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Mono(2-ethylhexyl) phthalate (MEHP) is an active metabolite of di(2-ethylhexyl) phthalate (DEHP), which is an endocrine-disrupting chemical. In the present study, MEHP glucuronidation in humans was studied using recombinant UDP-glucuronosyltransferases (UGTs) and microsomes of the liver and intestine. Among the recombinant UGTs examined, UGT1A3, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B4, and UGT2B7 glucuronidated MEHP. The kinetics of MEHP glucuronidation by UGT1A3, UGT1A7, UGT1A8, UGT1A10, UGT2B4, and UGT2B7 followed the Michaelis–Menten model, whereas that by UGT1A9 fit the negative allosteric model. CL int values were in the order of UGT1A9 > UGT2B7 > UGT1A7 > UGT1A8 ≥ UGT1A10 > UGT1A3 > UGT2B4. The kinetics of MEHP glucuronidation by liver microsomes followed the Michaelis–Menten model. Diclofenac (20 µM) and raloxifene (20 µM) decreased CL int values to 43 and 36 % that of native microsomes, respectively. The kinetics of MEHP glucuronidation by intestine microsomes fit the biphasic model. Diclofenac (150 and 450 µM) decreased CL int values to 32 and 13 % that of native microsomes for the high-affinity phase, and to 28 and 21 % for the low-affinity phase, respectively. Raloxifene (2.5 and 7.0 µM) decreased CL int values to 35 and 4.1 % that of native microsomes for the high-affinity phase, and to 48 and 53 % for the low-affinity phase, respectively. These results suggest that MEHP glucuronidation in humans is catalyzed by UGT1A3, UGT1A9, UGT2B4, and/or UGT2B7 in the liver, and by UGT1A7, UGT1A8, UGT1A9, UGT1A10, and/or UGT2B7 in the intestine, and also that these UGT isoforms play important and characteristic roles in the detoxification of DEHP.

Mono(2-ethylhexyl) phthalate (MEHP) is an active metabolite of di(2-ethylhexyl) phthalate (DEHP) and has endocrine-disrupting effects. MEHP is metabolized into glucuronide by UDP-glucuronosyltransferase (UGT) enzymes in mammals. In the present study, the hepatic and intestinal glucuronidation of MEHP in humans, dogs, rats, and mice was examined in an in vitro system using microsomal fractions. The kinetics of MEHP glucuronidation by liver microsomes followed the Michaelis–Menten model for humans and dogs, and the biphasic model for rats and mice. The K m and V max values of human liver microsomes were 110 µM and 5.8 nmol/min/mg protein, respectively. The kinetics of intestinal microsomes followed the biphasic model for humans, dogs, and mice, and the Michaelis–Menten model for rats. The K m and V max values of human intestinal microsomes were 5.6 µM and 0.40 nmol/min/mg protein, respectively, for the high-affinity phase, and 430 µM and 0.70 nmol/min/mg protein, respectively, for the low-affinity phase. The relative levels of V max estimated by Eadie–Hofstee plots were dogs (2.0) > mice (1.4) > rats (1.0) ≈ humans (1.0) for liver microsomes, and mice (8.5) > dogs (4.1) > rats (3.1) > humans (1.0) for intestinal microsomes. The percentages of the V max values of intestinal microsomes to liver microsomes were mice (120 %) > rats (57 %) > dogs (39 %) > humans (19 %). These results suggest that the metabolic abilities of UGT enzymes expressed in the liver and intestine toward MEHP markedly differed among species, and imply that these species differences are strongly associated with the toxicity of DEHP.

4- tert -Octylphenol (4-tOP) is an endocrine-disrupting chemical. It is mainly metabolized into glucuronide by UDP-glucuronosyltransferase (UGT) enzymes in mammals. In the present study, the glucuronidation of 4-tOP in humans, monkeys, rats, and mice was examined in an in vitro system using microsomal fractions. The kinetics of 4-tOP glucuronidation by liver microsomes followed the Michaelis–Menten model for humans and monkeys, and the biphasic model for rats and mice. The K m , V max , and CL int values of human liver microsomes were 0.343 µM, 11.6 nmol/min/mg protein, and 33.8 mL/min/mg protein, respectively. The kinetics of intestine microsomes followed the Michaelis–Menten model for humans, monkeys, and rats, and the biphasic model for mice. The K m , V max , and CL int values of human intestine microsomes were 0.743 µM, 0.571 nmol/min/mg protein, and 0.770 mL/min/mg protein, respectively. The CL int values estimated by Eadie–Hofstee plots were in the order of mice (high-affinity phase) (3.0) > humans (1.0) ≥ monkeys (0.9) > rats (high-affinity phase) (0.4) for liver microsomes, and monkeys (10) > mice (high-affinity phase) (5.6) > rats (1.4) > humans (1.0) for intestine microsomes. The percentages of the CL int values of intestine microsomes to liver microsomes were in the order of monkeys (27 %) > rats (high-affinity phase in liver microsomes) (7.9 %) > mice (high-affinity phase in liver and intestine microsomes) (4.2 %) > humans (2.3 %). These results suggest that the metabolic abilities of UGT enzymes expressed in the liver and intestine toward 4-tOP markedly differ among species and imply that species differences are strongly associated with the toxicities of alkylphenols.

A drug–drug interaction (DDI) study was conducted to evaluate the effect of icenticaftor (QBW251) on the pharmacokinetics (PK) of a 5‐probe cytochrome P450 (CYP) substrate cocktail, guided by in vitro studies in human hepatocytes and liver microsomes. Another DDI study investigated the effect of icenticaftor on the PK and pharmacodynamics (PD) of a monophasic oral contraceptive (OC) containing ethinyl estradiol (EE) and levonorgestrel (LVG) in premenopausal healthy female subjects. The static‐mechanistic DDI assessment indicated that icenticaftor may moderately induce the metabolic clearance of co‐medications metabolized by CYP3A4 (area under the concentration–time curve [AUC] ratio: 0.47) and potentially CYP2C; icenticaftor may also weakly inhibit the metabolic clearance of co‐medications metabolized by CYP1A2 and CYP3A4 (AUC ratio: 1.35 and 1.86, respectively) and moderately inhibit CYP2B6 (AUC ratio: 2.11). In the CYP substrate cocktail DDI study, icenticaftor 300 mg twice daily (b.i.d.) moderately inhibited CYP1A2 (AUC ratio: 3.35) and CYP2C19 (AUC ratio: 2.70). As expected from the results of the in vitro studies, weak induction was observed for CYP3A4 (AUC ratio: 0.51) and CYP2C8 (AUC ratio: 0.66). In the OC DDI study, co‐administration of icenticaftor 450 mg b.i.d. with monophasic OC containing 30‐μg EE and 150‐μg LVG once daily reduced the plasma exposure of both components by approximately 50% and led to increased levels of follicle‐stimulating hormone and luteinizing hormone. These results provide valuable guidance for the use of icenticaftor in patients taking concomitant medications that are substrates of CYP enzymes or patients using OCs.

Mallotus furetianus is traditionally used as folk medicine on Hainan Island, China. Given the significance of the pharmacokinetic interaction between herbs and drugs, we investigated the effects of Mallotus furetianus ethanol extract (MFE) on CYP3A activity in rats. The major bioactive constituents of MFE, gallic acid (GA) and epigallocatechin gallate (EGCG), were analyzed by Reverse Phase High-Performance Liquid Chromatography (RP-HPLC) for MFE standardization. Rats were orally administered 320 mg/10mL/kg MFE or water (control) once a day for 7 days. Two hours after the last MFE treatment, the rats were euthanized, and the livers and small intestines were excised. The activity of CYP3A was measured in hepatic and intestinal microsomes, and the expression in hepatic and intestinal tissues was assessed by qRT-PCR and western blot. In the pharmacokinetics experiment, rats were administered MFE as described above. Two hours after the final dose of MFE, a 15 mg/kg midazolam solution was orally administered. Blood samples were collected before and after midazolam administration. Midazolam plasma concentration and 1-hydroxymidazolam formation in microsomes were measured by LC-MS/MS methods.CYP3A activity in hepatic microsomes exhibited a significant decrease in MFE treatment group, while no change was observed in intestinal microsomes. MFE treatment significantly increased CYP3A1 activity, as well as mRNA and protein expression in the small intestine but reduced these parameters in the liver. However, CYP3A2 remained unaffected by MFE treatment. Pharmacokinetics study showed that administration of MFE for 7 days significantly reduced the Cmax of midazolam from 919 ± 70 ng / mL to 708 ± 91 ng /mL. Conversely, t1/2 was increased from 0.45 ± 0.08 h in the control group to 0.69 ± 0.15 h in the MFE group.Collectively, our study indicated that MFE could modulate CYP3A1 activity and expression of CYP3A1, subsequently changing the metabolism of midazolam in rats.

Drug metabolism studies are essential and necessary during the evaluation of drugs. This review discusses the in vitro human liver models to estimate the drug metabolic fates in vivo. Different approaches are provided and emphasis is placed on the potential of human liver microsomes for drug metabolism and inhibition studies. The methodology for these studies using human liver microsomes, applications of human liver microsomes, and the drugs studied by human liver microsomes are listed. Human liver microsomes represent a critical experimental model for the evaluation of drug metabolites with a high probability of clinical success.

Solid transplant recipients are at increased risk for invasive aspergillosis. Tacrolimus and Voriconazole is one of the most frequently utilized treatments for those recipients with invasive fungal infections. However, it is difficult to use them properly due to the interaction between them. This study aimed to investigate the potential drug-drug interaction between Tacrolimus and Voriconazole by multiple methods, including in vitro liver microsome method and the PBPK(Physiologically Based Pharmacokinetic) model. Midazolam and testosterone were used as probe substrates to evaluate individual differences in CYP3A4/5 metabolic activity. A comprehensive interaction analysis was also conducted based on the STITCH database and the DD-Inter system. Furthermore, a PBPK model was constructed by the data from the literature to simulate the real metabolic process in vivo. The research employed multiple methodologies to demonstrate that the co-administration of Voriconazole significantly enhances Tacrolimus concentrations, considering genotypes and the activity of CYP3A4/5 genotypes. The findings indicated a decrease in the relative percentages of midazolam and testosterone metabolites with increasing Voriconazole concentration. Moreover, the results for residual Tacrolimus in the 30-minute incubation group revealed that Voriconazole exerts a mild inhibitory effect on the in vitro metabolism of Tacrolimus. The STITCH database and DD-Inter system analysis also suggested that Tacrolimus and Voriconazole share a strong association in liver metabolism, most likely interacting with CYP3A4/5 and CYP2C19. Furthermore, the result of PBPK analysis indicated that Tacrolimus AUC increases with Voriconazole co-therapy. Moreover, the AUC of Tacrolimus in intermediate CYP2C19 metabolizers (IM) was the highest at 10.1 µmol·min/L, followed by poor metabolizers (PM) at 8.13 µmol·min/L, and extensive metabolizers (EM) at 2.18 µmol·min/L. And the genotype of CYP3A5 poor metabolizer (PM) had AUC of Tacrolimus at 3.13µmol·min/L and extensive metabolizer (EM) at 2.18µmol·min/L. Microsomal studies, PBPK models, and multiple other analyses have comprehensively elucidated the impact of Voriconazole on Tacrolimus concentrations. These findings can serve as a valuable point of reference for concurrently administering these two medications. These findings also indicate that the genotypes of CYP2C19 play an important role in the development of DDI during concurrent Tacrolimus and Voriconazole treatment, which may have some guidance for clinical medication.

(S)-N-(1-amino-3,3-dimethyl-1-oxobutan-2-yl)-1-butyl-1H-indazole-3carboxamide (ADB-BUTINACA) is an emerging synthetic cannabinoid that was first identified in Europe in 2019 and entered Singapore's drug scene in January 2020. Due to the unavailable toxicological and metabolic data, there is a need to establish urinary metabolite biomarkers for detection of ADB-BUTINACA consumption and elucidate its biotransformation pathways for rationalizing its toxicological implications. We characterized the metabolites of ADB-BUTINACA in human liver microsomes using liquid chromatography Orbitrap mass spectrometry analysis. Enzyme-specific inhibitors and recombinant enzymes were adopted for the reaction phenotyping of ADB-BUTINACA. We further used recombinant enzymes to generate a pool of key metabolites in situ and determined their metabolic stability. By coupling in vitro metabolism and authentic urine analyses, a panel of urinary metabolite biomarkers of ADB-BUTINACA was curated. Fifteen metabolites of ADB-BUTINACA were identified with key biotransformations being hydroxylation, N-debutylation, dihydrodiol formation, and oxidative deamination. Reaction phenotyping established that ADB-BUTINACA was rapidly eliminated via CYP2C19-, CYP3A4-, and CYP3A5-mediated metabolism. Three major monohydroxylated metabolites (M6, M12, and M14) were generated in situ, which demonstrated greater metabolic stability compared to ADB-BUTINACA. Coupling metabolite profiling with urinary analysis, we identified four urinary biomarker metabolites of ADB-BUTINACA: 3 hydroxylated metabolites (M6, M11, and M14) and 1 oxidative deaminated metabolite (M15). Our data support a panel of four urinary metabolite biomarkers for diagnosing the consumption of ADB-BUTINACA.

Many coronary heart disease patients fail to reach recommended LDL levels, either due to intolerance or inadequate response to available lipid-lowering therapy. Microsomal triglyceride transfer protein (MTP) inhibitors may provide a novel alternative pathway for LDL lowering. In this paper the authors tested the safety and LDL lowering efficacy of the MTP inhibitor, AEGR-733, alone and in combination with ezetimibe. Background Many patients with coronary heart disease do not achieve recommended LDL-cholesterol levels, due to either intolerance or inadequate response to available lipid-lowering therapy. Microsomal triglyceride transfer protein (MTP) inhibitors might provide an alternative way to lower LDL-cholesterol levels. We tested the safety and LDL-cholesterol-lowering efficacy of an MTP inhibitor, AEGR-733 (Aegerion Pharmaceuticals Inc., Bridgewater, NJ), alone and in combination with ezetimibe. Methods We performed a multicenter, double-blind, 12-week trial, which included 84 patients with hypercholesterolemia. Patients were randomly assigned ezetimibe 10 mg daily ( n = 29); AEGR-733 5.0 mg daily for the first 4 weeks, 7.5 mg daily for the second 4 weeks and 10 mg daily for the last 4 weeks ( n = 28); or ezetimibe 10 mg daily and AEGR-733 administered with the dose titration described above ( n = 28). Results Ezetimibe monotherapy led to a 20–22% decrease in LDL-cholesterol concentrations. AEGR-733 monotherapy led to a dose-dependent decrease in LDL-cholesterol concentration: 19% at 5.0 mg, 26% at 7.5 mg and 30% at 10 mg. Combined therapy produced similar but larger dose-dependent decreases (35%, 38% and 46%, respectively). The number of patients who discontinued study drugs owing to adverse events was five with ezetimibe alone, nine with AEGR-733 alone, and four with combined ezetimibe and AEGR-733. Discontinuations from AEGR-733 were due primarily to mild transaminase elevations. Conclusions Inhibition of LDL production with low-dose AEGR-733, either alone or in combination with ezetimibe, could be an effective therapeutic option for patients unable to reach target LDL-cholesterol levels. Key Points Many patients with coronary heart disease cannot achieve current target levels for LDL-cholesterol owing to either intolerance or inadequate response to conventional lipid-lowering therapy; new treatment strategies are required A possible therapeutic approach is inhibition of microsomal triglyceride transfer protein, which is essential for the assembly and secretion of apolipoprotein-B-containing lipoproteins AEGR-733, alone and in combination with ezetimibe, had notable LDL-cholesterol-lowering effects in patients with hyperlipidemia The main side effect associated with AEGR-733 was elevation in transaminase concentrations, which returned to baseline values after cessation of therapy; gastrointestinal side effects were minor Low-dose microsomal triglyceride transfer protein inhibitors, alone or in combination with ezetimibe, could be an effective therapeutic option for patients unable to reach target LDL levels with conventional therapy

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.