Cytochrome P450 Isoforms Research Articles

Probing the Role of CYP2 Enzymes in the Atropselective Metabolism of Polychlorinated Biphenyls Using Liver Microsomes from Transgenic Mouse Models.

Chiral polychlorinated biphenyls (PCB) are environmentally relevant developmental neurotoxicants. Because their hydroxylated metabolites (OH-PCBs) are also neurotoxic, it is necessary to determine how PCB metabolism affects the developing brain, for example, in mouse models. Because the cytochrome P450 isoforms involved in the metabolism of chiral PCBs remain unexplored, we investigated the metabolism of PCB 91 (2,2',3,4',6-pentachlorobiphenyl), PCB 95 (2,2',3,5',6-pentachlorobiphenyl), PCB 132 (2,2',3,3',4,6'-hexachlorobiphenyl), and PCB 136 (2,2',3,3',6,6'-hexachlorobiphenyl) using liver microsomes from male and female Cyp2a(4/5)bgs-null, Cyp2f2-null, and wild-type mice. Microsomes, pooled by sex, were incubated with 50 μM PCB for 30 min, and the levels and enantiomeric fractions of the OH-PCBs were determined gas chromatographically. All four PCB congeners appear to be atropselectively metabolized by CYP2A(4/5)BGS and CYP2F2 enzymes in a congener- and sex-dependent manner. The OH-PCB metabolite profiles of PCB 91 and PCB 132, PCB congeners with one para-chlorine substituent, differed between null and wild-type mice. No differences in the metabolite profiles were observed for PCB 95 and PCB 136, PCB congeners without a para-chlorine group. These findings suggest that Cyp2a(4/5)bgs-null and Cyp2f2-null mice can be used to study how a loss of a specific metabolic function (e.g., deletion of Cyp2a(4/5)bgs or Cyp2f2) affects the toxicity of chiral PCB congeners.

Metabolism of β-naphthoflavone by cytochrome P450 isoforms in three air breathing fish, Channa punctatus, Heteropneustes fossilis and Clarias batrachus

The crucial phase I xenobiotic metabolising enzyme cytochrome P450 (CYP 450) has a variety of isoforms and a wide range of substrate specificities. While the roles of mammalian CYP 450 are well established, those of non-mammalian species are much less defined. These roles are frequently extrapolated from mammalian data and assumed to be similar across vertebrate species. In the present study, three air breathing fish species, Channa punctatus, Heteropneustes fossilis and Clarias batrachus were injected intraperitoneally (IP) with 50 mg/kg β-naphthoflavone (β-NF) and its effects on hepatic CYP 450 system were evaluated. β-NF treatment resulted in significant increase (p<0.01) in CYP 450 content and CYP1A mediated EROD (Ethoxyresorufin O-deethylase) activity in all three treated fish groups compared to control. CYP2B mediated N,N-dimethylaniline demethylase activity and CYP2E1 mediated aniline hydroxylase activity showed no response suggesting insensitivity to β-NF treatment. All three fish species showed an increase in CYP3A4 mediated erythromycin N-demethylase activity, but only H. fossilis displayed a significant difference (p<0.05). Of all the activities investigated, EROD activity mediated by CYP1A responded towards β-NF treatment the best and could serve as a sensitive biomarker to gauge the effects of chemical pollutants in the aquatic environment.

In vitro effects and in silico analysis of newly synthetized pyrrole derivatives on the activity of different isoforms of Cytochrome P450: CYP1A2, CYP2D6 and CYP3A4

Four pyrrole based hydazide-hydrazones with established low hepatotoxicity and promising antiproliferative activity were evaluated (at 1µM concentration) for possible inhibitory activity on human isoforms of Cytochrome P450 CYP1A2, CYP3A4 and CYP2D6. The compounds didn’t exert any statistically significant inhibitory effects on CYP1A2 and CYP2D6. However on CYP3A4 only 12 resulted in low statistically significant inhibitory effect decreasing the enzyme activity by 20%, compared to the control (pure CYP3A4). In addition the potential interactions of 12 and the evaluated CYP isoforms were displayed after molecular docking with Glide (Schrödinger). Induced-fit simulations and binding free energy (MM/GBSA) calculations were applied to elucidate the accessibility in each CYP isoform. The most active CYP3A4 inhibitor 12 demonstrated good binding affinity and was in close vicinity to the het Fe ion (2.88 Å). Overall, good correlation between the in vitro results and the free binding MM/GBSA recalculations were observed.

A A Review on the Potential of Application of CRISPR/Cas9 Technology in CYP3A4 and CYP2D6 Genes: A Way to Move Forward in Identifying New Drug Targets

The cytochrome P450 (CYP) enzymes are of a ubiquitous heme–thiolate proteins family that plays an important role within the biotransformation of endogenous molecules and xenobiotics as today’s market is bio-transformed through CYP-mediated metabolism. Major CYP isoforms involved in most reactions are mainly CYP3A4 and CYP2D6. The CYP3A4 is found most extensively within the human liver and gut in which this CYP-isoform takes part in the phase I transformation of toxins, carcinogens, bile acids, and steroid hormones while CYP2D6 plays a central role in the oxidative metabolism of up to 25% of drugs in common clinical use, although it only accounts for 1-5% of the CYP liver content. With the help of vast gene editing technologies such as the CRISPR/Cas9 system, researchers have found its application to be useful and efficient in gene knockout studies involving genetic variants in association with drug metabolism due to its simplicity and affordability. Hence, this review aims to explore the potential use of CRISPR/Cas9 technology in gene editing in vitro studies particularly in knocking out the gene of interest, mainly drug metabolism enzymes. This article will explore how CRISPR/cas9 facilitates ex vivo assays of drug metabolism studies. The long-term goal for bio-editing is an in vivo treatment without the risk of causing harm to humans and providing a ready-to-be-used drug metabolism assay for ex vivo studies of drug development and pharmacokinetics study at a lower cost.

Design, synthesis, and biological evaluation of aminopyridine derivatives as novel tropomyosin receptor kinase inhibitors.

Tropomyosin receptor kinase (TRK) is a successful target for the treatment of various cancers caused by NTRK gene fusions. Herein, based on a rational drug design strategy, we designed and synthesized 35 aminopyrimidine derivatives that were shown to be TRKA inhibitors in the enzyme assay, among which compounds C3, C4, and C6 showed potent inhibitory activities against TRKA with IC50 values of 6.5, 5.0, and 7.0 nM, respectively. In vitro antiproliferative activity study showed that compound C3 significantly inhibited the proliferation of KM-12 cellsbut had weak inhibitory effect on MCF-7 cells and HUVEC cells. The preliminary druggability evaluation showed that compound C3 exhibited favorable liver microsomal and plasma stabilitiesand had weak or no inhibitory activity against cytochrome P450 isoforms at 10 µM. Compounds C3, C4, andC6 were also selected for ADME (absorption, distribution, metabolism, and elimination) properties prediction and molecular docking studies. Inhibition experiments showed that compound C3 was not selective for TRK subtypes. All results indicated that compound C3 was a useful candidate for the development of TRK inhibitors.

In vitro and in silico inhibition performance of choline against CYP1A2, CYP2D6 and CYP3A4

Choline is an essential nutrient involved in the synthesis of acetylcholine in the cholinergic neurons. The pharmacokinetic properties of choline are well described; however, there is a lack of data about its activity toward the CYP450 superfamily of enzymes. Therefore, the aim of this study was to conduct in silico and in vitro activity assessments of choline against three major CYP450 isoforms—CYP1A2, CYP2D6, and CYP3A4. Preliminary in silico modeling was performed on a specialized DL-CYP Prediction Server to evaluate the affinity of choline toward the enzymes. The in vitro study contained specific cytochrome P450 isoform inhibitors and substrates (for CYP1A2, CYP2D6, and CYP3A4) to determine the inhibition performance of choline at five different concentrations (0.150 − 1 µmol/L). The potential interactions of choline and CYPs were displayed after molecular docking with Glide (Schrödinger). In addition, induced-fit simulations and binding free energy calculations MM/GBSA (Molecular Mechanics-Generalized Born Surface Area) were applied to predict the accessibility in each CYP isoform. The initial in silico simulations revealed that choline lacks inhibition potency against the aforementioned enzymes. The in vitro evaluations confirmed that choline possessed no effect against CYP1A2; however, at 1 µmol/L choline exerted 22% and 27% blocking capacity against CYP2D6 and CYP3A4, respectively. Furthermore, there was good correlation between the in vitro results and the complexes’ free binding energy recalculations. Overall, the assessments indicated that choline is a weak CYP2D6 and CYP3A4 inhibitor. The latter results should be considered as a source of future unwanted drug–drug interactions.

A new insight into the aryl hydrocarbon receptor/cytochrome 450 signaling pathway in MG63, HOS, SAOS2, and U2OS cell lines

Osteosarcoma is the most common malignant tumor of bone, with rapid progressive growth, early distant metastases, and frequent recurrence after surgical treatment. Osteosarcoma is characterized by changes in the ratio and expression of different cytochrome P450 (CYP) isoforms that can affect the effectiveness of anticancer therapies. The inducible expression of CYP1 genes depends on the ligand-dependent functionality of the aryl hydrocarbon receptor (AHR). In this study, we examined the AHR/CYP1 signaling pathway in four osteosarcoma cell lines (MG63, HOS, SAOS2, and U2OS) induced by the known AHR ligands: indirubin, indole-3-carbinol, and beta-naphthoflavone. Using qPCR and Western blot analysis, we explored the effects of these ligands on the expression of the CYP1 genes and studied the correlation between these responses and the changes in the mRNA and protein levels of AHR and the AHR nuclear translocator (ARNT) in these osteosarcoma cell lines. The results show that the AHR/CYP1 signaling pathway retains its function only in MG63 and HOS cells, and is impaired in SAOS2 and U2OS cells. Our data should be taken into account when recommending new strategies for the treatment of osteosarcoma and when evaluating new drugs against osteosarcoma in vitro.

Effects of multi-walled carbon nanotubes in soil on earthworm growth and reproduction, enzymatic activities, and metabolomics

Increased production and environmental release of multi-walled carbon nanotubes (MWCNTs) increase soil exposure and potential risk to earthworms. However, MWCNT toxicity to earthworms remains unclear, with some studies identifying negative effects and others negligible effects. In this study, to determine whether exposure to MWCNTs negatively affects earthworms and to elucidate possible mechanisms of toxicity, earthworms were exposed to sublethal soil concentrations of MWCNTs (10, 50, and 100 mg/kg) for 28 days. Earthworm growth and reproduction, activities of cytochrome P450 (CYP) isoforms (1A2, 2C9, and 3A4) and antioxidant enzymes (superoxide dismutase (SOD), catalase (CAT), and glutathione-s-transferase (GST)), and metabolomics were determined. Effects of MWCNTs on earthworms depended on exposure concentration. Exposure to MWCNTs did not significantly affect growth and reproduction of individual earthworms. Exposure to 50 mg/kg MWCNTs significantly increased activities of CYP2C9, CYP3A4, SOD, CAT, and GST but clearly reduced levels of L-aspartate, L-asparagine, and glutamine. With exposure to 100 mg/kg MWCNTs, toxic effects on earthworms were observed, with significant inhibition in activities of CYP isoenzymes and SOD, significant reductions in L-aspartate, L-asparagine, glutamine, and tryptophan, and simultaneous accumulations of citrate, isocitrate, fumarate, 2-oxoglutarate, pyruvate, D-galactose, carbamoyl phosphate, formyl anthranilate, hypoxanthine, and xanthine. Results suggest that toxicity of MWCNTs to earthworms is associated with reduced detoxification capacity, excessive oxidative stress, and disturbance of multiple metabolic pathways, including amino acids metabolism, the tricarboxylic acid cycle, pyruvate metabolism, D-galactose metabolism, and purine metabolism. The study provides new insights to better understand and predict the toxicity of MWCNTs in soil.

Controversial Interactions of Tacrolimus with Dietary Supplements, Herbs and Food

Tacrolimus is an immunosuppressive calcineurin inhibitor used to prevent rejection in allogeneic organ transplant recipients, such as kidney, liver, heart or lung. It is metabolized in the liver, involving the cytochrome P450 (CYP3A4) isoform CYP3A4, and is characterized by a narrow therapeutic window, dose-dependent toxicity and high inter-individual and intra-individual variability. In view of the abovementioned facts, the aim of the study is to present selected interactions between tacrolimus and the commonly used dietary supplements, herbs and food. The review was based on the available scientific literature found in the PubMed, Scopus and Cochrane databases. An increase in the serum concentration of tacrolimus can be caused by CYP3A4 inhibitors, such as grapefruit, pomelo, clementine, pomegranate, ginger and turmeric, revealing the side effects of this drug, particularly nephrotoxicity. In contrast, CYP3A4 inducers, such as St. John’s Wort, may result in a lack of therapeutic effect by reducing the drug concentration. Additionally, the use of Panax ginseng, green tea, Schisandra sphenanthera and melatonin in patients receiving tacrolimus is highly controversial. Therefore, since alternative medicine constitutes an attractive treatment option for patients, modern healthcare should emphasize the potential interactions between herbal medicines and synthetic drugs. In fact, each drug or herbal supplement should be reported by the patient to the physician (concordance) if it is taken in the course of immunosuppressive therapy, since it may affect the pharmacokinetic and pharmacodynamic parameters of other preparations.

Adenosine N6-methylation upregulates the expression of human CYP2B6 by altering the chromatin status

N6-Methyladenosine (m6A) modification is the most prevalent RNA modification in mammals. We have recently demonstrated that inhibition of m6A modification by 3-deazaadenosine results in an increase in the expression of the cytochrome P450 (CYP) isoforms CYP1A2, CYP2B6, and CYP2C8 in human liver-derived cells. In the present study, we aimed to clarify the mechanism of m6A-mediated regulation of CYP2B6 expression. RNA immunoprecipitation using an anti-m6A antibody revealed that CYP2B6 mRNA in human liver and hepatocarcinoma-derived HepaRG cells was m6A-modified around the stop codon. In contrast to the treatment with 3-deazaadenosine, double knockdown of methyltransferase like (METTL) 3 and METTL14 (METTL3/14) resulted in a decrease in the levels of CYP2B6 mRNA in Huh-7 and HepaRG cells and a decrease in bupropion hydroxylase activity, a marker activity of CYP2B6, in HepaRG cells. The stability of CYP2B6 mRNA was not influenced by siMETTL3/14. Reporter assays using the plasmids containing the last exon or 5′-flanking region of CYP2B6 indicated that reporter activities were not influenced by knockdown of METTL3/14. The expression levels of the constitutive androstane receptor, pregnane X receptor, and retinoid X receptor, which are the nuclear receptors regulating the transcription of CYP2B6, were not influenced by siMETTL3/14. The chromatin immunoprecipitation and formaldehyde-assisted enrichment of regulatory elements assays revealed that H3K9me2, a repressive histone marker, was enriched in the vicinity of the upstream region of CYP2B6, and knockdown of METTL3/14 induced the condensation of the chromatin structure in this region. In conclusion, we demonstrated that METTL3/14 upregulated CYP2B6 expression by altering the chromatin status.

Computational Prediction of Inhibitors and Inducers of the Major Isoforms of Cytochrome P450.

Human cytochrome P450 enzymes (CYPs) are heme-containing monooxygenases. This superfamily of drug-metabolizing enzymes is responsible for the metabolism of most drugs and other xenobiotics. The inhibition of CYPs may lead to drug–drug interactions and impair the biotransformation of drugs. CYP inducers may decrease the bioavailability and increase the clearance of drugs. Based on the freely available databases ChEMBL and PubChem, we have collected over 70,000 records containing the structures of inhibitors and inducers together with the IC50 values for the inhibitors of the five major human CYPs: 1A2, 3A4, 2D6, 2C9, and 2C19. Based on the collected data, we developed (Q)SAR models for predicting inhibitors and inducers of these CYPs using GUSAR and PASS software. The developed (Q)SAR models could be applied for assessment of the interaction of novel drug-like substances with the major human CYPs. The created (Q)SAR models demonstrated reasonable accuracy of prediction. They have been implemented in the web application P450-Analyzer that is freely available via the Internet.

In Vitro Metabolic Fate of the Synthetic Cannabinoid Receptor Agonists 2F-QMPSB and SGT-233 Including Isozyme Mapping and Carboxylesterases Activity Testing.

Quinolin-8-yl 3-(4,4-difluoropiperidine-1-sulfonyl)-4-methylbenzoate (2F-QMPSB) and 3-(4,4-difluoropiperidine-1-sulfonyl)-4-methyl-N-(2-phenylpropan-2-yl)benzamide (SGT-233) belong to a new group of synthetic cannabinoid receptor agonists containing a sulfamoyl benzoate or sulfamoyl benzamide core structure. 2F-QMPSB was identified in herbal material seized in Europe in 2018. The aims of this study were the identification of in vitro Phase I and II metabolites of 2F-QMPSB and SGT-233 to find analytical targets for toxicological screenings. Furthermore, the contribution of different monooxygenases and human carboxylesterases to Phase I metabolism was investigated. Liquid chromatography coupled to high-resolution tandem mass spectrometry was used for analysis. Ester hydrolysis was found to be an important step in the metabolism of 2F-QMPSB, which was catalyzed mainly by human carboxylesterases (hCES)1 isoforms. Additionally, nonenzymatic ester hydrolysis was observed in case of 2F-QMPSB. Notably, the carboxylic acid product derived from ester hydrolysis and metabolites thereof were only detectable in negative ionization mode. In case of SGT-233, mono- and dihydroxy metabolites were identified, as well as glucuronides. The cytochrome P450 (CYP) isozymes CYP2C8, CYP2C9, CYP2C19, CYP3A4 and CYP3A5 were found to be involved in the hydroxylation of both compounds. The results of these in vitro experiments suggest that the ester hydrolysis products of 2F-QMPSB and their glucuronides are suitable targets for toxicological screenings. In the case of SGT-233, the mono- and dihydroxy metabolites were identified as suitable screening targets. The involvement of various CYP isoforms in the metabolism of both substances reduces the likelihood of drug-drug interactions due to CYP inhibition.

Evaluation of Zuo-Gui Yin Decoction Effects on Six CYP450 Enzymes in Rats Using a Cocktail Method by UPLC-MS/MS.

Background Zuo-Gui Yin Decoction (ZGYD), a traditional Chinese prescription, is mainly used in various kinds of andrology and gynecology diseases. However, the study on the interaction of ZGYD and drugs has not been reported. Therefore, evaluating the interaction between ZGYD and metabolic enzymes is helpful to guide rational drug use. Objective This study was conducted to explore the effects of ZGYD on the activity and mRNA expressions of six Cytochrome P450 (CYP450) enzymes in rats and to provide a basis for its rational clinical use. Methods Sprague-Dawley rats were randomly divided into control, ZGYD high, medium, and low-dose group (n = 6). The concentrations of six probe substrates in plasma of rats in each group were determined by UPLC-MS/MS. In addition, RT-PCR and Western blot were used to determine the effects of ZGYD on the expression of CYP450 isoforms in the liver. Results Compared with the control group, the main pharmacokinetic parameters AUC(0-t), AUC (0~∞), of omeprazole, dextromethorphan, and midazolam in the high-dose group were significantly decreased, while the CL of these were significantly increased. The gene expressions of CYP2C11 and CYP3A1 were upregulated in the ZGYD medium, high-dose group. The protein expression of CYP2C11 was upregulated in the high-dose group, and the protein expression of CYP3A1 was upregulated in the medium, high-dose group. Conclusion The results showed that ZGYD exhibited the induction effects on CYP2C11 and CYP3A1 (CYP2C19 and CYP3A4 in humans) in rats. However, no significant change in CYP1A2, CYP2B1, CYP2C7, and CYP2D2 activities was observed. It would be useful for the safe and effective usage of ZGYD in clinic.

Simultaneous absolute protein quantification of seven cytochrome P450 isoforms in rat liver microsomes by LC-MS/MS-based isotope internal standard method.

The cytochrome P450 (CYP) enzymes play a pivotal role in drug metabolism. LC-MS/MS-based targeting technology has been applied to the analysis of CYP enzymes, promoting drug development and drug-drug interaction studies. Rat is one of the most commonly used models for drug metabolism assessment, but LC-MS/MS assay quantifying the abundance of CYP enzymes in rats is rarely reported. Herein, an accurate and stable LC-MS/MS based method was developed and validated for the simultaneous quantification of seven major rat CYP isoforms (CYP1A2, 2B1, 2C6, 2C11, 2D1, 2E1, and 3A1) in liver microsomes. The careful optimization of trypsin digestion and chromatography combined with isotope-labeled peptide as internal standard improved the efficiency and accuracy of the analysis. Highly specific surrogate peptides were obtained by a procedure including trypsin digestion for six hours and separated on a Hypersil Gold C18 column (100 × 2.1 mm, 3 μm) using gradient elution for 15 min with a mobile phase of water containing 0.1% formic acid and acetonitrile. In the method validation, linearity, matrix effect, recovery, stability, accuracy, and precision all meet the requirements. Subsequently, this method was applied to detect seven enzymes in rat liver microsomes from four different sources, and the correlation between the abundance and activity of CYP enzymes was further analyzed. The high-throughput detection method provided in this study will provide support for pertinent pharmaceutical research based on rat models.

Investigation of Radiotracer Metabolic Stability In Vitro with CYP-Overexpressing Hepatoma Cell Lines.

The characterization of novel radiotracers toward their metabolic stability is an essential part of their development. While in vitro methods such as liver microsome assays or ex vivo blood or tissue samples provide information on overall stability, little or no information is obtained on cytochrome P450 (CYP) enzyme and isoform-specific contribution to the metabolic fate of individual radiotracers. Herein, we investigated recently established CYP-overexpressing hepatoblastoma cell lines (HepG2) for their suitability to study the metabolic stability of radiotracers in general and to gain insight into CYP isoform specificity. Wildtype HepG2 and CYP1A2-, CYP2C19-, and CYP3A4-overexpressing HepG2 cells were incubated with radiotracers, and metabolic turnover was analyzed. The optimized protocol, covering cell seeding in 96-well plates and analysis of supernatant by radio thin-layer-chromatography for higher throughput, was transferred to the evaluation of three 18F-labeled celecoxib-derived cyclooxygenase-2 inhibitors (coxibs). These investigations revealed time-dependent degradation of the intact radiotracers, as well as CYP isoform- and substrate-specific differences in their metabolic profiles. HepG2 CYP2C19 proved to be the cell line showing the highest metabolic turnover for each radiotracer studied here. Comparison with human and murine liver microsome assays showed good agreement with the human metabolite profile obtained by the HepG2 cell lines. Therefore, CYP-overexpressing HepG2 cells provide a good complement for assessing the metabolic stability of radiotracers and allow the analysis of the CYP isoform-specific contribution to the overall radiotracer metabolism.

Recent Advances in Molecular Fluorescent Probes for CYP450 Sensing and Imaging

Cytochrome P450 (CYP450) is a major drug-metabolizing enzyme system mainly distributed in liver microsomes and involved in the metabolism of many endogenous substances (such as fatty acids and arachidonic acids), and exogenous compounds (such as drugs, toxicants, carcinogens, and procarcinogens). Due to the similarity in structures and catalytic functions between CYP450 isoforms, the lack of effective selective detection tools greatly limits the understanding and the research of their respective physiological roles in living organisms. Until now, several small-molecular fluorescent probes have been employed for selective detection and monitoring of CYP450s (Cytochrome P450 enzymes) in vitro or in vivo owing to the tailored properties, biodegradability, and high temporal and spatial resolution imaging in situ. In this review, we summarize the recent advances in fluorescent probes for CYP450s (including CYP1, CYP2, and CYP3 families), and we discuss and focus on their identification mechanisms, general probe design strategies, and bioimaging applications. We also highlight the potential challenges and prospects of designing new generations of fluorescent probes in CYP450 studies, which will further enhance the diversity, practicality, and clinical feasibility of research into CYP450.

Defining the Selectivity of Chemical Inhibitors Used for Cytochrome P450 Reaction Phenotyping: Overcoming Selectivity Limitations with a Six-Parameter Inhibition Curve-Fitting Approach.

The utility of chemical inhibitors in cytochrome P450 (CYP) reaction phenotyping is highly dependent on their selectivity and potency for their target CYP isoforms. In the present study, seventeen inhibitors of CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6, and 3A4/5 commonly used in reaction phenotyping were evaluated for their cross-enzyme selectivity in pooled human liver microsomes. The data were evaluated using a statistical desirability analysis to identify (1) inhibitors of superior selectivity for reaction phenotyping and (2) optimal concentrations for each. Among the inhibitors evaluated, α-naphthoflavone, furafylline, sulfaphenazole, tienilic acid, N-benzylnirvanol, and quinidine were most selective, such that their respective target enzymes were inhibited by ~95% without inhibiting any other CYP enzyme by more than 10%. Other commonly employed inhibitors, such as ketoconazole and montelukast, among others, were of insufficient selectivity to yield a concentration that could adequately inhibit their target enzymes without affecting other CYP enzymes. To overcome these shortcomings, an experimental design was developed wherein dose response data from a densely sampled multi-concentration inhibition curve are analyzed by a six-parameter inhibition curve function, allowing accounting of the inhibition of off-target CYP isoforms inhibition and more reliable determination of maximum targeted enzyme inhibition. The approach was exemplified using rosiglitazone N-demethylation, catalyzed by both CYP2C8 and 3A4, and was able to discern the off-target inhibition by ketoconazole and montelukast from the inhibition of the targeted enzyme. This methodology yields more accurate estimates of CYP contributions in reaction phenotyping. Significance Statement Isoform-selective chemical inhibitors are important tools for identifying and quantifying enzyme contributions as part of a CYP reaction phenotyping assessment for projecting drug-drug interactions. However, currently employed practices fail to adequately compensate for shortcomings in inhibitor selectivity and the resulting confounding impact on estimates of the CYP enzyme contribution to drug clearance. In this report, we describe a detailed IC50 study design with 6-parameter modeling approach that yields more accurate estimates of enzyme contribution.

Pediatric AIDS-Therapeutic Successes Built on a Foundation of Pediatric Clinical Pharmacology with Pharmacokinetic-Pharmacodynamic Modeling.

Pediatric AIDS-Therapeutic Successes Built on a Foundation of Pediatric Clinical Pharmacology with Pharmacokinetic-Pharmacodynamic Modeling.

Endoplasmic Reticulum-Targeting Near-Infrared Fluorescent Probe for CYP2J2 Activity and Its Imaging Application in Endoplasmic Reticulum Stress and Tumor

CYP2J2 as an endoplasmic reticulum (ER)-expressed vital cytochrome P450 isoform participates in the metabolism of endogenous polyunsaturated fatty acids. Its abnormal expression and function are closely related to the progress of cancer and cardiovascular diseases. Herein, an ER-targeting near-infrared (NIR) fluorescent probe ER-BnXPI was developed for monitoring CYP2J2 activity, which possessed a high selectivity and sensitivity toward CYP2J2 among various CYP450 isoforms and exhibited excellent subcellular localization for ER. Then, the CYP2J2 variation behavior under the ER stress model was imaged by ER-BnXPI in living cells and successfully used for the in vivo imaging in different tumors that well distinguished tumor tissues from para-cancerous tissues. All these findings fully demonstrated that ER-BnXPI could be used as a promising tool for exploring the physiological function of CYP2J2 and provided some novel approach for the diagnosis and therapy of CYP2J2-related vascular inflammation and cancer.

Candidate Proficiency Test Chemicals to Address Industrial Chemical Applicability Domains for in vitro Human Cytochrome P450 Enzyme Induction.

Cytochrome P450 (CYP) enzymes play a key role in the metabolism of both xenobiotics and endogenous chemicals, and the activity of some CYP isoforms are susceptible to induction and/or inhibition by certain chemicals. As CYP induction/inhibition can bring about significant alterations in the level of in vivo exposure to CYP substrates and metabolites, CYP induction/inhibition data is needed for regulatory chemical toxicity hazard assessment. On the basis of available human in vivo pharmaceutical data, a draft Organisation for Economic Co-operation and Development Test Guideline (TG) for an in vitro CYP HepaRG test method that is capable of detecting the induction of four human CYPs (CYP1A1/1A2, 2B6, and 3A4), has been developed and validated for a set of pharmaceutical proficiency chemicals. However to support TG adoption, further validation data was requested to demonstrate the ability of the test method to also accurately detect CYP induction mediated by industrial and pesticidal chemicals, together with an indication on regulatory uses of the test method. As part of “GOLIATH”, a European Union Horizon-2020 funded research project on metabolic disrupting chemical testing approaches, work is underway to generate supplemental validated data for an additional set of chemicals with sufficient diversity to allow for the approval of the guideline. Here we report on the process of proficiency chemical selection based on a targeted literature review, the selection criteria and considerations required for acceptance of proficiency chemical selection for OECD TG development (i.e. structural diversity, range of activity, relevant chemical sectors, global restrictions etc). The following 13 proposed proficiency chemicals were reviewed and selected as a suitable set for use in the additional validation experiments: tebuconazole, benfuracarb, atrazine, cypermethrin, chlorpyrifos, perfluorooctanoic acid, bisphenol A, N,N-diethyl-m-toluamide, benzo-[a]-pyrene, fludioxonil, malathion, triclosan, and caffeine. Illustrations of applications of the test method in relation to endocrine disruption and non-genotoxic carcinogenicity are provided.