Mechanism-Based Insights into Removing the Mutagenicity of Aromatic Amines by Small Structural Alterations.

Aromatic and heteroaromatic amines (ArNH2) are activated by cytochrome P450 monooxygenases, primarily CYP1A2, into reactive N-arylhydroxylamines that can lead to covalent adducts with DNA nucleobases. Hereby, we give hands-on mechanism-based guidelines to design mutagenicity-free ArNH2. The mechanism of N-hydroxylation of ArNH2 by CYP1A2 is investigated by density functional theory (DFT) calculations. Two putative pathways are considered, the radicaloid route that goes via the classical ferryl-oxo oxidant and an alternative anionic pathway through Fenton-like oxidation by ferriheme-bound H2O2. Results suggest that bioactivation of ArNH2 follows the anionic pathway. We demonstrate that H-bonding and/or geometric fit of ArNH2 to CYP1A2 as well as feasibility of both proton abstraction by the ferriheme-peroxo base and heterolytic cleavage of arylhydroxylamines render molecules mutagenic. Mutagenicity of ArNH2 can be removed by structural alterations that disrupt geometric and/or electrostatic fit to CYP1A2, decrease the acidity of the NH2 group, destabilize arylnitrenium ions, or disrupt their pre-covalent transition states with guanine.

Identification of the contact region responsible for the formation of the homomeric CYP1A2•CYP1A2 complex.

Previous studies showed that cytochrome P450 1A2 (CYP1A2) forms a homomeric complex that influences its metabolic characteristics. Specifically, CYP1A2 activity exhibits a sigmoidal response as a function of NADPH-cytochrome P450 reductase (POR) concentration and is consistent with an inhibitory CYP1A2•CYP1A2 complex that is disrupted by increasing [POR] (Reed et al. (2012) Biochem. J. 446, 489-497). The goal of this study was to identify the CYP1A2 contact regions involved in homomeric complex formation. Examination of X-ray structure of CYP1A2 implicated the proximal face in homomeric complex formation. Consequently, the involvement of residues L91-K106 (P1 region) located on the proximal face of CYP1A2 was investigated. This region was replaced with the homologous region of CYP2B4 (T81-S96) and the protein was expressed in HEK293T/17 cells. Complex formation and its disruption was observed using bioluminescence resonance energy transfer (BRET). The P1-CYP1A2 (CYP1A2 with the modified P1 region) exhibited a decreased BRET signal as compared with wild-type CYP1A2 (WT-CYP1A2). On further examination, P1-CYP1A2 was much less effective at disrupting the CYP1A2•CYP1A2 homomeric complex, when compared with WT-CYP1A2, thereby demonstrating impaired binding of P1-CYP1A2 to WT-CYP1A2 protein. In contrast, the P1 substitution did not affect its ability to form a heteromeric complex with CYP2B4. P1-CYP1A2 also showed decreased activity as compared with WT-CYP1A2, which was consistent with a decrease in the ability of P1-CYP1A2 to associate with WT-POR, again implicating the P1 region in POR binding. These results indicate that the contact region responsible for the CYP1A2•CYP1A2 homomeric complex resides in the proximal region of the protein.

In silico screening and analysis of nonsynonymous SNPs in human CYP1A2 to assess possible associations with pathogenicity and cancer susceptibility.

Cytochrome P450 1A2 (CYP1A2) is one of the main hepatic CYPs involved in metabolism of carcinogens and clinically used drugs. Nonsynonymous single nucleotide polymorphisms (nsSNPs) of this enzyme could affect cancer susceptibility and drug efficiency. Hence, identification of human CYP1A2 pathogenic nsSNPs could be of great importance in personalized medicine and pharmacogenetics. Here, 176 nsSNPs of human CYP1A2 were evaluated using a variety of computational tools, of which 18 nsSNPs were found to be associated with pathogenicity. Further analysis suggested possible association of 9 nsSNPs (G73R, G73W, R108Q, R108W, E168K, E346K, R431W, F432S and R456H) with the risk of hepatocellular carcinoma. Molecular dynamics simulations revealed higher overall flexibility, decreased intramolecular hydrogen bonds and lower content of regular secondary structures for both cancer driver variants G73W and F432S when compared to the wild-type structure. In case of F432S, loss of the conserved hydrogen bond between Arg137 and heme propionate oxygen may affect heme stability and the observed significant rise in fluctuation of the CD loop could modify CYP1A2 interactions with its redox partners. Together, these findings propose CYP1A2 as a possible candidate for hepatocellular carcinoma and provide structural insights into how cancer driver nsSNPs could affect protein structure, heme stability and interaction network.

Active Ingredients and Anti-Arthritic Mechanisms of Ba-Wei-Long-Zuan Granule Revealed by 1 H-NMR-Based Metabolomics Combined with Network Pharmacology Analysis.

Ba-Wei-Long-Zuan granule (BWLZ) is a traditional herbal preparation. It has been widely used for the treatment of rheumatoid arthritis (RA). However, its active ingredients and mechanisms of action are still unclear. The present study aims to reveal the active compounds and anti-arthritic mechanisms of BWLZ against collagen-induced arthritis (CIA) by using 1 H-NMR-based metabolomics, molecular docking and network pharmacology methods. After 30 days of administration, BWLZ could effectively improve the metabolic disorders in CIA rats. The anti-arthritic effect of BWLZ was related to its restoration of 16 disturbed serum metabolites. Molecular docking and network analysis showed that 20 compounds present in BWLZ could act on multiple targets. Among them, coclaurine and hesperidin showed the highest hit rates for target proteins related to both metabolic regulation and RA, indicating that these two compounds might be potential active ingredients of BWLZ. Moreover, pathway enrichment analysis suggested that the anti-arthritic mechanisms of BWLZ might be attributed to its network regulation of several biological processes, such as steroid hormone biosynthesis, mTOR signaling pathway, alanine, aspartate and glutamate metabolism, and synthesis and degradation of ketone bodies. These results provide further evidence for the anti-arthritic properties of BWLZ and are beneficial for its quality control and clinical application. The potential targets and biological processes found in this study may provide valuable information for further studying the molecular mechanisms of BWLZ against RA. In addition, our work provides new insights for revealing the active ingredients and regulatory mechanisms of complex herbal preparations.

Inhibition of human CYP1 enzymes by a classical inhibitor α-naphthoflavone and a novel inhibitor N-(3, 5-dichlorophenyl)cyclopropanecarboxamide: An in vitro and in silico study.

Enzymes in the cytochrome P450 family 1 (CYP1) catalyze metabolic activation of procarcinogens and deactivation of certain anticancer drugs. Inhibition of these enzymes is a potential approach for cancer chemoprevention and treatment of CYP1-mediated drug resistance. We characterized inhibition of human CYP1A1, CYP1A2, and CYP1B1 enzymes by the novel inhibitor N-(3,5-dichlorophenyl)cyclopropanecarboxamide (DCPCC) and α-naphthoflavone (ANF). Depending on substrate, IC50 values of DCPCC for CYP1A1 or CYP1B1 were 10-95 times higher than for CYP1A2. IC50 of DCPCC for CYP1A2 was 100-fold lower than for enzymes in CYP2 and CYP3 families. DCPCC IC50 values were 10-680 times higher than the ones of ANF. DCPCC was a mixed-type inhibitor of CYP1A2. ANF was a competitive tight-binding inhibitor of CYP1A1, CYP1A2, and CYP1B1. CYP1A1 oxidized DCPCC more rapidly than CYP1A2 or CYP1B1 to the same metabolite. Molecular dynamics simulations and binding free energy calculations explained the differences of binding of DCPCC and ANF to the active sites of all three CYP1 enzymes. We conclude that DCPCC is a more selective inhibitor for CYP1A2 than ANF. DCPCC is a candidate structure to modulate CYP1A2-mediated metabolism of procarcinogens and anticancer drugs.

Design, synthesis, and enzymatic characterization of quinazoline-based CYP1A2 inhibitors.

Cytochrome P450 isozyme 1A2 (CYP1A2) is one main xenobiotic metabolizing enzyme in humans. It has been associated with the bioactivation of procarcinogens, including 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a tobacco specific and potent pulmonary carcinogen. This work describes the computational design and in-silico screening of potential CYP1A2 inhibitors, their chemical synthesis, and enzymatic characterization with the ultimate aim of assessing their potential as cancer chemopreventive agents. To achieve this, a combined classifiers model was used to screen a library of quinazoline-based molecules against known CYP1A2 inhibitors, non-inhibitors, and substrates to predict which quinazoline candidates had a better probability as an inhibitor. Compounds with high probability of CYP1A2 inhibition were further computationally evaluated via Glide docking. Candidates predicted to have selectivity and high binding affinity for CYP1A2 were synthesized and assayed for their enzymatic inhibition of CYP1A2, leading to the discovery of novel and potent quinazoline-based CYP1A2 inhibitors.

Phe-125 and Phe-226 of pig cytochrome P450 1A2 stabilize the binding of aflatoxin B1 and 7-ethoxyresorufin through the key CH/π interactions.

Cytochrome P450 1A2 (CYP1A2) plays important roles in the metabolism of many planar and aromatic drugs and also contributes to the bioactivation of aflatoxin B1 (AFB1) in vivo. To date, the structural basis for CYP1A2's preference to the planar substrates remains unclear. Herein, we investigated the structure-activity relationships for pig CYP1A2 catalyzing AFB1 and 7-ethoxyresorufin (7-ER). A molecular docking study was performed based on a constructed model of pig CYP1A2, which predicted the contributions of Thr-118, Thr-124, Phe-125, Phe-226, Leu-260, and Asp-313 to the substrate catalysis. Site-directed mutagenesis and kinetic analyses exhibited the common grounds: Phe-125, Phe-226 and Asp-313 were vital to AFB1 oxidation (including exo-epoxidation and 9A-hydroxylation) and ethoxyresorufin O-deethylation. Meanwhile, Phe-125 and Phe-226 formed CH/π interactions with AFB1/7-ER, and Asp-313 formed hydrogen bonds with them. Based on other published reports, this study further emphasizes the critical roles of Phe-125 and Phe-226 in recognizing the planar substrates. Our findings highlight the structural basis of pig CYP1A2 specifically catalyzing AFB1 and 7-ER, and may help to elucidate the underlying mechanism of CYP1A2's metabolic preference to the planar and aromatic substrates.

Binding Modes and Metabolism of Caffeine.

A correct estimate of ligand binding modes and a ratio of their occupancies is crucial for calculations of binding free energies. The newly developed method BLUES combines molecular dynamics with nonequilibrium candidate Monte Carlo. Nonequilibrium candidate Monte Carlo generates a plethora of possible binding modes and molecular dynamics enables the system to relax. We used BLUES to investigate binding modes of caffeine in the active site of its metabolizing enzyme Cytochrome P450 1A2 with the aim of elucidating metabolite-formation profiles at different concentrations. Because the activation energies of all sites of metabolism do not show a clear preference for one metabolite over the others, the orientations in the active site must play a key role. In simulations with caffeine located in a spacious pocket above the I-helix, it points N3 and N1 to the heme iron, whereas in simulations where caffeine is in close proximity to the heme N7 and C8 are preferably oriented toward the heme iron. We propose a mechanism where at low caffeine concentrations caffeine binds to the upper part of the active site, leading to formation of the main metabolite paraxanthine. On the other hand, at high concentrations two molecules are located in the active site, forcing one molecule into close proximity to the heme and yielding metabolites theophylline and trimethyluretic acid. Our results offer an explanation of previously published experimental results.

Prediction of regioselectivity and preferred order of metabolisms on CYP1A2-mediated reactions part 3: Difference in substrate specificity of human and rodent CYP1A2 and the refinement of predicting system.

Differences in CYP1A2-mediated metabolisms in human, rat and mouse have been analyzed with Template of human CYP1A2 established in our previous studies (Drug Metab Pharmacokinet 31:363, 2016 and 32:229, 2017). Using more than 25 chemicals including phenanthrene, MeIQx, PhIP, caffeine and furafylline, Template for human CYP1A2 was found to be applicable for rat and mouse CYP1A2 reactions with the consideration of five distinct regional interactions: 1) Expanded use of Ring D region of pro-metabolized molecules and also of trigger molecules, 2) acceptance of secondary amino groups at Position 31 of Ring eC1, 3) overlapping of pro-metabolized and trigger molecules at Ring eC4, 4) restricted maneuvering of substrates into Bay 1 region, and 5) allowance of passage of slightly large ligands in Thin-Area. These distinction points were found to be mutual for both substrates and inhibitors. In the present study, the decision-tree for substrates entering from Thin-Area has been reevaluated in consideration of species differences in human and rodent CYP1A2 forms. As the results, five steps of verification procedures have been introduced to predict the occurrence order of the regioselective metabolisms.

Physiologically-Based Pharmacokinetic Models for CYP1A2 Drug-Drug Interaction Prediction: A Modeling Network of Fluvoxamine, Theophylline, Caffeine, Rifampicin, and Midazolam.

This study provides whole-body physiologically-based pharmacokinetic models of the strong index cytochrome P450 (CYP)1A2 inhibitor and moderate CYP3A4 inhibitor fluvoxamine and of the sensitive CYP1A2 substrate theophylline. Both models were built and thoroughly evaluated for their application in drug-drug interaction (DDI) prediction in a network of perpetrator and victim drugs, combining them with previously developed models of caffeine (sensitive index CYP1A2 substrate), rifampicin (moderate CYP1A2 inducer), and midazolam (sensitive index CYP3A4 substrate). Simulation of all reported clinical DDI studies for combinations of these five drugs shows that the presented models reliably predict the observed drug concentrations, resulting in seven of eight of the predicted DDI area under the plasma curve (AUC) ratios (AUC during DDI/AUC control) and seven of seven of the predicted DDI peak plasma concentration (Cmax ) ratios (Cmax during DDI/Cmax control) within twofold of the observed values. Therefore, the models are considered qualified for DDI prediction. All models are comprehensively documented and publicly available, as tools to support the drug development and clinical research community.

Time-dependent inhibition (TDI) of CYP1A2 by a CYP3A4-mediated reactive metabolite: proposal for a novel mechanism of irreversible TDI by a non-suicide substrate.

1. The purpose of this study was to clarify the mechanism of DSP-1053 time-dependent inhibition (TDI) for CYP1A2. 2. DSP-1053 inhibited time- and concentration-dependently CYP1A2 activity in human liver microsomes even in a dilution assay. However, DSP-1053 was not metabolized by recombinant human CYP1A2. These findings indicate that the inhibitory effect of DSP-1053 on CYP1A2 does not follow a general mechanism-based inhibition (MBI) because it did not seem to be a suicide substrate. 3. In fact, CYP1A2 was not inhibited with DSP-1053 pre-incubation in recombinant human CYP1A2. On the other hand, CYP1A2 was potently inhibited after pre-incubation with DSP-1053 in a mixture of human recombinant CYP1A2 and CYP3A4. In addition, DSP-1053 TDI of CYP1A2 in human liver microsomes was drastically reduced not only by addition of a CYP3A4 inhibitor, but also by addition of potassium cyanide (KCN), which is a trapping agent for iminium ions. We also confirmed in this study that CYP1A2 suicide inhibition by DSP-1053 metabolites generated by CYP3A4 had only minimal role in DSP-1053 TDI of CYP1A2. 4. In conclusion, a possible mechanism for DSP-1053 TDI of CYP1A2 is that DSP-1053 iminium ion, which is generated by CYP3A4, departs from CYP3A4 without inhibiting it and covalently binds to CYP1A2.

Highly sensitive fluorescent bioassay of 2,3,7,8-tetrachloro-dibenzo-p-dioxin based on abnormal expression of cytochrome P450 1A2 in human cells.

Current in vitro bioassays of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, a major threat carcinogen) are relied on murine cells and fluorescent probe 7-ethoxyresorufin (7-ER), in which TCDD mostly causes abnormal expression of cytochrome P450 1A1 (CYP1A1). However, for human cells, TCDD mainly leads to a distinct abnormal expression of cytochrome P450 1A2 (CYP1A2). The poor response of 7-ER to CYP1A2 limits the traditional bioassay for human cells. Herein, we report a fluorescent probe N-(3-hydroxybutyl)-4-methoxy-1,8-naphthalimide (HBMN) for in vitro bioassay of TCDD with human cells. HBMN had ca. 60 times higher affinity to CYP1A2 than 7-ER. As such, the sensing sensitivity increased by 10 times, and different expression of CYP1A2 by TCDD induction in different human cells was found. Besides, HBMN was also feasible in rapid screening of TCDD concentration by naked eye. It would open a new way to highly sensitive detect TCDD and understand the pathogenesis of TCDD in different human organs.

Design and synthesis of selective CYP1B1 inhibitor via dearomatization of α-naphthoflavone.

Selective cytochrome P450 (CYP) 1B1 inhibition has potential as an anticancer strategy that is unrepresented in the current clinical arena. For development of a selective inhibitor, we focused on the complexity caused by sp3-hybridized carbons and synthesized a series of benzo[h]chromone derivatives linked to a non-aromatic B-ring using α-naphthoflavone (ANF) as the lead compound. Ring structure comparison suggested compound 37 as a suitable cyclohexyl-core with improved solubility. Structural evolution of 37 produced the azide-containing cis-49a, which had good properties in three important respects: (1) selectivity for CYP1B1 over CYP1A1 and CYP1A2 (120-times and 150-times, respectively), (2) greater inhibitory potency of >2 times that of ANF, and (3) improved solubility. The corresponding aromatic B-ring compound 59a showed low selectivity and poor solubility. To elucidate the binding mode, we performed X-ray crystal structure analysis, which revealed the interaction mode and explained the subtype selectivity of cis-49a.

Cytochromes P450 1A2 and 3A4 Catalyze the Metabolic Activation of Sunitinib.

Sunitinib is a multitargeted tyrosine kinase inhibitor associated with idiosyncratic hepatotoxicity. The mechanisms of this toxicity are unknown. We hypothesized that sunitinib undergoes metabolic activation to form chemically reactive, potentially toxic metabolites which may contribute to development of sunitinib-induced hepatotoxicity. The purpose of this study was to define the role of cytochrome P450 (P450) enzymes in sunitinib bioactivation. Metabolic incubations were performed using individual recombinant P450s, human liver microsomal fractions, and P450-selective chemical inhibitors. Glutathione (GSH) and dansylated GSH were used as trapping agents to detect reactive metabolite formation. Sunitinib metabolites were analyzed by liquid chromatography-tandem mass spectrometry. A putative quinoneimine-GSH conjugate (M5) of sunitinib was detected from trapping studies with GSH and dansyl-GSH in human liver microsomal incubations, and M5 was formed in an NADPH-dependent manner. Recombinant P450 1A2 generated the highest levels of defluorinated sunitinib (M3) and M5, with less formation by P450 3A4 and 2D6. P450 3A4 was the major enzyme forming the primary active metabolite N-desethylsunitinib (M1). In human liver microsomal incubations, P450 3A inhibitor ketoconazole reduced formation of M1 by 88%, while P450 1A2 inhibitor furafylline decreased generation of M5 by 62% compared to control levels. P450 2D6 and P450 3A inhibition also decreased M5 by 54 and 52%, respectively, compared to control. In kinetic assays, recombinant P450 1A2 showed greater efficiency for generation of M3 and M5 compared to that of P450 3A4 and 2D6. Moreover, M5 formation was 2.7-fold more efficient in human liver microsomal preparations from an individual donor with high P450 1A2 activity compared to a donor with low P450 1A2 activity. Collectively, these data suggest that P450 1A2 and 3A4 contribute to oxidative defluorination of sunitinib to generate a reactive, potentially toxic quinoneimine. Factors that alter P450 1A2 and 3A activity may affect patient risk for sunitinib toxicity.

Increased Phenacetin Oxidation upon the L382V Substitution in Cytochrome P450 1A2 is Associated with Altered Substrate Binding Orientation.

Leucine382 of cytochrome P450 1A2 (CYP1A2) plays an important role in binding and O-dealkylation of phenacetin, with the L382V mutation increasing substrate oxidation (Huang and Szklarz, 2010, Drug Metab. Dispos. 38:1039⁻1045). This was attributed to altered substrate binding orientation, but no direct experimental evidence had been available. Therefore, in the current studies, we employed nuclear magnetic resonance (NMR) longitudinal (T1) relaxation measurements to investigate phenacetin binding orientations within the active site of CYP1A2 wild type (WT) and mutants. Paramagnetic relaxation time (T1P) for each proton of phenacetin was calculated from the T1 value obtained from the enzymes in ferric and ferrous-CO state in the presence of phenacetin, and used to model the orientation of phenacetin in the active site. All aromatic protons of phenacetin were nearly equidistant from the heme iron (6.34⁻8.03 Å). In contrast, the distance between the proton of the ⁻OCH2⁻ group, which is abstracted during phenacetin oxidation, and the heme iron, was much shorter in the L382V (5.93 Å) and L382V/N312L (5.96 Å) mutants compared to the N312L mutant (7.84 Å) and the wild type enzyme (6.55 Å), consistent with modeling results. These studies provide direct evidence for the molecular mechanism underlying increased oxidation of phenacetin upon the L382V mutation.

The citrus flavonone hesperetin attenuates the nuclear translocation of aryl hydrocarbon receptor.

The environmental polycyclic aromatic hydrocarbons (PAH) and dioxins are carcinogens and their adverse effects have been largely attributed to the activation of AhR. Hesperetin is a flavonone found abundantly in citrus fruits and has been shown to be a biologically active agent. In the present study, the effect of hesperetin on the nuclear translocation of AhR and the downstream gene expression was investigated in MCF-7 cells. Confocal microscopy indicated that 7, 12-dimethylbenz[α]anthracene (DMBA) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) -induced nuclear translocation of AhR was deterred by hesperetin treatment. The reduced nuclear translocation could also be observed in Western analysis. Reporter-gene assay further illustrated that the induced XRE transactivation was weakened by the treatment of hesperetin. Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) assay demonstrated that the gene expressions of CYP1A1, 1A2, and 1B1 followed the same pattern of AhR translocation. These results suggested that hesperetin counteracted AhR transactivation and suppressed the downstream gene expression.

Determination of the human cytochrome P450 monooxygenase catalyzing the enantioselective oxidation of 2,2',3,5',6-pentachlorobiphenyl (PCB 95) and 2,2',3,4,4',5',6-heptachlorobiphenyl (PCB 183).

2,2',3,5',6-Pentachlorobiphenyl (PCB 95) and 2,2',3,4,4',5',6-heptachlorobiphenyl (PCB 183) possess axial chirality and form the aS and aR enantiomers. The enantiomers of these congeners have been reported to accumulate in the human body enantioselectively via unknown mechanisms. In this study, we determined the cytochrome P450 (CYP) monooxygenase responsible for the enantioselective oxidization of PCB 95 and PCB 183, using a recombinant human CYP monooxygenase. We evaluated 13 CYP monooxygenases, namely CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2B6, CYP2C8, CYP2C19, CYP2E1, CYP2J2, CYP3A4, CYP3A5, CYP4F2, and aromatase (CYP19), and revealed that CYP2A6 preferably oxidizes aS-PCB 95 enantioselectively; however, it did not oxidize PCB 183. The enantiomer composition was elevated from 0.5 (racemate) to 0.54. In addition, following incubation with CYP2A6, the enantiomer fraction (EF) of PCB 95 demonstrated a time-dependent increase.

Accumulation properties of polychlorinated biphenyl congeners in Yusho patients and prediction of their cytochrome P450-dependent metabolism by in silico analysis.

In what has become known as the Yusho incident, thousands of people in western Japan were poisoned by the accidental ingestion of rice bran oil contaminated with polychlorinated biphenyls (PCBs) and various dioxins and dioxin-like compounds. In this study, we investigated the accumulation patterns of 69 PCB congeners in the blood of Yusho patients in comparison with those of non-exposed controls. The blood samples were collected at medical check-ups in 2004 and 2005. To compare the patterns of PCB congeners, we calculated the concentration ratio of each congener relative to the 2,2',4,4',5,5'-hexaCB (CB153) concentration. The concentration ratios of tetra- and penta-chlorinated congeners in the blood of Yusho patients were significantly lower than those of controls. To examine the cytochrome P450 (CYP)-dependent metabolic potential of the 2,3',4,4'5-pentaCB (CB118), CB153, and 2,3,3',4,4'5-hexaCB (CB156) congeners, we conducted PCB-CYP (CYP1A1, CYP1A2, CYP2A6, and CYP2B6) docking simulation by in silico analysis. The docking models showed that human CYP1A1, CYP2A6, and CYP2B6 isozymes have the potential to metabolize CB118 and CB153. On the other hand, it was inferred that CB156 is difficult to be metabolized by these four CYP isozymes. These results indicate that CYP1 and CYP2 isozymes may be involved in the characteristic accumulation patterns of PCB congeners in the blood of Yusho patients.

The inhibitory effects of ß-caryophyllene, ß-caryophyllene oxide and α-humulene on the activities of the main drug-metabolizing enzymes in rat and human liver in vitro.

Sesquiterpenes, the main components of plant essential oils, are often taken in the form of folk medicines and dietary supplements. Several sesquiterpenes possess interesting biological activities but they could interact with concurrently administered drugs via inhibition of drug-metabolizing enzymes. Therefore, the present study was designed to test the potential inhibitory effect of tree structurally relative sesquiterpenes ß-caryophyllene (CAR), ß-caryophyllene oxide (CAO) and α-humulene (HUM) on the activities of the main drug-metabolizing enzymes. For this purpose, rat and human hepatic subcellular fractions were incubated with CAR, CAO or HUM together with specific substrates for oxidation, reduction and conjugation enzymes and their coenzymes. HPLC, spectrophotometric and spectrofluorimetric analyses of product formations were used. All tested sesquiterpenes significantly inhibited cytochromes P4503A (CYP3A) activities in rats as well as in human hepatic microsomes, with CAO being the strongest inhibitor. A non-competitive type of inhibition was found. On the other hand, none of the tested sesquiterpenes significantly affected the activities of carbonyl-reducing enzymes (CBR1, AKRs, NQO1) or conjugation enzymes (UGTs, GSTs, SULTs, COMT). As CYP3A enzymes metabolize many drugs, their inhibition by CAO, CAR and HUM might affect the pharmacokinetics of concurrently administered drugs. Similar results obtained in rat and human hepatic microsomes indicate that rats could be used for further testing of possible drug-sesquiterpenes interactions in vivo.

Prediction of regioselectivity and preferred order of metabolisms on CYP1A2-mediated reactions. Part 2: Solving substrate interactions of CYP1A2 with non-PAH substrates on the template system.

In our previous paper (Drug Metabolism Parmacokinet31: 363, 2016), a simulation system for ligand interactions of human CYP1A2 was developed using "Template" composed of hexagonal grids focusing on polyaromatic hydrocarbons (PAHs). The system has been expanded for the application of non-PAH chemicals including medical drugs, industrial chemicals and natural products in the present study. Additions of two Templates C and D around Ring C/E and Ring B, respectively, perpendicular each to Template A, offered the accommodation of non-PAH substrates. The size and shape of Templates C and D were defined from the reciprocal comparison of experimental data of ligands with simulation on Templates. The requirements of occupancies at Trigger region (Ring B) and at region of Facial-side Movement (Ring C) as well as Site of Oxidation were found to be mutual throughout CYP1A2 good substrates tested for over the 450 reactions, irrespective of their shapes and flexibilities. The CYP1A2 Template system was also verified with distinct types of poor substrates (47 chemicals) and inhibitors (37 inhibitors) and found to offer the information on probable structural causes. Present CYP1A2 Template system offers a unified evaluation of human CYP1A2 ligands, which aids for toxicological assessments as well as drug metabolism studies.