Discovery of a Novel Lidocaine Metabolite by Human Liver Microsome and Identification of Microbial Species Which Produces the Same Metabolite.

Preparation of drug metabolites at the milligram scale is essential for determining the structure and toxicity of drug metabolites. However, their preparation using recombinant proteins and human liver microsomes (HLM) is often difficult because of technical and ethical issues. Reproducing human drug metabolism in food-derived microorganisms may be useful for overcoming these challenges. In this study, we identified an unknown metabolite of the anaesthetic drug lidocaine, which is metabolised by HLM. By screening for lidocaine metabolic activity in five types of foods (blue cheese, shiitake mushroom, natto, yoghurt, and dry yeast), we found that bacteria isolated from natto reproduced the lidocaine metabolic reaction that occurs in HLM. A fraction containing the unknown lidocaine metabolite was prepared through mass cultivation of a Bacillus subtilis standard strain, ethyl acetate extraction, open column chromatography, and HPLC purification. We identified the unknown metabolite as 3-(2,6-dimethylphenyl)-1-ethyl-2-methyl-4-imidazolidinone using NMR. Our results showed that food-derived microorganisms can produce large amounts of human drug metabolites via large-scale cultivation. Additionally, food microorganisms that can reproduce drug metabolism in humans can be used to examine drug metabolites at a low cost and without ethical issues.

Molecular docking and in vitro evaluations reveal the role of human cytochrome P450 3A4 in the cross-coupling metabolism of phenolic xenobiotics.

Metabolism generally transforms xenobiotics into more polar and hydrophilic products, facilitating their elimination from the body. Recently, a new metabolic pathway that transforms phenolic xenobiotics into more lipophilic and bioactive dimer products was discovered. To elucidate the role of cytochrome P450 (CYP) enzymes in mediating this cross-coupling metabolism, we used high-throughput screening to identify the metabolites generated from the coupling of 20 xenobiotics with four endogenous metabolites in liver microsomes. Endogenous vitamin E (VE) was the most reactive metabolite, as VE reacted with seven phenolic xenobiotics containing various structures (e.g., an imidazoline ring or a diphenol group) to generate novel lipophilic ethers such as bakuchiol-O-VE, phentolamine-O-VE, phenylethyl resorcinol-O-VE, 2-propanol-O-VE, and resveratrol-O-VE. Seven recombinant CYP enzymes were successfully expressed and purified in Escherichia coli. Integration of the results of recombinant human CYP incubation and molecular docking identified the central role of CYP3A4 in the cross-coupling metabolic pathway. Structural analysis revealed the π-π interactions, hydrogen bonds, and hydrophobic interactions between reactive xenobiotics and VE in the malleable active sites of CYP3A4. The consistency between the molecular docking results and the in vitro human cytochrome P450 evaluation shows that docking calculations can be used to screen molecules participating in cross-coupling metabolism. The results of this study provide supporting evidence for the overlooked toxicological effects induced by direct reactions between xenobiotics and endogenous metabolites during metabolic processes.

Enzyme-Mediated Reactions of Phenolic Pollutants and Endogenous Metabolites as an Overlooked Metabolic Disruption Pathway.

It is generally recognized that phenol-containing molecules mainly undergo phase II metabolic reactions, whereas glucuronide and sulfate are conjugated to form water-soluble products. Here, we report direct reactions of phenolic pollutants (triclosan, alkylphenol, bisphenol A [BPA], and its analogues) and some endogenous metabolites (vitamin E [VE] and estradiol) to generate new lipophilic ether products (e.g., BPA-O-VEs and alkylphenol-O-estradiol). A nontargeted screening strategy was used to identify the products in human liver microsome incubations, and the most abundant products (BPA-O-VEs) were confirmed via in vivo exposure in mice. BPA-O-VEs were frequently detected in sera from the general population at levels comparable to those of glucuronide/sulfate-conjugated BPA. Recombinant human cytochrome P450s were applied to find that CYP3A4 catalyzed the formation of these newly discovered ether metabolites by linking the VE hydroxyl group to the BPA phenolic ring, leading to the significantly reduced antioxidative activities of BPA-O-VEs compared to VEs. The effects of the reaction on the homeostasis of reacted biomolecules were finally assessed by in vitro assay and in vivo mice exposures. The generation of BPA-O-VEs decreased the VE concentrations and increased the reactive oxygen species generation after exposure to BPA at environmentally relevant concentrations. The identified reactions provided an overlooked metabolic disruption pathway for phenolic pollutants.

Study on the metabolic process of synthetic cannabinoids 4F-MDMB-BINACA and 4F-MDMB-BICA in human liver microsome and zebrafish model via UHPLC-QE Orbitrap MS.

In order to address the increasing abuse of synthetic cannabinoids, on July 1, 2021, China listed the whole category of synthetic cannabinoids in the Supplementary Catalog for the Control of Non-medicinal Narcotic Drugs and Psychotropic Substances. Because synthetic cannabinoids metabolize rapidly, techniques are urgently needed to identify the phase I metabolites of new synthetic cannabinoids, as well as the symbol metabolites, which can be used for detection in real cases. In this study, we used pooled human liver microsome (pHLM) and zebrafish combined with ultra-high-performance liquid chromatography (UHPLC) Q Exactive Orbitrap MS to identify the phase I metabolites of two new synthetic cannabinoids 4F-MDMB-BICA and 4F-MDMB-BINACA in vitro and in vivo, respectively. We studied the toxicokinetics of 4F-MDMB-BICA and 4F-MDMB-BINACA by sampling from a pHLM incubation system at different time points to study the change in metabolites over time. We detected a total of 14 metabolites of 4F-MDMB-BINACA and 16 metabolites of 4F-MDMB-BICA in this study. Metabolites of 4F-MDMB-BICA were detected in vitro for the first time. One metabolite of 4F-MDMB-BINACA, M05, was discovered for the first time. Based on the toxicokinetics results, we recommend three metabolites (M03, M11, M12) of 4F-MDMB-BINACA and three metabolites (M10, M12, M14) of 4F-MDMB-BICA as their symbol metabolites. The results showed that these two structurally similar synthetic cannabinoids 4F-MDMB-BINACA and 4F-MDMB-BICA had similar metabolic processes, as well as similar structures of their main symbol metabolites.

Species Difference in the Metabolism of Mulberrin in Vitro and Its Inhibitory Effect on Cytochrome P450 and UDP-Glucuronosyltransferase Enzymes.

This study aimed to evaluate the interspecies difference in metabolism of mulberrin and examine the interaction between mulberrin and CYP enzymes or recombinant human uridine 5'-diphosphate (UDP)-glucuronosyltransferase (UGT) enzymes. Liver microsomes from human (HLMs), Beagle dog (DLMs), minipig (PLMs), monkey (MLMs), rabbit (RLMs), rat (RAMs), and mouse (MIMs) were used to investigate metabolic diversity among different species. Additionally, recombinant human supersomes were used to confirm that metabolic enzymes are involved in the biotransformation of mulberrin. We also evaluated the influence of mulberrin on protein expression by Western blot analysis. Mulberrin metabolism showed significant interspecies differences. We found four and two metabolites in phase I and II reaction systems, respectively. In phase I metabolism profiles of mulberrin for HLMs, PLMs and MLMs conformed to the classic Michaelis-Menten kinetics, RAMs and MIMs followed biphasic kinetics; phase II reaction of mulberrin in HLMs, DLMs, PLMs, MLMs, RLMs, RAMs and MIMs followed biphasic kinetics. UGT1A1 were the major CYP isoforms responsible for the metabolism of mulberrin. Mulberrin showed potent inhibitory effects against CYP3A4, CYP2C9, CYP2E1, UGT1A1, UGT1A3 and UGT2B7 with IC50 values of 54.21, 9.93, 39.12, 3.84, 2.01, 16.36 µM, respectively. According to Western blot analysis, mulberrin can upregulate the protein expression of CYP2C19, and downregulate the expression levels of CYP3A5 and CYP2C9 in HepG2 cells as concentration increased. The interspecies comparisons can help find other species with metabolic pathways similar to those in humans for future in vivo studies.

Biotransformation of Liquiritigenin into Characteristic Metabolites by the Gut Microbiota.

The bioavailability of flavonoids is generally low after oral administration. The metabolic transformation of flavonoids by the gut microbiota may be one of the main reasons for this, although these metabolites have potential pharmacological activities. Liquiritigenin is an important dihydroflavonoid compound found in Glycyrrhiza uralensis that has a wide range of pharmacological properties, such as antitumor, antiulcer, anti-inflammatory, and anti-AIDS effects, but its mechanism of action remains unclear. This study explored the metabolites of liquiritigenin by examining gut microbiota metabolism and hepatic metabolism in vitro. Using LC-MS/MS and LC/MSn-IT-TOF techniques, three possible metabolites of liquiritigenin metabolized by the gut microbiota were identified: phloretic acid (M3), resorcinol (M4), and M5. M5 is speculated to be davidigenin, which has antitumor activity. By comparing these two metabolic pathways of liquiritigenin (the gut microbiota and liver microsomes), this study revealed that there are three main metabolites of liquiritigenin generated by intestinal bacteria, which provides a theoretical basis for the study of pharmacologically active substances in vivo.

Characterization of in vitro phase I metabolites of methamnetamine in human liver microsomes by liquid chromatography-quadrupole time-of-flight mass spectrometry.

N-Methyl-1-(naphthalen-2-yl)propan-2-amine (methamnetamine, PAL-1046) is an amphetamine-based new psychoactive substance (NPS). Methamnetamine has been reported to cause excessive release of serotonin, and it is classified as an empathogen or entactogen. It is not regulated as a controlled substance in most countries, and there are no studies on its metabolism. In this study, in vitro phase I metabolism of methamnetamine in human liver microsomes (HLM) and flavin-containing monooxygenase (FMO) was investigated by liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-Q-TOF/MS). Eight metabolites of methamnetamine were identified and were structurally characterized achieved by a combination of accurate mass analysis and tandem mass spectrometry. The identified metabolic processes include N-demethylation, N-hydroxylation, aromatic hydroxylation, and a combination of these processes. N-Hydroxylated metabolites were confirmed based on expressed FMOs. The major metabolite was formed from methamnetamine via hydroxylation of the naphthalene ring after the in vitro phase I process. These results could help detect methamnetamine ingestion by NPS abusers.

Evaluation of MTBH, a novel hesperetin derivative, on the activity of hepatic cytochrome P450 isoform in vitro and in vivo using a cocktail method by HPLC-MS/MS.

1. 8-methylene-tert-butylamine-3',5,7-trihydroxy-4'-methoxyflavanone (MTBH), a novel hesperidin derivative, has potential in the prevention of hepatic disease, however, its effects on cytochrome P450 isoforms (CYP450s) remains unexplored. The purpose was to investigate the effects of MTBH on the mRNA, protein levels, and activities of six CYP450s (1A2, 2C11/9, 2D2/6, 3A1/4, 2C13/19, and 2E1) in vitro and in vivo.2. In vitro study, rat and human liver microsomes were adopted to elucidate the inhibitory effect of MTBH on six CYP450s using probe drugs. In vivo study, Sprague-Dawley male rats were treated with MTBH (25, 50, or 100 mg/kg for 28 consecutive days), phenobarbital (80 mg/kg for 12 consecutive days), or 0.5% CMC-Na solution (control group) by intragastric administration, then, the mRNA, protein levels and activities of liver CYP450s were analysed by real-time PCR, western blotting and probe-drug incubation systems, respectively.3. The in vitro study indicated that MTBH inhibits the activities of CYP3A1/4 and CYP2E1 in rat and human liver microsomes. In vivo data showed that MTBH inhibits mRNA, protein levels, and activities of CYP3A1 and CYP2E1 in medium- and high-dose MTBH groups.4. MTBH has the potential to cause drug-drug interactions when co-administered with drugs that are metabolised by CYP3A1/4 and CYP2E1.

Evaluation of the effect of Bovis Calculus Artifactus on eight rat liver cytochrome P450 isozymes using LC-MS/MS and cocktail approach.

Bovis Calculus Artifactus (BCA) is the main substitute for natural Calculus bovis, a traditional drug in China used to treat high fever, convulsion, and sore throat. The effect of BCA on cytochrome P450 (CYP) activities is unknown. This study was to investigate the effect of BCA on eight rat hepatic microsomal CYPisozymes to evaluate the potential drug interactions using the cocktail approach.Metabolites of the eight isoform probe substrates of CYP isozymes were quantified by LC-MS/MS. The method was validated by incubating known CYP inhibitors α-naphthoflavone (CYP1A2), thiotepa (CYP2B1), quercetin (CYP2C7), sulfaphenazole (CYP2C6), ticlopidine (CYP2C11), quinidine (CYP2D1), ketoconazole (CYP3A1),4-methylpyrazole (CYP2E1) with individual probe substrate and rat liver microsomes. The formation rates of the corresponding metabolites of the eight probe substrates were determined to evaluate the activity of each isozyme.The results showed that BCA has different degrees of inhibitory effect on four CYP450 isoforms (CYP2C6, CYP2C11, CYP2D1, CYP3A1) (p < 0.05), but no significant influence on CYP1A2, 2B1, 2C7 or 2E1 (p > 0.05). Attention should be paid to the BCA-drug interactions by careful monitoring and appropriate dosage adjustments in the concurrent use of the drugs which are metabolized by CYP1A2, CYP2C19, and CYP3A4. Abbreviations: BCA, bovis calculus artifactus; CYP, cytochrome P450; DDIs, drug-drug interactions; ESI, electrospray ionization; MRM, multiple reaction monitoring; NBC, Natural Bovis Calculus; QC, quality control; T CM, traditional Chinese medicine.

Anthraquinones inhibit cytochromes P450 enzyme activity in silico and in vitro.

Anthraquinones exhibit various pharmacological activities (e.g., antioxidant and laxative) and are commonly found in consumer products including foods, dietary supplements, drugs, and traditional medicines. Despite their widespread use, there are limited data available on their toxicokinetic properties. Cytochrome P450 enzymes (CYPs) in the liver play major roles in metabolizing exogenous chemicals (e.g., pharmaceuticals, food ingredients, and environmental pollutants) and endogenous biomolecules (e.g., steroid hormones and cholesterol). Inhibition of CYP activities may lead to serious interactions among these compounds. Here, in silico (quantitative structure-activity relationship modeling) and in vitro (human recombinant enzymes and liver microsomes) methods were used to identify inhibitors of five major CYP isoforms (CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4) among 22 anthraquinones. First, in silico prediction and in vitro human recombinant enzyme assays were conducted for all compounds, and results showed that most of the anthraquinones were potent CYP1A2 inhibitors. Second, five selected anthraquinones (emodin, aloe-emodin, rhein, purpurin, and rubiadin) were further evaluated in human liver microsomes. Finally, plasma concentrations of the five anthraquinones in animal and humans were identified in the literature and compared to their in vitro inhibition potency (IC50 values) towards CYP activities. Emodin, rhein, and aloe-emodin inhibited activities of multiple CYPs in human liver microsomes and potential in vivo inhibition may occur due to their high plasma concentrations. These in silico and in vitro results enabled rapid identification of potential CYP inhibitors and prioritized future in-depth studies.

Identification of the metabolites of n-butylidenephthalide in rat and human liver microsomes by liquid chromatography-high-resolution mass spectrometry.

n-Butylidenephthalide (NBDP) is one of the bioactive constituents originally isolated from Ligusticum chuanxiong Hort. The aim of this study was to study the metabolic profiles of NBDP in rat and human liver microsomes. NBDP was individually incubated with liver microsomes of rat and human at 37°C for 1 h and the samples incubated were analyzed by ultra-high-performance liquid chromatography combined with high-resolution mass spectrometry. The identities of the metabolites were identified by accurate masses, product ions and retention times. Under the current conditions, a total of 14 metabolites were detected and identified. M12, M13 and M14 were biosynthesized and unambiguously characterized by nuclear magnetic resonance spectroscopy. All the metabolites can be detected in rat liver microsomes, whereas in human liver microsomes, M1, M3, M4, M5, M6 and M7 were not detected. Our results demonstrated that the metabolic pathways of NBDP included hydroxylation, hydration, hydrolysis and glutathione conjugation. This study provides an overview of the metabolic profiles of NBDP in vitro, which is helpful to understand the action of this compound.

Tubulin colchicine site binding agent LL01 displays potent antitumor efficiency both in vitro and in vivo with suitable drug-like properties.

Through rational drug design, we previously identified an indenoprazole derivative, 2-(6-ethoxy-3-(3-ethoxyphenylamino)-1-methyl-1,4-dihydroindeno[1,2-c]pyrazol-7-yloxy)acetamide (LL01), as a potent tubulin polymerization inhibitor targeting the tubulin colchicine binding site. In this study, we further demonstrated that LL01 was not a P-gp substrate. It potently inhibited the growth of a variety of tumor cells, including those with multidrug resistance, with GI50 values in the low nanomole ranges. In vitro liver microsome stability assay, LL01 was modest stable in the liver microsomes of human, mouse and rat, but was fast metabolized in dog. After single oral administration of LL01 at a dose of 10 mg/kg in SD male rats, LL01 showed acceptable PK properties with a mean bioavailability of 41%. In human HepG2 hepatoma xenograft, at the oral doses of 25 mg/kg/day and 12.5 mg/kg/day, LL01 inhibited the tumor growth by 61.27%, and 43.74%, respectively, which is much better than the positive drug sorafenib (29.45%; 30 mg/kg/day). Therefore, LL01 might be a potential drug candidate for further investigation for hepatocellular carcinoma therapy.

Osthole inhibited the activity of CYP2C9 in human liver microsomes and influenced indomethacin pharmacokinetics in rats.

Osthol, a pharmacologically active ingredient in various traditional Chinese medicines, is predominantly metabolized by CYP2C9. It may be co-administered with other drugs which are metabolized by CYP2C9 in clinical medicine. However, CYP2C9*1/*2/*3 genotype on the pharmacokinetics of osthole and its metabolic diversity between rat and human are unclear.In this study, we investigated the effects of osthole on enzyme activity of CYP2C11/CYP2C9 in rat liver microsomes (RLMs) and human liver microsomes (HLMs), to distinguish metabolic manner of osthole in different species. Interestingly, we found that osthole inhibits the activity of CYP2C11 in a non-competitive manner in RLMs, while inhibits CYP2C9 activity in a competitive manner in pooled HLMs. Then, the effects of CYP2C9*1/*2/*3 allele on the pharmacokinetics of osthole were identified. In human CYP2C9 isoform, the Ki value of 21.93 µM (CYP2C9*1), 18.10 µM (CYP2C9*2), 13.12 µM (CYP2C9*3) indicate that there are individual differences in the inhibition of osthole on CYP2C9 activity.We investigated how the indomethacin pharmacokinetics was affected by osthole in SD rat. To estimate the area under the curve (AUC), maximum plasma concentration (Cmax) and apparent clearance (CL/F), indomethacin (10 mg/kg) was given orally combined with osthole (20 mg/kg) in adult SD rat. We found the value of PK on indomethacin, such as the AUC0-∞, was from 176.40 ± 17.29 to 173.74 ± 27.69 µg/ml h-1, Cmax from 9.02 ± 1.24 to 9.89 ± 0.82 µg/ml and CL/F from 0.11 ± 0.01 to 0.12 ± 0.04 mg/kg/h which were unsignificantly changed compared with the control groups. However, the Tmax was prolonged from 2.00 ± 0.00 h to 7.33 ± 1.15 h, and T1/2 increased from 8.38 ± 2.30 h to 11.37 ± 2.11 h. These results indicate that osthole could potentially affect the metabolism of indomethacin in vivo.

Discovery of a widespread metabolic pathway within and among phenolic xenobiotics.

Metabolism is an organism's primary defense against xenobiotics, yet it also increases the production of toxic metabolites. It is generally recognized that phenolic xenobiotics, a group of ubiquitous endocrine disruptors, undergo rapid phase II metabolism to generate more water-soluble glucuronide and sulfate conjugates as a detoxification pathway. However, the toxicological effects of the compounds invariably point to the phase I metabolic cytochrome P450 enzymes. Here we show that phenolic xenobiotics undergo an unknown metabolic pathway to form more lipophilic and bioactive products. In a nontargeted screening of the metabolites of a widely used antibacterial ingredient: triclosan (TCS), we identified a metabolic pathway via in vitro incubation with weever, quail, and human microsomes and in vivo exposure in mice, which generated a group of products: TCS-O-TCS. The lipophilic metabolite of TCS was frequently detected in urine samples from the general population, and TCS-O-TCS activated the constitutive androstane receptor with the binding activity about 7.2 times higher than that of the parent compound. The metabolic pathway was mediated mainly by phase I enzymes localized on the microsomes and widely observed in chlorinated phenols, phenols, and hydroxylated aromatics. The pathway was also present in different phenolic xenobiotics and formed groups of unknown pollutants in organisms (e.g., TCS-O-bisphenol A and TCS-O-benzo(a)pyrene), thus providing a cross-talk reaction between different phenolic pollutants during metabolic processes in organisms.

Transforming of Triptolide into Characteristic Metabolites by the Gut Microbiota.

The importance of the gut microbiota in drug metabolism, especially in that of nonabsorbable drugs, has become known. The aim of this study was to explore the metabolites of triptolide by the gut microbiota. With high-performance liquid chromatography coupled with tandem mass spectrometry and ion trap time-of-flight multistage mass spectrometry (LC-MS/MS and LC/MSn-IT-TOF), four metabolites of triptolide (M1, M2, M3, and M4) were found in the intestinal contents of rats. M1 and M2, were isomeric monocarbonyl-hydroxyl-substituted metabolites with molecular weights of 390. M3 and M4 were isomeric dehydrogenated metabolites with molecular weights of 356. Among the four metabolites, the dehydrogenated metabolites (M3 and M4) were reported in the gut microbiota for the first time. The metabolic behaviors of triptolide in the gut microbiota and liver microsomes of rats were further compared. The monocarbonyl-hydroxyl-substituted metabolites (M1 and M2) were generated in both systems, and another monohydroxylated metabolite (M5) was found only in the liver microsomes. The combined results suggested that the metabolism of triptolide in the gut microbiota was specific, with two characteristic, dehydrogenated metabolites. This investigation might provide a theoretical basis for the elucidation of the metabolism mechanism of triptolide and guide its proper application in clinical administration.

Intraindividual Variation and Correlation of Cytochrome P450 Activities in Human Liver Microsomes.

We systematically studied and explored intraindividual variation and correlation in the activities of cytochrome P450 (CYP) using 105 normal liver samples. The intraindividual percentage coefficients of variation of relative Km, Vmax, and CLint were 45.9% (18.3-140%), 44.0% (16.8-127%), and 52.0% (24.2-134%). Overall values of relative Km, Vmax, and CLint for 10 CYPs were sorted. The order, from highest to lowest, was CYP2D6, 3A4/5, 2C19, 2C9, 2B6, 1A2, 2A6, 2C8, and 2E1 for Km; CYP3A4/5, 2C19, 2D6, 2C8, 2E1, 2B6, 1A2, 2A6, and 2C9 for Vmax; and CYP2D6, 2B6, 3A4/5, 2C19, 2C9, 1A2, 2E1, 2C8, and 2A6 for CLint. CYP2A6*4, 2B6 785A>G, and 2D6 100C>T contributed to intraindividual variation of CYP activities. The correlations and similarities among 10 CYP activities were analyzed, and it was found that the highest correlation and similarity for Vmax, Km, and CLint occurred between CYP2C9 and 2C8, CYP2C9 and 1A2, and CYP2C9 and 2C8, respectively. In conclusion, CYP activities exhibit considerable intraindividual variation, with some notable correlations, which might be helpful to comprehensively understand the internal connection among CYP activities and to guide the rational use of drugs.

Enzyme Kinetics and Molecular Docking Studies on Cytochrome 2B6, 2C19, 2E1, and 3A4 Activities by Sauchinone.

Sauchinone, an active lignan isolated from the aerial parts of Saururus chinensis (Saururaceae), exhibits anti-inflammatory, anti-obesity, anti-hyperglycemic, and anti-hepatic steatosis effects. As herb-drug interaction (HDI) through cytochrome P450s (CYPs)-mediated metabolism limits clinical application of herbs and drugs in combination, this study sought to explore the enzyme kinetics of sauchinone towards CYP inhibition in in vitro human liver microsomes (HLMs) and in vivo mice studies and computational molecular docking analysis. In in vitro HLMs, sauchinone reversibly inhibited CYP2B6, 2C19, 2E1, and 3A4 activities in non-competitive modes, showing inhibition constant (Ki) values of 14.3, 16.8, 41.7, and 6.84 µM, respectively. Also, sauchinone time-dependently inhibited CYP2B6, 2E1 and 3A4 activities in vitro HLMs. Molecular docking study showed that sauchinone could be bound to a few key amino acid residues in the active site of CYP2B6, 2C19, 2E1, and 3A4. When sibutramine, clopidogrel, or chlorzoxazone was co-administered with sauchinone to mice, the systemic exposure of each drug was increased compared to that without sauchinone, because sauchinone reduced the metabolic clearance of each drug. In conclusion, when sauchinone was co-treated with drugs metabolized via CYP2B6, 2C19, 2E1, or 3A4, sauchinone-drug interactions occurred because sauchinone inhibited the CYP-mediated metabolic activities.

Hepatic glucuronidation of 4-tert-octylphenol in humans: inter-individual variability and responsible UDP-glucuronosyltransferase isoforms.

4-tert-Octylphenol (4-tOP) is an endocrine-disrupting chemical. It is mainly metabolized into glucuronide by UDP-glucuronosyltransferase (UGT) enzymes in humans. The purpose of this study was to assess inter-individual variability in and the possible roles of UGT isoforms in hepatic 4-tOP glucuronidation in the humans. 4-tOP glucuronidation activities in the liver microsomes and recombinant UGTs of humans were assessed at broad substrate concentrations, and kinetics were analyzed. Correlation analyses between 4-tOP and diclofenac or 4-hydroxybiphenyl activities in pooled and individual human liver microsomes were also performed. Typical CLint values were 17.8 mL/min/mg protein for the low type, 25.2 mL/min/mg protein for the medium type, and 47.7 mL/min/mg protein for the high type. Among the recombinant UGTs (13 isoforms) examined, UGT2B7 and UGT2B15 were the most active of catalyzing 4-tOP glucuronidation. Although the K m values of UGT2B7 and UGT2B15 were similar (0.36 and 0.42 µM, respectively), the CLint value of UGT2B7 (6.83 mL/min/mg protein) >UGT2B15 (2.35 mL/min/mg protein). Strong correlations were observed between the glucuronidation activities of 4-tOP and diclofenac (a probe for UGT2B7) or 4-hydroxybiphenyl (a probe for UGT2B15) with 0.79-0.88 of Spearman correlation coefficient (r s) values. These findings demonstrate that 4-tOP glucuronidation in humans is mainly catalyzed by hepatic UGT2B7 and UGT2B15, and suggest that these UGT isoforms play important and characteristic roles in the detoxification of 4-tOP.

[Effect of clinical doses of Realgar-Indigo Naturalis formula and large-dose of realgar on CYP450s of rat liver].

To investigate the effect of clinical dose of Realgar-Indigo Naturais formula (RIF) and large-dose of Realgar on main drug-metabolizing enzymes CYP450s of rat liver, as well as its regulatory effect on mRNA expression. Wistar rats were administrated orally with tested drugs for 14 days. A Cocktail method combined with HPLC-MS/MS was used in the determination of 4 cytochrome P450 isozymes (CYP1A2, CYP2B, CYP3A and CYP2C) in liver of the rats, and the mRNA expression levels of the above subtypes were detected by real-time fluorescent quantitative PCR. The results showed that RIF can significantly induce CYP1A2 and CYP2B enzyme activity, and inhibit CYP3A enzyme activity. This result was consistent with the mRNA expression. However, its single compound showed weaker or even contrary phenomenon. Different doses of Realgar also showed significant inconsistencies on CYP450 enzymes activity and mRNA expression. These phenomena may be relevant with RIF compatibility synergies or toxicity reduction. The results can also prompt drug interactions when RIF is combined with other medicines in application.

Metabolic interactions of magnolol with cytochrome P450 enzymes: uncompetitive inhibition of CYP1A and competitive inhibition of CYP2C.

Magnolol (MAG; 5,5'-diallyl-2,2'-biphenyldiol) is a major bioactive component of Magnolia officinalis. We investigated the metabolic interactions of MAG with hepatic cytochrome P450 monooxygenase (CYP) through in vitro microsomal metabolism study using human (HLM) and rat liver microsomes (RLM). CYP2C and 3A subfamilies were significantly involved in the metabolism of MAG, while CYP1A subfamily was not in HLM and RLM. The relative contribution of phase I enzymes including CYP to the metabolism of MAG was comparable to that of uridine diphosphate glucuronosyltransferase (UGT) in RLM. Moreover, MAG potently inhibited the metabolic activity of CYP1A (IC50 of 1.62 µM) and 2C (IC50 of 5.56 µM), while weakly CYP3A (IC50 of 35.0 µM) in HLM and RLM. By the construction of Dixon plot, the inhibition type of MAG on CYP activity in RLM was determined as follows: uncompetitive inhibitor for CYP1A (Ki of 1.09-12.0 µM); competitive inhibitor for CYP2C (Ki of 10.0-15.2 µM) and 3A (Ki of 93.7-183 µM). Based on the comparison of the current IC50 and Ki values with a previously reported liver concentration (about 13 µM) of MAG after its seven times oral administration at a dose of 50 mg/kg in rats, it is suggested that MAG could show significant inhibition of CYP1A and 2C, but not CYP3A, in the in vivo rat system. These results could lead to further studies in clinically significant metabolism-mediated MAG-drug interactions.