CYP3A4 Activity Research Articles

The Intervention and Mechanism of Action for Aloin against Subchronic Aflatoxin B1 Induced Hepatic Injury in Rats.

As a class of difurancoumarin compounds with similar structures, aflatoxins (AF) are commonly found in the environment, soil, and food crops. AF pose a serious threat to the health of humans, poultry, and livestock. This study aimed to investigate the neuroprotective effect and detailed mechanism of aloin on hepatic injury induced by subchronic AFB1 in rats. The result showed that aloin could significantly inhibit the decrease in food intake, body weight growth, immune organ index, and serum albumin content caused by long-term AFB1 exposure. Meanwhile, aloin reduced the level of serum liver function and improved renal swelling and pathological changes of liver tissue. Aloin could also inhibit liver lipid peroxidation and improve liver antioxidant capacity. Further investigation revealed that aloin inhibited the activity and expression of hepatic CYP1A2 and CYP3A4 and down-regulated IL-1β expression in subchronic AFB1-induced liver injury rats. The above study demonstrated that aloin played an important role in blocking or delaying the development process of subchronic AFB1-induced hepatotoxicity. Therefore, aloin is considered to have a potential role as a protective agent against AFB1.

Impact of rifampicin on the pharmacokinetics of clarithromycin and 14-hydroxy clarithromycin in patients with multidrug combination therapy for pulmonary Mycobacterium avium complex infection

IntroductionClarithromycin (CAM), ethambutol (EB), and rifampicin (RFP) combination therapy is used to treat pulmonary Mycobacterium avium complex (MAC) infection; however, serum CAM concentration decreases due to RFP-mediated induction of CYP3A activity. Therefore, we investigated the pharmacokinetics of CAM, 14-hydroxy clarithromycin (14-OH CAM), EB, and RFP in patients receiving this three-drug combination therapy. MethodsCAM monotherapy was started, EB was added 2 weeks later, and RFP was added 2 weeks after that. Serum CAM, 14-OH CAM, EB, and RFP concentrations were measured before and at 2, 4, 6, and 12 or 24 h after administration on days 14, 28, and 42, and pharmacokinetic parameters were calculated. ResultsMedian area under the curve (AUC) of CAM decreased by 92.1% from 0 to 12 h after concomitant administration of RFP compared with CAM monotherapy [1.7 (interquartile range [IQR], 1.4–1.8) μg·h/mL vs. 21.5 (IQR, 17.7–32.3) μg·h/mL, respectively]. In contrast, median AUC of 14-OH CAM was not significantly different between concomitant administration of RFP [9.1 (IQR, 7.9–10.9) μg·h/mL] and CAM monotherapy [8.2 (IQR, 6.3–9.3) μg·h/mL]. AUCs of CAM and 14-OH CAM did not change in CAM+EB combination therapy. ConclusionsWhen RFP is combined with CAM in the treatment of pulmonary MAC infection, the blood concentration of CAM significantly decreased and that of the active metabolite 14-OH CAM increased, but not significantly. Our results suggest that combination therapy with CAM and RFP needs to be reconsidered and may require dose modification in the treatment of pulmonary MAC infection.

ABCB1 and ABCG2 Control Brain Accumulation and Intestinal Disposition of the Novel ROS1/TRK/ALK Inhibitor Repotrectinib, While OATP1A/1B, ABCG2, and CYP3A Limit Its Oral Availability.

Repotrectinib shows high activity against ROS1/TRK/ALK fusion-positive cancers in preclinical studies. We explored the roles of multidrug efflux transporters ABCB1 and ABCG2, the OATP1A/1B uptake transporter(s), and the CYP3A complex in pharmacokinetics and tissue distribution of repotrectinib in genetically modified mouse models. In vitro, human ABCB1 and ABCG2, and mouse Abcg2 efficiently transported repotrectinib with efflux transport ratios of 13.5, 5.6, and 40, respectively. Oral repotrectinib (10 mg/kg) showed higher plasma exposures in Abcg2-deficient mouse strains. Brain-to-plasma ratios were increased in Abcb1a/1b−/− (4.1-fold) and Abcb1a/1b;Abcg2−/− (14.2-fold) compared to wild-type mice, but not in single Abcg2−/− mice. Small intestinal content recovery of repotrectinib was decreased 4.9-fold in Abcb1a/1b−/− and 13.6-fold in Abcb1a/1b;Abcg2−/− mice. Intriguingly, Abcb1a/1b;Abcg2−/− mice displayed transient, mild, likely CNS-localized toxicity. Oatp1a/1b deficiency caused a 2.3-fold increased oral availability and corresponding decrease in liver distribution of repotrectinib. In Cyp3a−/− mice, repotrectinib plasma AUC0–h was 2.3-fold increased, and subsequently reduced 2.0-fold in humanized CYP3A4 transgenic mice. Collectively, Abcb1 and Abcg2 restrict repotrectinib brain accumulation and possibly toxicity, and control its intestinal disposition. Abcg2 also limits repotrectinib oral availability. Oatp1a/1b mediates repotrectinib liver uptake, thus reducing its systemic exposure. Systemic exposure of repotrectinib is also substantially limited by CYP3A activity. These insights may be useful to optimize the therapeutic application of repotrectinib.

The Selective NMDA Receptor GluN2B Subunit Antagonist CP-101,606 with Antidepressant Properties Modulates Cytochrome P450 Expression in the Liver.

Recent research indicates that selective NMDA receptor GluN2B subunit antagonists may become useful for the treatment of major depressive disorders. We aimed to examine in parallel the effect of the selective NMDA receptor GluN2B subunit antagonist CP-101,606 on the pituitary/serum hormone levels and on the regulation of cytochrome P450 in rat liver. CP-101,606 (20 mg/kg ip. for 5 days) decreased the activity of CYP1A, CYP2A, CYP2B, CYP2C11 and CYP3A, but not that of CYP2C6. The alterations in enzymatic activity were accompanied by changes in the CYP protein and mRNA levels. In parallel, a decrease in the pituitary growth hormone-releasing hormone, and in serum growth hormone and corticosterone (but not T3 and T4) concentration was observed. After a 3-week administration period of CP-101,606 less changes were found. A decrease in the CYP3A enzyme activity and protein level was still maintained, though no change in the mRNA level was found. A slight decrease in the serum concentration of corticosterone was also maintained, while GH level returned to the control value. The obtained results imply engagement of the glutamatergic system in the neuroendocrine regulation of cytochrome P450 and potential involvement of drugs acting on NMDA receptors in metabolic drug–drug interactions.

Inhibition of phase-1 biotransformation and cytostatic effects of diphenyleneiodonium on hepatoblastoma cell line HepG2 and a CYP3A4-overexpressing HepG2 cell clone.

Cell-based in vitro liver models are an important tool in the development and evaluation of new drugs in pharmacological and toxicological drug assessment. Hepatic microsomal enzyme complexes, consisting of cytochrome P450 oxidoreductase (CPR) and cytochrome P450 monooxygenases (CYPs), play a decisive role in catalysing phase-1 biotransformation of pharmaceuticals and xenobiotics. For a comprehensive understanding of the phase-1 biotransformation of drugs, the availability of well-characterized substances for the targeted modulation of in vitro liver models is essential. In this study, we investigated diphenyleneiodonium (DPI) for its ability to inhibit phase-1 enzyme activity and further its toxicological profile in an in vitro HepG2 cell model with and without recombinant expression of the most important drug metabolization enzyme CYP3A4.Aim of the study was to identify effective DPI concentrations for CPR/CYP activity modulation and potentially associated dose and time dependent hepatotoxic effects. The cells were treated with DPI doses up to 5,000nM (versus vehicle control) for a maximum of 48 h and subsequently examined for CYP3A4 activity as well as various toxicological relevant parameters such as cell morphology, integrity and viability, intracellular ATP level, and proliferation. Concluding, the experiments revealed a time- and concentration-dependent DPI mediated partial and complete inhibition of CYP3A4 activity in CYP3A4 overexpressing HepG2-cells (HepG2-CYP3A4). Other cell functions, including ATP synthesis and consequently the proliferation were negatively affected in both in vitro cell models. Since neither cell integrity nor cell viability were reduced, the effect of DPI in HepG2 can be assessed as cytostatic rather than cytotoxic.

Intrauterine growth restriction alters the activity of drug metabolising enzymes in the maternal-placental-fetal unit

PurposeTen percent of pregnancies are affected by intrauterine growth restriction (IUGR), and evidence suggests that affected neonates have reduced activity of hepatic cytochrome P450 (CYP) drug metabolising enzymes. Given that almost all pregnant individuals take medications and additional medications are often required during an IUGR pregnancy, we aimed to determine the impact of IUGR on hepatic CYP activity in sheep fetuses and pregnant ewes. MethodsSpecific probes were used to determine the impact of IUGR on the activity of several CYP isoenzymes (CYP1A2, CYP2C19, CYP2D6 and CYP3A) in sheep fetuses and pregnant ewes. Probes were administered intravenously to the ewe at 132 days (d) gestation (term 150 d), followed by blood sampling from the maternal and fetal circulation over 24 h. Maternal and fetal liver tissue was collected at 139–140 d gestation, from which microsomes were isolated and incubated with probes. Metabolite and maternal plasma cortisol concentrations were measured using Liquid Chromatography – tandem mass spectrometry (LC-MS/MS). ResultsMaternal plasma cortisol concentration and maternal hepatic CYP1A2 and CYP3A activity was significantly higher in IUGR pregnancies. Maternal hepatic CYP activity was higher than fetal hepatic CYP activity for all CYPs tested, and there was minimal CYP1A2 or CYP3A activity in the late gestation fetus when assessed using in vitro methods. ConclusionsThe physiological changes to the maternal-placental-fetal unit in an IUGR pregnancy have significant effects on maternal drug metabolism, suggesting changes in medications and/or doses may be required to optimise maternal and fetal health.

Impact of Tacrolimus Daily Dose Limitation in Renal Transplant Recipients Expressing CYP3A5: A Retrospective Study.

The pharmacokinetic variability of tacrolimus can be partly explained by CYP3A5 activity. Our objective was to evaluate a tacrolimus sparing policy on renal graft outcome according to CYP3A5 6986A>G genetic polymorphism. This retrospective study included 1114 recipients with a median follow-up of 6.3 years. Genotyping of the 6986A>G allelic variant corresponding to CYP3A5*3 was systematically performed. One year after transplantation, tacrolimus blood trough concentration (C0) target range was 5–7 ng/mL. However, daily dose was capped to 0.10 mg/kg/day regardless of the CYP3A5 genotype. A total 208 CYP3A5*1/- patients were included. Despite a higher daily dose, CYP3A5*1/- recipients exhibited lower C0 during follow-up (p < 0.01). Multivariate analysis did not show any significant influence of CYP3A5*1/- genotype (HR = 0.70, 0.46–1.07, p = 0.10) on patient-graft survival. Glomerular Filtration Rate (GFR) decline was significantly lower for the CYP3A5*1/- group (p = 0.02). The CYP3A5*1/- genotype did not significantly impact the risk of biopsy-proven acute rejection (BPAR) (HR = 1.01, 0.68–1.49, p = 0.97) despite significantly lower C0. Based on our experience, a strategy of tacrolimus capping is associated with a better GFR evolution in CYP3A5*1/- recipients without any significant increase of BPAR incidence. Our study raised some issues about specific therapeutic tacrolimus C0 targets for CYP3A5*1/- patients and suggests to set up randomized control studies in this specific population.

Characterization of fluorescent probe substrates to develop an efficient high-throughput assay for neonatal hepatic CYP3A7 inhibition screening

CYP3A7 is a member of the cytochrome P450 (CYP) 3A enzyme sub-family that is expressed in the fetus and neonate. In addition to its role metabolizing retinoic acid and the endogenous steroid dehydroepiandrosterone sulfate (DHEA-S), it also has a critical function in drug metabolism and disposition during the first few weeks of life. Despite this, it is generally ignored in the preclinical testing of new drug candidates. This increases the risk for drug-drug interactions (DDI) and toxicities occurring in the neonate. Therefore, screening drug candidates for CYP3A7 inhibition is essential to identify chemical entities with potential toxicity risks for neonates. Currently, there is no efficient high-throughput screening (HTS) assay to assess CYP3A7 inhibition. Here, we report our testing of various fluorescent probes to assess CYP3A7 activity in a high-throughput manner. We determined that the fluorescent compound dibenzylfluorescein (DBF) is superior to other compounds in meeting the criteria considered for an efficient HTS assay. Furthermore, a preliminary screen of an HIV/HCV antiviral drug mini-library demonstrated the utility of DBF in a HTS assay system. We anticipate that this tool will be of great benefit in screening drugs that may be used in the neonatal population in the future.

Lactobacillus rhamnosus induces CYP3A and changes the pharmacokinetics of verapamil in rats

Verapamil, a calcium channel blocker, has been approved as the first-line drug for treatment of angina pectoris, hypertension and supraventricular tachycardia. Lactobacillus rhamnosus, one of the normal strains in human intestinal tract, is very popular in the probiotic market for conferring a health benefit on the host. This report investigated the potential of gut microbiota-drug interactions between lactobacillus rhamnosus and verapamil via using wild type (WT) and Cyp3a1/2 knockout (KO) rats. In WT rats, administration of Lactobacillus rhamnosus for 14 days decreased systemic exposure of verapamil and increased its metabolite norverapamil in vivo, and resulted in gut microbiota-drug interactions. In Cyp3a1/2 KO rats, however, this interaction disappeared. Further studies found that Lactobacillus rhamnosus induced CYP3A activity and expression, and changed the composition of gut microbiota, thus changing the pharmacokinetics of verapamil. These results demonstrated the interaction between lactobacillus rhamnosus and verapamil, and indicated that the effect of gut microbiota on metabolic enzymes cannot be ignored.

Effect of CYP3A4 metabolism on sex differences in the pharmacokinetics and pharmacodynamics of zolpidem

To investigate pharmacokinetic and pharmacodynamic differences of zolpidem between males and females and their causes, including CYP3A4 activity. A single oral dose of zolpidem (10 mg) was administered to 15 male and 15 female healthy subjects. Blood samples were collected up to 12 h post-dose to determine plasma zolpidem concentrations. Pharmacokinetic parameters were obtained using non-compartmental analysis. Digit symbol substitution test, choice reaction time, and visual analog scale of sleepiness were used to evaluate pharmacodynamics. We measured CYP3A4 activity using 4β-hydroxycholesterol, an endogenous metabolite. Mean maximum plasma concentration and area under the plasma concentration–time curve were higher for females than for males (9.9% and 32.5%, respectively); other pharmacokinetic parameters showed no significant differences. Pharmacodynamic scores for females showed delayed recovery compared with that for males. CYP3A4 activity was higher in females than in males (p = 0.030). There was no serious adverse event, and adverse event incidence was not different between the sexes. Zolpidem exposure was about 30% higher in females than in males. Delayed pharmacodynamic score recovery in females could be related to higher zolpidem concentrations. Although apparent clearance was lower in females, systemic clearance might not be the cause of the different exposures to zolpidem.

Functional characterization of novel rare CYP2A6 variants and potential implications for clinical outcomes.

CYP2A6 activity, phenotyped by the nicotine metabolite ratio (NMR), is a predictor of several smoking behaviors, including cessation and smoking‐related disease risk. The heritability of the NMR is 60–80%, yet weighted genetic risk scores (wGRSs) based on common variants explain only 30–35%. Rare variants (minor allele frequency <1%) are hypothesized to explain some of this missing heritability. We present two targeted sequencing studies where rare protein‐coding variants are functionally characterized in vivo, in silico, and in vitro to examine this hypothesis. In a smoking cessation trial, 1687 individuals were sequenced; characterization measures included the in vivo NMR, in vitro protein expression, and metabolic activity measured from recombinant proteins. In a human liver bank, 312 human liver samples were sequenced; measures included RNA expression, protein expression, and metabolic activity from extracted liver tissue. In total, 38 of 47 rare coding variants identified were novel; characterizations ranged from gain‐of‐function to loss‐of‐function. On a population level, the portion of NMR variation explained by the rare coding variants was small (~1%). However, upon incorporation, the accuracy of the wGRS was improved for individuals with rare protein‐coding variants (i.e., the residuals were reduced), and approximately one‐third of these individuals (12/39) were re‐assigned from normal to slow metabolizer status. Rare coding variants can alter an individual’s CYP2A6 activity; their integration into wGRSs through precise functional characterization is necessary to accurately assess clinical outcomes and achieve precision medicine for all. Investigation into noncoding variants is warranted to further explain the missing heritability in the NMR.

High resolution stereolithography fabrication of perfusable scaffolds to enable long-term meso-scale hepatic culture for disease modeling

Microphysiological systems (MPS), comprising human cell cultured in formats that capture features of the three-dimensional (3D) microenvironments of native human organs under microperfusion, are promising tools for biomedical research. Here we report the development of a mesoscale physiological system (MePS) enabling the long-term 3D perfused culture of primary human hepatocytes at scales of over 106 cells per MPS. A central feature of the MePS, which employs a commercially-available multiwell bioreactor for perfusion, is a novel scaffold comprising a dense network of nano- and micro-porous polymer channels, designed to provide appropriate convective and diffusive mass transfer of oxygen and other nutrients while maintaining physiological values of shear stress. The scaffold design is realized by a high resolution stereolithography fabrication process employing a novel resin. This new culture system sustains mesoscopic hepatic tissue-like cultures with greater hepatic functionality (assessed by albumin and urea synthesis, and CYP3A4 activity) and lower inflammation markers compared to comparable cultures on the commercial polystyrene scaffold. To illustrate applications to disease modeling, we established an insulin-resistant phenotype by exposing liver cells to hyperglycemic and hyperinsulinemic media. Future applications of the MePS include the co-culture of hepatocytes with resident immune cells and the integration with multiple organs to model complex liver-associated diseases

Time-dependent inhibition of CYP3A4-mediated midazolam metabolism by macrolide antibiotics in CYP3A4 genetic variants: Comparison with testosterone metabolism.

Objectives: The present study aimed to evaluate the effects of CYP3A4 genetic variation on the kinetics of mechanism-based inhibition (MBI) of both inhibitors using midazolam as a substrate for comparison with our previous study, as midazolam and testosterone have different binding sites. Background: The genetic variation of cytochrome P450 (CYP) 3A4 affects MBI, expressed as the maximum inactivation rate constant (k inact,max) and the inhibitor concentration required to achieve half-maximal inactivation (K I). We previously showed, using testosterone as a substrate, that the MBI kinetics of erythromycin and clarithromycin differ among CYP3A4 variants. Materials and methods: Midazolam 1’-hydroxylation inactivation profiles of erythromycin and clarithromycin were assessed using recombinant CYP3A4.1, .2, .7, .16, and .18 expressed in Escherichia coli. MBI parameters were calculated from changes in the inactivation rate constant (Δk obs) by the inhibitors. Results: Both inhibitors increased Δk obs value in a concentration- and preincubation time-dependent manner, and MBI kinetics differed among variants. Trends of differences in MBI parameters among variants were similar to those assessed using testosterone as a substrate; K I decreased for CYP3A4.7, and k inact,max decreased for CYP3A4.2, .7, and .16. Conclusion: The genetic variation of recombinant CYP3A4 affects the MBI profile of CYP3A4 by erythromycin and clarithromycin, while the influence of genetic variation was similarly observed regardless of substrates. Our findings are of clinical relevance because the residual enzyme activity of CYP3A4 in the presence of inhibitor was estimated to vary among genetic variants.

CYP3A Activity in End-of-Life Cancer Patients Measured by 4β-Hydroxycholesterol/cholesterol Ratio, in Men and Women.

Simple SummaryThe elimination of drugs by enzymes in the liver may be impaired in cancer patients that are close to death (end-of-life). This could cause unwanted side effects or lack of effect of drugs and compromise the quality of life in patients. Blood samples collected in 137 deceased end-of-life cancer patients were analyzed for the marker 4β-hydroxycholesterol/cholesterol (4β-OHC/C), representing the activity of the most important drug eliminating enzyme, CYP3A. In addition, samples from young (n = 280) and elderly (n = 30) controls were analyzed for 4β-OHC/C. The average 4β-OHC/C was higher in male and female end-of-life cancer patients than in young and elderly controls without cancer. This finding may suggest that the ability to eliminate drugs by CYP3A is maintained until end of life and that drugs metabolized by CYP3 may not need dose adjustment or discontinuation in cancer patients close to death.More than 50% of all drugs are metabolized by the cytochrome P450 3A enzyme (CYP3A). The aim of this study was to investigate if the CYP3A activity, measured by the endogenous marker 4β-hydroxycholesterol/cholesterol ratio (4β-OHC/C), is changed during the last weeks and days of life in men and women. To this end, serum samples from 137 deceased patients (median age 70 years) collected at a single time point 1–60 days before death, were analyzed and compared to 280 young (median 27 years), and 30 elderly (median age 70 years) non-cancer controls. There were no significant differences in the 4β-OHC/C ratio between men and women in end-of-life patients (p < 0.25). The median 4β-OHC/C was significantly higher in end-of-life male patients compared to both young (p < 0.0001) and elderly (p < 0.05) male controls. In a similar manner, 4β-OHC/C in end-of-life female patients was significantly higher compared to young and elderly female controls, p < 0.0001 and p < 0.001, respectively. There was no significant correlation between 4β-OHC/C and survival time. The results from this study suggest maintained CYP3A activity to the very last days of life and even a capacity of induction of the enzyme in end-of-life cancer patients.

Quantification of CYP3A and Drug Transporters Activity in Healthy Young, Healthy Elderly and Chronic Kidney Disease Elderly Patients by a Microdose Cocktail Approach.

Background: Ageing and chronic kidney disease (CKD) affect pharmacokinetic (PK) parameters. Since mechanisms are related and remain unclear, cytochrome P450 (CYP) 3A and drug transporter activities were investigated in the elderly with or without CKD and compared to healthy adults using a microdose cocktail.Methods: Healthy young participants (n = 20), healthy elderly participants (n = 16) and elderly patients with CKD (n = 17) received, in study period 1, a single dose of microdose cocktail probe containing 30 µg midazolam, 750 µg dabigatran etexilate, 100 µg atorvastatin, 10 µg pitavastatin, and 50 µg rosuvastatin. After a 14-day wash-out period, healthy young participants continued to study period 2 with the microdose cocktail plus rifampicin. PK parameters including area under the plasma concentration-time curve (AUC), maximum plasma drug concentration (Cmax), and half-life were estimated before making pairwise comparisons of geometric mean ratios (GMR) between groups.Results: AUC and Cmax GMR (95% confidence interval; CI) of midazolam, a CYP3A probe substrate, were increased 2.30 (1.70–3.09) and 2.90 (2.16–3.88) fold in healthy elderly and elderly patients with CKD, respectively, together with a prolonged half-life. AUC and Cmax GMR (95%CI) of atorvastatin, another CYP3A substrate, was increased 2.14 (1.52–3.02) fold in healthy elderly and 4.15 (2.98–5.79) fold in elderly patients with CKD, indicating decreased CYP3A activity related to ageing. Associated AUC changes in the probe drug whose activity could be modified by intestinal P-glycoprotein (P-gp) activity, dabigatran etexilate, were observed in patients with CKD. However, whether the activity of pitavastatin and rosuvastatin is modified by organic anion transporting polypeptide 1B (OATP1B) and of breast cancer resistance protein (BCRP), respectively, in elderly participants with or without CKD was inconclusive.Conclusions: CYP3A activity is reduced in ageing. Intestinal P-gp function might be affected by CKD, but further confirmation appears warranted.Clinical Trial Registration:http://www.thaiclinicaltrials.org/ (TCTR 20180312002 registered on March 07, 2018)

Megestrol acetate is a specific inducer of CYP3A4 mediated by human pregnane X receptor.

Megestrol acetate is a synthetic progestogen used to treat some cancers and cancer-associated cachexia, but its potential interactions with other drugs are not well known. This study aims to determine the regulation of drug metabolizing enzymes by megestrol acetate. Primary human hepatocytes were treated and analyzed by PCR array to identify genes involved in drug metabolism that are impacted by megestrol acetate. P450 3A4 (CYP3A4) reporter gene assay and HPLC analyses of nifedipine metabolites were used to determine CYP3A4 gene expression and activities. Competitive ligand binding assay was used to determine the affinity of megestrol acetate toward human pregnane x receptor (hPXR). Electrophoretic mobility shift assay and mammalian two hybrid assay were used to determine the mechanism of megestrol to activate hPXR. The levels and activities of CYP3A4 were significantly induced (> 4-folds) by megestrol acetate in human hepatocytes and HepG2 cells. Megestrol treatment induced CYP3A4 through the activation of hPXR, a ligand-activated transcription factor that plays a role in drug metabolism and transport. Other tested nuclear receptors showed no response. The mechanism studies showed that megestrol activated hPXR by binding to the ligand binding domain (LBD) of hPXR and increasing the recruitment of the cofactors such as steroid receptor cofactor (SRC-1). The results suggest that megestrol acetate is a specific inducer of CYP3A4 mediated by hPXR and therefore has the potential to cause drug interactions, especially in the co-administration with drugs that are substrates of CYP3A4.

Nicotine metabolism and its association with CYP2A6 genotype among Indigenous people in Alaska who smoke.

Prevalence of smoking is higher in Alaska Native and American Indian (ANAI) populations living in Alaska than the general US population. Genetic factors contribute to smoking and cessation rates. The objective of this study was to compare CYP2A6 genetic variation and CYP2A6 enzyme activity toward nicotine in an ANAI population. ANAI (N = 151) people trying to quit smoking were recruited. DNA samples were genotyped for CYP2A6 variants *1X2A, *1B, *2, *4, *9, *10, *12, and *35. Multiple nicotine metabolites were measured in plasma and urine samples, including cotinine and 3′‐hydroxycotinine used to determine CYP2A6 activity (e.g., nicotine metabolite ratio [NMR]). We calculated summary statistics for all of the genotypes and metabolites and assigned CYP2A6 activity scores based on known information. We studied the association of CYP2A6 variants with the NMR and smoking histories. The overall frequency of the CYP2A6*1B gain of function allele was high in the ANAI versus non‐ANAI populations in other studies. Both *4 null and *9 decrease of function alleles had frequencies similar to previous studies of ANAI populations. In a multivariate analysis, the genotype‐inferred CYP2A6 activity score was associated with both plasma and urine NMR (p value = 8.56E‐08 and 4.08E‐13, respectively). Plasma NMR was also associated with duration of smoking (p value < 0.01) but not urinary total nicotine equivalents uncorrected for creatinine (TNE9uc) or biological sex. Urine NMR was significantly associated (p value < 0.01) with TNE9uc. Variation in NMR in this ANAI population is explained in part by CYP2A6 genetic variation.

The Reduction of the Combined Effects of Aflatoxin and Ochratoxin A in Piglet Livers and Kidneys by Dietary Antioxidants

The purpose of this study was to investigate the combined effects of aflatoxin B1 and ochratoxin A on protein expression and catalytic activities of CYP1A2, CYP2E1, CYP3A29 and GSTA1 and the preventive effect of dietary byproduct antioxidants administration against these mycotoxin damage. Three experimental groups (E1, E2, E3) and one control group (C) of piglets after weaning (TOPIGS-40 hybrid) were fed with experimental diets for 30 days. A basal diet containing normal compound feed for starter piglets was used as a control treatment and free of mycotoxin. The experimental groups were fed as follows: E1—basal diet plus a mixture (1:1) of two byproducts (grapeseed and sea buckthorn meal), E2—the basal diet experimentally contaminated with mycotoxins (479 ppb OTA and 62ppb AFB1) and E3—basal diet containing 5% of the mixture (1:1) of grapeseed and sea buckthorn meal and contaminated with the mix of OTA and AFB1. After 4 weeks, the animals were slaughtered, and tissue samples were taken from liver and kidney in order to perform microsomal fraction isolation, followed by protein expression and enzymatic analyses. The protein expressions of CYP2E1 and CYP3A29 were up-regulated in an insignificant manner in liver, whereas in kidney, those of CYP1A2, CYP2E1 and CYP3A29 were down-regulated. The enzymatic activities of CYP1A2, CYP2E1 and CYP3A29 decreased in liver, in a significant manner, whereas in kidney, these increased significantly. The co-presence of the two mycotoxins and the mixture of grape seed and sea buckthorn meal generated a tendency to return to the control values, which suggest that grapeseed and sea buckthorn meal waste represent a promising source in counteracting the harmful effect of ochratoxin A and aflatoxin B.

Development of a 3D Cell Culture System Using Amphiphilic Polydepsipeptides and Its Application to Hepatic Differentiation.

Various three-dimensional (3D) culture systems are available to provide more accurate in vivo mimicry than two-dimensional (2D) cultures. Synthetic and/or xeno-free biomaterials are desired, as they would provide lower batch-to-batch variability and high repeatability. Here, we introduce a 3D culture system using nanofibers composed of an amphiphilic polydepsipeptide-based polymer named HYDROX, which turns into 3D nanofibers after hydration. Our system produces a large amount of cell aggregates and requires only the seeding of a cell mixture. In addition, cells cultured with HYDROX can be collected with only a centrifugation procedure, and analytical assays can then be performed. Here, we applied HYDROX to hepatic differentiation from induced pluripotent stem cells. The cells cultured with HYDROX formed aggregates and HYDROX strongly promoted hepatic differentiation and maturation in terms of functions such as the positive ratio of alpha-1 antitrypsin, the production of albumin, the cytochrome P450 (CYP) 3A4 activity, and the low-density-lipoprotein uptake ability. In addition, primary human hepatocytes cultured with HYDROX showed significantly improved CYP3A4 gene expression and activity. The viscoelasticity and stiffness of HYDROX can be modulated by varying the concentration of the synthetic polymer, thereby providing a suitable microenvironment for the differentiation of cells with various characteristics toward a target cell type. Our findings demonstrated that HYDROX is a promising biomaterial for 3D cultures in research fields ranging from basic cell research to drug discovery.

Comprehensive Investigation of Stereoselective Food Drug Interaction Potential of Resveratrol on Nine P450 and Six UGT Isoforms in Human Liver Microsomes.

The stereoselectivity of the food drug inhibition potential of resveratrol on cytochrome P450s and uridine 5′-diphosphoglucuronosyl transferases was investigated in human liver microsomes. Resveratrol enantiomers showed stereoselective inhibition of CYP2C9, CYP3A, and UGT1A1. The inhibitions of CYP1A2, CYP2B6, and CYP2C19 by resveratrol were stereo-nonselective. The estimated Ki values determined for CYP1A2 were 13.8 and 9.2 μM for trans- and cis-resveratrol, respectively. Trans-resveratrol noncompetitively inhibited CYP3A and UGT1A1 activities with Ki values of 23.8 and 27.4 μM, respectively. Trans-resveratrol inhibited CYP1A2, CYP2C19, CYP2E1, and CYP3A in a time-dependent manner with Ki shift values >2.0, while cis-resveratrol time-dependently inhibited CYP2C19 and CYP2E1. The time-dependent inhibition of trans-resveratrol against CYP3A4, CYP2E1, CYP2C19, and CYP1A2 was elucidated using glutathione as a trapping reagent. This information helped the prediction of food drug interaction potentials between resveratrol and co-administered drugs which are mainly metabolized by UGT1A1, CYP1A2, CYP2C19, CYP2E1, and CYP3A.