as new P450 inhibitors or drug candidates.

In Vitro Metabolism of Pyrethroid Pesticides by Rat and Human Hepatic Microsomes and Cytochrome P450 Isoforms

Species differences in the intrinsic clearance (CL(int)) and the enzymes involved in the metabolism of pyrethroid pesticides were examined in rat and human hepatic microsomes. The pyrethroids bifenthrin, S-bioallethrin, bioresmethrin, beta-cyfluthrin, cypermethrin, cis-permethrin, and trans-permethrin were incubated in rat and human hepatic microsomes in the presence or absence of NADPH. Metabolism was measured using a parent depletion approach. The CL(int) of the pyrethroids was 5- to 15-fold greater in rat relative to human microsomes except for trans-permethrin, which was approximately 45% greater in human microsomes. The metabolism of bifenthrin, S-bioallethrin, and cis-permethrin in rat and human hepatic microsomes was solely the result of oxidative processes. The metabolism of bioresmethrin and cypermethrin in human hepatic microsomes was solely the result of hydrolytic processes. Bioresmethrin and cypermethrin in rat hepatic microsomes and beta-cyfluthrin and trans-permethrin in microsomes from both species were metabolized by both oxidative and hydrolytic pathways. The metabolism of trans-permethrin was reduced when incubated with its diastereomer, cis-permethrin, in both rat and human hepatic microsomes. Rat cytochrome P450 (P450) isoforms that showed activity toward several pyrethroids included CYP1A1, CYP1A2, CYP2C6, CYP2C11, CYP3A1, and CYP3A2. Human P450 isoforms that showed activity toward multiple pyrethroids were CYP2C8, CYP2C9, CYP2C19, and CYP3A4. Species-specific differences in metabolism may result in variable detoxification of pyrethroids, which may in turn result in divergent neurotoxic outcomes. These species differences and isomer interactions in metabolism of pyrethroids should be considered when assessing the potential adverse health effects of pyrethroid pesticides.

Identification of Cytochrome P450 Enzymes Involved in the Metabolism of the Designer Drugs N-(1-Phenylcyclohexyl)-3-ethoxypropanamine and N-(1-Phenylcyclohexyl)-3-methoxypropanamine

The involvement of human hepatic cytochrome P450 isoenzymes (P450s) in the metabolism of the designer drugs N-(1-phenylcyclohexyl)-3-ethoxypropanamine (PCEPA) and N-(1-phenylcyclohexyl)-3-methoxypropanamine (PCMPA) to the common metabolite N-(1-phenylcyclohexyl)-3-hydroxypropanamine (PCHPA) was studied using insect cell microsomes with cDNA-expressed human P450s and human liver microsomes (HLMs). Incubation samples were analyzed by gas chromatography-mass spectrometry or liquid chromatography-mass spectrometry. Among the tested isoenzymes, P450 2B6, P450 2C19, P450 2D6, and P450 3A4 catalyzed PCEPA O-deethylation, and P450 2B6, P450 2C19, and P450 2D6 catalyzed PCMPA O-demethylation. According to the relative activity factor approach, these enzymes accounted for 22, 3, 30, and 45% of the net clearance for PCEPA and 51, 8, and 40% of the net clearance for PCMPA, respectively. At 1 microM PCEPA, the chemical inhibitors 4-(4-chlorobenzyl)pyridine for P450 2B6 and quinidine for P450 2D6 reduced metabolite formation in pooled HLMs by 37 and 73%, respectively, and at 10 microM PCEPA, they reduced metabolite formation by 57 and 26%, respectively. At 1 microM PCMPA, 4-(4-chlorobenzyl)pyridine and quinidine reduced metabolite formation in pooled HLMs by 25 and 39%, respectively, and at 10 microM PCMPA, they reduced metabolite formation by 62 and 27%, respectively. The experiments with the MAB inhibitory to P450 3A4 and the chemical inhibitor ketoconazole for P450 3A4 showed no inhibitory effect concerning PCEPA O-dealkylation. Experiments with HLMs from P450 2D6 poor metabolizers showed a reduction of metabolite formation as compared to pooled HLM of 73 and 25% (1 microM and 10 microM PCEPA) and 40 and 38% (1 microM and 10 microM PCMPA), respectively. In conclusion, the main metabolic step was catalyzed by different P450s.