Cytochrome P450 3A4 Activity Research Articles

Stir-fried Semen Armeniacae Amarum Suppresses Aristolochic Acid I-Induced Nephrotoxicity and DNA Adducts.

To investigate the protective effects of stir-fried Semen Armeniacae Amarum (SAA) against aristolochic acid I (AAI)-induced nephrotoxicity and DNA adducts and elucidate the underlying mechanism involved for ensuring the safe use of Asari Radix et Rhizoma. In vitro, HEK293T cells overexpressing Flag-tagged multidrug resistance-associated protein 3 (MRP3) were constructed by Lentiviral transduction, and inhibitory effect of top 10 common pairs of medicinal herbs with Asari Radix et Rhizoma in clinic on MRP3 activity was verified using a self-constructed fluorescence screening system. The mRNA, protein expressions, and enzyme activity levels of NAD(P)H quinone dehydrogenase 1 (NQO1) and cytochrome P450 1A2 (CYP1A2) were measured in differentiated HepaRG cells. Hepatocyte toxicity after inhibition of AAI metabolite transport was detected using cell counting kit-8 assay. In vivo, C57BL/6 mice were randomly divided into 5 groups according to a random number table, including: control (1% sodium bicarbonate), AAI (10 mg/kg), stir-fried SAA (1.75 g/kg) and AAI + stir-fried SAA (1.75 and 8.75 g/kg) groups, 6 mice in each group. After 7 days of continuous gavage administration, liver and kidney damages were assessed, and the protein expressions and enzyme activity of liver metabolic enzymes NQO1 and CYP1A2 were determined simultaneously. In vivo, combination of 1.75 g/kg SAA and 10 mg/kg AAI suppressed AAI-induced nephrotoxicity and reduced dA-ALI formation by 26.7%, and these detoxification effects in a dose-dependent manner (P<0.01). Mechanistically, SAA inhibited MRP3 transport in vitro, downregulated NQO1 expression in vivo, increased CYP1A2 expression and enzymatic activity in vitro and in vivo, respectively (P<0.05 or P<0.01). Notably, SAA also reduced AAI-induced hepatotoxicity throughout the detoxification process, as indicated by a 41.3% reduction in the number of liver adducts (P<0.01). Stir-fried SAA is a novel drug candidate for the suppression of AAI-induced liver and kidney damages. The protective mechanism may be closely related to the regulation of transporters and metabolic enzymes.

UHPLC-MS/MS standardized extract of Vernonia amygdalina leaf inhibits CYP2C9 and CYP3A4 activities in hepatic cells of control and streptozotocin-induced diabetic rats.

Vernonia amygdalina Del. is a perennial tropical shrub from Asteraceae. The fresh leaf of V.amygdalina is consumed as a vegetable due to its medicinal and nutritional properties. The present study focused on the quantification of bioactive compounds, luteolin-7-O-glucoside, luteolin-7-O-glucuronide, and 1,5-O-dicaffeoylquinic acid from aqueous leaf extract of V.amygdalina. The study also aims to investigate the effects of the aqueous leaf extract of V.amygdalina on cytochrome P450 2C9 (CYP2C9), and cytochrome P450 3A4 (CYP3A4) in hepatic cells of control and diabetic rats. The quantification of the bioactive compounds was conducted using ultra-high-performance liquid chromatography multiple reactions monitoring tandem mass spectrometry (UHPLC-MS/MS-MRM) technique. The effect of the extract on CYP2C9 and CYP3A4 activities was determined using a fluorometric screening kit according to the manufacturer's instructions. The three bioactive compounds were detected and quantified in the aqueous leaf extract. Results showed that the content of luteolin-7-O-glucuronide (47 μg/mg) was the highest followed by luteolin-7-O-glucoside (3.5 μg/mg) and 1,5-O-dicaffeoylquinic acid (1.07 μg/mg). The extract showed an inhibitory effect on CYP3A4 and CYP2C9 enzyme activities in control and diabetic rats. The UHPLC-MS/MS-MRM method is sensitive and reliable for the quality control of V.amygdalina leaf extract. The inhibitory effect of the extract suggests that concomitant use of V.amygdalina leaf preparations with conventional drugs metabolized and eliminated from the body by CYP3A4 and CYP2C9 enzymes may lead to possible interaction.

The Effects of a Grape Seed Procyanidin Extract on Cytochrome P450 3A4 Activity and Inflammatory Mediators in the Lungs of Heavy Active and Former Smokers.

Grape seed procyanidin extract (GSE) is widely used to promote cardiovascular health and has purported anti-inflammatory properties. Chronic inflammation in the lungs caused by environmental toxins such as tobacco smoking plays a pivotal role in lung cancer development. In a modified phase I lung cancer chemoprevention study conducted in heavy active and former smokers using leucoselect phytosome (LP), a standardized grape seed procyanidin extract complexed with soy phospholipids to enhance bioavailability, three months of LP treatment favorably modulated a variety of surrogate endpoint biomarkers, including markers of cell proliferation. In this correlative study, we further analyzed the effects of LP on cytochrome P450 3A4 (CYP3A4) activities by comparing the endogenous conversions of cortisol and cortisone to 6-beta-hydroxycortisol and 6-beta-hydroxycortisone, respectively, before and after LP treatment and the anti-inflammatory effects of LP in the lung microenvironment of these participants by comparing a profile of inflammatory cytokines and chemokines in matched pre- and post-treatment bronchoalveolar lavage (BAL) fluids. LP treatment did not significantly alter CYP3A4 activity, and three months of LP treatment significantly decreased tumor necrosis factor (TNF), C-C Motif Chemokine Ligand 3 (CCL3) and granzyme B in BAL fluids. Furthermore, post-LP-treatment BAL fluids significantly reduced migration/invasion of various human lung neoplastic cells in vitro. Our findings support the anti-inflammatory effects of GSE/LP in the lung microenvironment and its potential utility for reducing cancerizing forces, as well as driving forces for other common respiratory diseases such as chronic obstructive pulmonary disease and asthma, in the lungs of heavy former and active smokers.

Impact of Missense Mutations on AFB1 Metabolism in Bovine Cytochrome P4503A Isoforms: A Computational Mutagenesis and Molecular Docking Analysis.

Cytochrome P450 3A (CYP3A) enzymes catalyze the metabolism of a wide range of endogenous and exogenous compounds. Genetic variations in the 3 CYP3A isoforms (CYP3A28, CYP3A74, and CYP3A76) may influence their expression and activity, leading to inter-individual differences in xenobiotic metabolism. In domestic cattle, understanding how genetic variations modulate CYP3A activity is crucial for both its therapeutic implications (clinical efficacy and adverse drug effects) and food safety (residues in foodstuff). Here, we updated the variant calling of CYP3As in 300 previously sequenced Piedmontese beef cattle, using the most recent reference genome, which contains an updated, longer sequence for CYP3A28. All but one previously identified missense variants were confirmed and a new variant, R105W in CYP3A28, was discovered. Through computational mutagenesis and molecular docking, we computationally predicted the impact of all identified CYP3A variant enzymes on protein stability and their affinity for aflatoxin B1 (AFB1), a potent carcinogen and food contaminant. For CYP3A28, we also computationally predicted its affinity for the probe substrate nifedipine (NIF). We found that CYP3A28 with R105W variant cannot accommodate NIF nor AFB1 in the binding pocket, thus affecting their metabolism. Our work provides computational foundation and prioritized ranking of CYP3A variants for future experimental validations.

Evidence that cadmium aggravate the toxicity of triphenyl phosphate in aquatic sediments to Corbicula fluminea

The ubiquitous co-existence of triphenyl phosphate (TPhP) and heavy metals in sediments raises significant biotoxicity concerns. However, uncertainty still exists regarding their combined toxicity to benthic organisms. Therefore, this research was conducted to elucidate the influences of cadmium (Cd) on TPhP toxicity to Corbicula fluminea (C. fluminea) in sediments. As a result, Cd promoted the accumulation of TPhP in C. fluminea and enhanced TPhP toxicity, manifested by damaged cell membranes and pronounced histological alterations. Molecular docking revealed that TPhP-Cd complexes exhibit greater binding affinity to cytochrome P4501A1 (CYP1A1) compared to TPhP alone. With the activity of CYP1A1 increasing, the biotransformation of TPhP was promoted in low-TPhP+Cd treatments (T5C0/T5C5/T5C35). Additionally, metabolites related to antioxidant defence and repair processes were reinforced to alleviate the toxicity of TPhP and Cd. However, excessive oxidative stress impaired the CYP1A1 activity in high-TPhP+Cd treatments (T35C0/T35C5/T35C35). Furthermore, metabolic pathway analysis revealed significant perturbations in the citrate cycle, alanine, aspartate and glutamate metabolism, purine metabolism, and pyrimidine metabolism. These disruptions weakened the repair capacity and aggravated apoptosis in digestive glands, potentially contributing to the synergistic toxicity of TPhP and Cd. The results highlight the ecological risks posed by TPhP in combination with heavy metals to benthic organisms.

Development of novel CDK9 and CYP3A4 inhibitors for cancer therapy through field and computational approaches.

Cyclin-dependent kinase 9 (CDK9) and cytochrome P450 3A4 (CYP3A4) have emerged as promising targets in the development of anticancer drugs, presenting a consistent challenge in the quest for potent inhibitors. CDK9 inhibitors can selectively target fast-growing cancer cells by disrupting transcription elongation, which in turn hinders the production of proteins essential for cell cycle progression and survivaŚ. Understanding how CYP3A4 metabolizes specific chemotherapy drugs allows for personalized treatment plans, optimizing drug dosages according to a patient's metabolic profile. Since many cancer patients undergo combination therapies, and CYP3A4 is vital in drug metabolism, its inhibition or induction by one drug can alter the plasma levels of others, potentially leading to treatment failure or increased toxicity. Therefore, managing CYP3A4 activity is critical for effective cancer treatment. Employing a range of computational methodologies, this study systematically investigated the binding mechanisms of pyrimidine derivatives against CDK9 and CYP3A4. The field-based model demonstrated high R 2 values (0.99), with Q2 (0.66), demonstrating its ability to predict in silico inhibitory activity against the target of this study. The screening process followed in this work led to the discovery of powerful new inhibitor compounds. Of the 15 new compounds designed, three have a high affinity with the target (ranging from -8 to -9kcal/molkcal/mol) and were singled out through docking filtration for more detailed investigation. As well as, a reference compound with a substantial pIC50 value of 8.4, serving as the foundation for the development of the new compounds, was included for comparative analysis. To elucidate the essential features of CDK9 and CYP3A4 inhibitor design, a comparative analysis was conducted between 3D-QSAR-generated contours and molecular docking conformations of ligands. Molecular dynamics simulations were carried out for a duration of 100 ns on selected docked complexes, specifically those involving novel compounds with CDK9 and CYP3A4 enzymes. Additionally, the binding free energy for these complexes was assessed using the MM/PBSA method, which evaluates the free energy landscape of protein-ligand interactions. The results of MM/PBSA highlighted the strength of the new compounds in enhancing interactions with the target protein, which favors the results of molecular docking and MD simulation. These insights contribute to a deeper understanding of the mechanisms underlying CDK9 and CYP3A4 inhibition, offering potential avenues for the development of innovative and effective CDK9 inhibitors.

Thyme and Oregano Oil Potential Therapeutics against Malathion Toxicity through Biochemical, Histological, and Cytochrome P450 1A2 Activities in Male Wistar Rats.

The widespread use of malathion may offer several hazards to humans and animals; additionally, many medicinal plants provide what is known as a broad antitoxicity treatment. This study was carried out to investigate hazardous biochemical and histological reactions to MOP and evaluate the effectiveness of TEO and OEO essential oils in restoring normal physiological conditions after MOP exposure by measuring enzyme-specific activity for Cytochrome P450 1A2 (CYP1A2). One hundred and twenty rats were divided into six groups of twenty animals each: (i) C - MOP served as the control group, (ii) C + MOP treated with 5 mg/kg/BW of Malathion-D10, (iii) TEO treated with 100 mg/kg/BW of oregano essential oil, (iv) TEO treated with 100 mg/kg/BW of thyme essential oil, (v) MOP + OEO treated with 5 mg/kg/BW of Malathion-D10 and 100 mg/kg/BW of oregano essential oil, and (vi) MOP + TEO treated with 5 mg/kg/BW of Malathion-D10 and 100 mg/kg/BW of thyme essential oil. The results indicated the protective effects of OEO and TEO against MOP-induced weight loss. Additionally, there was a significant improvement in ALT, AST, and ALK-Ph after being treated with OEO and TEO, either alone or after MOP exposure. Also, treatment with OEO and TEO ameliorated these oxidative stress parameters, indicating their antioxidative properties. A histopathological examination of liver tissues showed reduced hepatocellular damage and improved liver architecture in the OEO and TEO, both alone and in combination with MOP, and protective effects were more pronounced in the TEO-treated groups. However, the results indicated that TEO was more effective than OEO in increasing CYP1A2 expression and alleviating MOP-induced toxicity. Specifically, TEO showed higher protein expression and therapeutic action in reducing liver damage. In conclusion, these findings suggest that OEO and TEO may be potent therapeutic agents against MOP toxicity, offering protective effects by enhancing CYP1A2 activity and mitigating organ damage. Such knowledge would be an important step toward developing potentially unique treatment options for natural antitoxins.

Nicotine Gum in Thai Smokers with Different CYP2A6 Enzymes: A Population Pharmacokinetic Analysis

Objective: Despite the popularity of nicotine gum in Thailand, population pharmacokinetics of nicotine gum in the Thai population has not been investigated. This study aimed to develop a population pharmacokinetic (POPPK) model of nicotine and to quantify the effects of genetic and non-genetic factors to nicotine pharmacokinetics. Materials and Methods: Secondary data collected from a previous clinical trial assessing cytochrome P450 2A6 (CYP2A6) genotypes in Thai smokers was investigated. Eighteen participants who had received a single dose of 2 mg nicotine gum were included. Blood samples were collected before, at 0.25, 0.5, 1, 1.5, 2, 2.5, 3, 4.5 and 6 hours after nicotine administration. POPPK analysis was performed using nonlinear mixed effect modelling. Results: One-compartment with 1st order elimination and absorption with 6-transit compartments best described the data. CYP2A6 enzyme activity was a significant covariate on the nicotine clearance. Apparent elimination clearance (CL/F) for a person with 100% CYP2A6 activity was 266.0 L/h. CL/F would be 36.0 L/h in a subject with 0% CYP2A6 activity. However, the impact of non-genetic factors (monthly alcohol consumption, Fagerstrom Test for Nicotine Dependence score and the number of cigarettes per day) on pharmacokinetics of nicotine were not found. Conclusion: This first report on population pharmacokinetics of nicotine gum in Thai smokers provided the pharmacokinetic model and quantified CL/F for smokers with different CYP2A6 genotypes. A markedly lower exposure to nicotine in the Thai population compared to others highlights the need for more studies to ensure the efficacy of nicotine gum in the Thai population.

Intrasubject Variability in Intravenous and Oral Probes for Hepatic and First-Pass CYP3A Activity.

Clearances and the area under the plasma concentration-time curve extrapolated to infinity (AUC0-∞) of intravenous (IV) and oral midazolam and alfentanil are probes for hepatic and first-pass cytochrome P450 3A (CYP3A) activity, drug interactions, and phenotyping. Single-time plasma concentrations are also used as a proxy for clearance and AUC0-∞. Pupil diameter change is a noninvasive surrogate for plasma alfentanil. An ideal probe should have minimal intrasubject (interday) variability. Despite their widespread use, the intrasubject variability of CYP3A probes is not well characterized. This investigation determined the intrasubject (interday) variability of midazolam and alfentanil metrics of hepatic and first-pass CYP3A. Twelve volunteers were studied in a four-period protocol, with each period identical and separated by approximately 2 weeks. In each period, participants received 1 mg IV midazolam then 15 μg/kg IV alfentanil 1 h later. The next day, they received 3 mg oral midazolam then 60 μg/kg oral alfentanil. Plasma drug concentrations were determined by liquid chromatography-mass spectrometry (LCMS). Dark-adapted pupil diameters were measured coincident with blood sampling. Plasma concentrations and pupil effects (miosis) were analyzed using noncompartmental methods. The results were the coefficient of variation (%CV, mean ± SD) across four sessions in 12 participants. For IV midazolam: AUC0-∞, clearance, and 5 h concentration, the %CVs were 12 ± 3, 12 ± 3, and 18 ± 8. For IV alfentanil AUC0-∞, clearance, 2 h concentration, and area under the effect curve from time zero to infinity (AUEC0-∞), the %CVs were 16 ± 5, 15 ± 4, 22 ± 7, and 50 ± 28. For oral midazolam AUC0-∞, clearance, and 5 h concentration, %CVs were 19 ± 5, 18 ± 4, and 28 ± 11. For oral alfentanil: AUC0-∞, clearance, 4 h concentration, and AUEC0-∞, %CVs were 20 ± 4, 21 ± 4, 42 ± 26, and 37 ± 14. Midazolam and alfentanil had comparable intrasubject variabilities of clearance and AUC0-∞. Single-time point metrics had greater intrasubject variability than AUC0-∞ and clearance. Miosis was a surrogate for alfentanil concentrations and provided real-time results, but intrasubject variability was greater than that of clearances and AUC0-∞.

Relevance of plasma lenvatinib concentrations and endogenous urinary cytochrome P450 3A activity biomarkers in clinical practice.

Lenvatinib (LEN), a multitarget tyrosine kinase inhibitor used in various cancer treatments, is mainly metabolized by cytochrome P450 3A (CYP3A) enzymes. The importance of therapeutic drug monitoring (TDM) in patients administered LEN has been proposed. Although some biomarkers of endogenous CYP3A activity have been reported, their utility in dosage adjustments has not been well evaluated. This study investigated the correlation between plasma LEN concentrations and endogenous urinary CYP3A biomarkers in clinical practice. Concentrations of plasma LEN (N = 225) and CYP3A biomarkers (cortisol, 6β-hydroxycortisol, deoxycholic acid, and 1β-hydroxydeoxycholic acid) in urine (N = 214) from 20 patients (hepatocellular carcinoma, N = 6; thyroid cancer, N = 3; endometrial cancer, N = 8; and renal cell carcinoma, N = 3) collected for consultation for up to 1 year were evaluated using liquid chromatography-tandem mass spectrometry. Moreover, plasma trough LEN concentrations were predicted using a three-compartment model with linear elimination for outpatients administered LEN before sample collection. Moderate correlations were observed between the quantified actual concentrations and the predicted trough concentrations of LEN, whereas there was no correlation with endogenous urinary CYP3A biomarkers. The utility of endogenous urinary CYP3A biomarkers could not be determined. However, TDM for outpatients administered orally available medicines may be predicted using a nonlinear mixed effect model (NONMEM). This study investigated the utility of endogenous urinary CYP3A biomarkers for personalized medicine and NONMEM for predicting plasma trough drug concentrations. These findings will provide important information for further clinical investigation and detailed TDM.

Evaluation of the Effects of Extracts Containing Valeriana officinalis and Piper methysticum on the Activities of Cytochrome P450 3A and P-Glycoprotein.

This work investigated interactions ascribed to the administration of phytomedicines containing Valeriana officinalis and Piper methysticum with conventional drugs. The phytomedicines were characterized by HPLC and administered per os to male Wistar rats, either concomitantly or not with the CYP3A substrate midazolam. To distinguish between the presystemic or systemic effect, midazolam was given orally and intravenously. The effects on the P-gp substrate fexofenadine uptake by Caco-2 cells were examined. The valerenic acid content was 1.6 ± 0.1 mg per tablet, whereas kavain was 13.7 ± 0.3 mg/capsule. Valerian and kava-kava extracts increased the maximum plasma concentration (Cmax) of midazolam 2- and 4-fold compared to the control, respectively. The area under the plasma concentrations versus time curve (AUC(0-∞)) was enhanced from 994.3 ± 152.3 ng.h/mL (control) to 3041 ± 398 ng.h/mL (valerian) and 4139 ± 373 ng.h/mL (kava-kava). The half-life of midazolam was not affected. These changes were attributed to the inhibition of midazolam metabolism by the enteric CYP3A since the i. v. pharmacokinetic of midazolam remained unchanged. The kava-kava extract augmented the uptake of fexofenadine by 3.5-fold compared to the control. Although Valeriana increased the uptake of fexofenadine, it was not statistically significant to that of the control (12.5 ± 3.7 ng/mg protein vs. 5.4 ± 0.3 ng/mg protein, respectively). Therefore, phytomedicines containing V.officinalis or P. methysticum inhibited the intestinal metabolism of midazolam in rats. Conversely, the P-gp-mediated transport of fexofenadine was preferably affected by kava-kava.

Efficient hepatocyte differentiation of primary human hepatocyte-derived organoids using three dimensional nanofibers (HYDROX) and their possible application in hepatotoxicity research

Human liver organoids are in vitro three dimensionally (3D) cultured cells that have a bipotent stem cell phenotype. Translational research of human liver organoids for drug discovery has been limited by the challenge of their low hepatic function compared to primary human hepatocytes (PHHs). Various attempts have been made to develop functional hepatocyte-like cells from human liver organoids. However, none have achieved the same level of hepatic functions as PHHs. We here attempted to culture human liver organoids established from cryopreserved PHHs (PHH-derived organoids), using HYDROX, a chemically defined 3D nanofiber. While the proliferative capacity of PHH-derived organoids was lost by HYDROX-culture, the gene expression levels of drug-metabolizing enzymes were significantly improved. Enzymatic activities of cytochrome P450 3A4 (CYP3A4), CYP2C19, and CYP1A2 in HYDROX-cultured PHH-derived organoids (Org-HYDROX) were comparable to those in PHHs. When treated with hepatotoxic drugs such as troglitazone, amiodarone and acetaminophen, Org-HYDROX showed similar cell viability to PHHs, suggesting that Org-HYDROX could be applied to drug-induced hepatotoxicity tests. Furthermore, Org-HYDROX maintained its functions for up to 35 days and could be applied to chronic drug-induced hepatotoxicity tests using fialuridine. Our findings demonstrated that HYDROX could possibly be a novel biomaterial for differentiating human liver organoids towards hepatocytes applicable to pharmaceutical research.

Induction of hepatic CYP3A4 expression by cholesterol and cholic acid: Alterations of gene expression, microsomal activity, and pharmacokinetics.

Human cytochrome P450 3A4 (CYP3A4) is a drug-metabolizing enzyme that is abundantly expressed in the liver and intestine. It is an important issue whether compounds of interest affect the expression of CYP3A4 because more than 30% of commercially available drugs are metabolized by CYP3A4. In this study, we examined the effects of cholesterol and cholic acid on the expression level and activity of CYP3A4 in hCYP3A mice that have a human CYP3A gene cluster and show human-like regulation of the coding genes. A normal diet (ND, CE-2), CE-2 with 1% cholesterol and 0.5% cholic acid (HCD) or CE-2 with 0.5% cholic acid was given to the mice. The plasma concentrations of cholesterol, cholic acid and its metabolites in HCD mice were higher than those in ND mice. In this condition, the expression levels of hepatic CYP3A4 and the hydroxylation activities of triazolam, a typical CYP3A4 substrate, in liver microsomes of HCD mice were higher than those in liver microsomes of ND mice. Furthermore, plasma concentrations of triazolam in HCD mice were lower than those in ND mice. In conclusion, our study suggested that hepatic CYP3A4 expression and activity are influenced by the combination of cholesterol and cholic acid invivo.

Human Keratinocyte Responses to Woodsmoke Chemicals.

Air pollution consists of complex mixtures of chemicals with serious deleterious health effects from acute and chronic exposure. To help understand the mechanisms by which adverse effects occur, the present work examines the responses of cultured human epidermal keratinocytes to specific chemicals commonly found in woodsmoke. Our earlier findings with liquid smoke flavoring (aqueous extract of charred wood) revealed that such extracts stimulated the expression of genes associated with oxidative stress and proinflammatory response, activated the aryl hydrocarbon receptor, thereby inducing cytochrome P4501A1 activity, and induced cross-linked envelope formation, a lethal event ordinarily occurring during terminal differentiation. The present results showed that furfural produced transcriptional responses resembling those of liquid smoke, cyclohexanedione activated the aryl hydrocarbon receptor, and several chemicals induced envelope formation. Of these, syringol permeabilized the cells to the egress of lactate dehydrogenase at a concentration close to that yielding envelope formation, while furfural induced envelope formation without permeabilization detectable in this way. Furfural (but not syringol) stimulated the incorporation of amines into cell proteins in extracts in the absence of transglutaminase activity. Nevertheless, both chemicals substantially increased the amount of cellular protein incorporated into envelopes and greatly altered the envelope protein profile. Moreover, the proportion of keratin in the envelopes was dramatically increased. These findings are consistent with the chemically induced protein cross-linking in the cells. Elucidating mechanisms by which this phenomenon occurs may help understand how smoke chemicals interact with proteins to elicit cellular responses, interpret bioassays of complex pollutant mixtures, and suggest additional sensitive ways to monitor exposures.

Induction of human hepatic cytochrome P-450 3A4 expression by antifungal succinate dehydrogenase inhibitors

Succinate dehydrogenase inhibitors (SDHIs) are widely-used fungicides, to which humans are exposed and for which putative health risks are of concern. In order to identify human molecular targets for these agrochemicals, the interactions of 15 SDHIs with expression and activity of human cytochrome P-450 3A4 (CYP3A4), a major hepatic drug metabolizing enzyme, were investigated in vitro. 12/15 SDHIs, i.e., bixafen, boscalid, fluopyram, flutolanil, fluxapyroxad, furametpyr, isofetamid, isopyrazam, penflufen, penthiopyrad, pydiflumetofen and sedaxane, were found to enhance CYP3A4 mRNA expression in human hepatic HepaRG cells and primary human hepatocytes exposed for 48 h to 10 µM SDHIs, whereas 3/15 SDHIs, i.e., benzovindiflupyr, carboxin and thifluzamide, were without effect. The inducing effects were concentrations-dependent for boscalid (EC50=22.5 µM), fluopyram (EC50=4.8 µM) and flutolanil (EC50=53.6 µM). They were fully prevented by SPA70, an antagonist of the Pregnane X Receptor, thus underlining the implication of this xenobiotic-sensing receptor. Increase in CYP3A4 mRNA in response to SDHIs paralleled enhanced CYP3A4 protein expression for most of SDHIs. With respect to CYP3A4 activity, it was directly inhibited by some SDHIs, including bixafen, fluopyram, fluxapyroxad, isofetamid, isopyrazam, penthiopyrad and sedaxane, which therefore appears as dual regulators of CYP3A4, being both inducer of its expression and inhibitor of its activity. The inducing effect nevertheless predominates for these SDHIs, except for isopyrazam and sedaxane, whereas boscalid and flutolanil were pure inducers of CYP3A4 expression and activity. Most of SDHIs appear therefore as in vitro inducers of CYP3A4 expression in cultured hepatic cells, when, however, used at concentrations rather higher than those expected in humans in response to environmental or dietary exposure to these agrochemicals.

In Vitro Hepatotoxicity of Routinely Used Opioids and Sedative Drugs.

A hepatocyte cell line was used to determine the hepatotoxicity of sedatives and opioids, as the hepatotoxicity of these drugs has not yet been well characterized. This might pose a threat, especially to critically ill patients, as they often receive high cumulative doses for daily analgosedation and often already have impaired liver function due to an underlying disease or complications during treatment. A well-established biosensor based on HepG2/C3A cells was used for the determination of the hepatotoxicity of commonly used sedatives and opioids in the intensive care setting (midazolam, propofol, s-ketamin, thiopental, fentanyl, remifentanil, and sufentanil). The incubation time was 2 × 3 days with clinically relevant (Cmax) and higher concentrations (C5× and C10×) of each drug in cell culture medium or human plasma. Afterward, we measured the cell count, vitality, lactate dehydrogenase (LDH), mitochondrial dehydrogenase activity, cytochrome P 450 1A2 (CYP1A2), and albumin synthesis. All tested substances reduced the viability of hepatocyte cells, but sufentanil and remifentanil showed more pronounced effects. The cell count was diminished by sufentanil in both the medium and plasma and by remifentanil only in plasma. Sufentanil and remifentanil also led to higher values of LDH in the cell culture supernatant. A reduction of mitochondrial dehydrogenase activity was seen with the use of midazolam and s-ketamine. Microalbumin synthesis was reduced in plasma after its incubation with higher concentrations of sufentanil and remifentanil. Remifentanil and s-ketamine reduced CYP1A2 activity, while propofol and thiopental increased it. Our findings suggest that none of the tested sedatives and opioids have pronounced hepatotoxicity. Sufentanil, remifentanil, and s-ketamine showed moderate hepatotoxic effects in vitro. These drugs should be given with caution to patients vulnerable to hepatotoxic drugs, e.g., patients with pre-existing liver disease or liver impairment as part of their underlying disease (e.g., hypoxic hepatitis or cholestatic liver dysfunction in sepsis). Further studies are indicated for this topic, which may use more complex cell culture models and global pharmacovigilance reports, addressing the limitation of the used cell model: HepG2/C3A cells have a lower metabolic capacity due to their low levels of CYP enzymes compared to primary hepatocytes. However, while the test model is suitable for parental substances, it is not for toxicity testing of metabolites.

Changes in Perampanel Pharmacokinetics and Cytochrome P450 3A4 Activity Before, During, and After Pregnancy.

This study evaluated perampanel pharmacokinetics and cytochrome P450 3A4 (CYP3A4) activity, assessed using the level of 4β-hydroxycholesterol (4β-OHC) as an endogenous biomarker of CYP3A4, before, during, and after pregnancy in a woman with epilepsy and compared these measurements with those from a control group of nonpregnant women with epilepsy. A 21-year-old pregnant woman was being treated with perampanel (serum concentration: 1120 ng/mL), lacosamide, and lamotrigine. After the first trimester, the lamotrigine concentration decreased markedly; however, the perampanel concentration remained almost unchanged (range, 1130-1320 ng/mL). Similarly, serum 4β-OHC levels did not change during pregnancy (before pregnancy, 78.2 ng/mL; during pregnancy, 62.2-83.2 ng/mL). To compare these measurements with those in nonpregnant women, we enrolled 27 nonpregnant women with epilepsy (age range, 16-40 years). In the control patients, we found a strong negative correlation between the concentration-to-dose ratio of perampanel and the 4β-OHC level ( r = -0.78, P < 0.001). As there was no significant change in CYP3A4 activity, we concluded that the serum perampanel concentration did not change significantly before, during, or after pregnancy. More patients need to be studied to confirm these early results.

Gene Polymorphisms of NLRP3 Associated With Plasma Levels of 4β-Hydroxycholesterol, an Endogenous Marker of CYP3A Activity, in Patients With Asthma.

Inflammation decreases the activity of cytochrome P450 3A (CYP3A). Nucleotide-binding oligomerization domain (NOD)-like receptor family pyrin domain containing 3 (NLRP3) is responsible for regulating the inflammatory response, and its genetic polymorphisms have been linked to inflammatory diseases such as asthma. However, there have been few studies on the effect of NLRP3 on CYP3A activity. We aimed to investigate the association between polymorphisms in the NLRP3 gene and plasma 4β-hydroxycholesterol (4βOHC), an endogenous marker of CYP3A activity, in patients with asthma. In this observational study including 152 adult asthma patients, we analyzed 10 NLRP3 gene single-nucleotide polymorphisms (SNPs). Plasma 4βOHC levels were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The results showed that five SNPs were associated with significantly lower plasma 4βOHC concentrations. Among these SNPs, rs3806265, rs4612666, rs1539019, and rs10733112 contributed to a significant increase in plasma IL-6 concentrations. Moreover, a multivariate regression model showed that the rs3806265 TT, rs4612666 CC, rs1539019 AA, and rs10733112 TT genotypes were significant factors for decreased plasma 4βOHC, even after including patient background factors and CYP3A5*3 (rs776746) gene polymorphisms as covariates. These results were also observed when plasma 4βOHC concentrations were corrected for cholesterol levels. We conclude that NLRP3 gene polymorphisms are involved in increasing plasma IL-6 concentrations and decreasing plasma 4βOHC concentrations in patients with asthma. Therefore, NLRP3 gene polymorphisms may be a predictive marker of CYP3A activity in inflammatory diseases such as asthma.

Validation of Population Pharmacokinetic Models for Clozapine Dosage Prediction.

Clozapine is unique in its capacity to ameliorate severe schizophrenia but at high risk of toxicity. A relationship between blood concentration and clinical response and evidence for concentration-response relationships to some adverse effects justify therapeutic drug monitoring of clozapine. However, the relationship between drug dose and blood concentration is quite variable. This variability is, in part, due to inductive and inhibitory interactions varying the activity of cytochrome P450 1A2 (CYP1A2), the principal pathway for clozapine elimination. Several population pharmacokinetic models have been presented to facilitate dose selection and to identify poor adherence in individual patients. These models have faced little testing for validity in independent populations or even for persisting validity in the source population. Therefore, we collected a large population of clozapine-treated patients (127 patients, 1048 timed plasma concentrations) in whom dosing and covariate information could be obtained with high certainty. A population pharmacokinetic model was constructed with data collected in the first 6 weeks from study enrolment (448 plasma concentrations), to estimate covariate influences and to allow alignment with previously published models. The model was tested for its performance in predicting the concentrations observed at later time intervals up to 5 years. The predictive performances of 6 published clozapine population models were then assessed in the entire population. The population pharmacokinetic model based on the first 6 weeks identified significant influences of sex, smoking, and cotreatment with fluvoxamine on clozapine clearance. The model built from the first 6 weeks had acceptable predictive performance in the same patient population up to the first 26 weeks using individual parameters, with a median predictive error (PE) of -0.1% to -15.9% and median absolute PE of 22.9%-27.1%. Predictive performance fell progressively with time after 26 weeks. Bayesian addition of plasma concentration observations within each prediction period improved individual predictions. Three additional observations extended acceptable predictive performance into the second 6 months of therapy. When the published models were tested with the entire data set, median PE ranged from -8% to +35% with a median absolute PE of >39% in all models. Thus, none of the tested models was successful in external validation. Bayesian addition of single patient observations improved individual predictions from all models but still without achieving acceptable performances. We conclude that the relationship between covariates and blood clozapine concentrations differs between populations and that relationships are not stable over time within a population. Current population models for clozapine are not capturing influential covariates.

Mechanistic Framework to Predict Maternal-Placental-Fetal Pharmacokinetics of Nifedipine Employing Physiologically Based Pharmacokinetic Modeling Approach.

Nifedipine is used for treating mild to severe hypertension and preventing preterm labor in pregnant women. Nevertheless, concerns about nifedipine fetal exposure and safety are always raised. The aim of this study was to develop and validate a maternal-placental-fetal nifedipine physiologically based pharmacokinetic (PBPK) model and apply the model to predict maternal, placental, and fetal exposure to nifedipine at different pregnancy stages. A nifedipine PBPK model was verified with nonpregnant data and extended to the pregnant population after the inclusion of the fetoplacental multicompartment model that accounts for the placental tissue and different fetal organs within the Simcyp Simulator version 22. Model parametrization involved scaling nifedipine transplacental clearance based on Caco-2 permeability, and fetal hepatic clearance was obtained from in vitro to in vivo extrapolation encompassing cytochrome P450 3A7 and 3A4 activities. Predicted concentration profiles were compared with in vivo observations and the transplacental transfer results were evaluated using 2-fold criteria. The PBPK model predicted a mean cord-to-maternal plasma ratio of 0.98 (range, 0.86-1.06) at term, which agrees with experimental observations of 0.78 (range, 0.59-0.93). Predicted nifedipine exposure was 1.4-, 2.0-, and 3.0-fold lower at 15, 27, and 39 weeks of gestation when compared with nonpregnant exposure, respectively. This innovative PBPK model can be applied to support maternal and fetal safety assessment for nifedipine at various stages of pregnancy.