Assessment of the Effects of Abrocitinib on the Pharmacokinetics of Probe Substrates of Cytochrome P450 1A2, 2B6 and 2C19 Enzymes and Hormonal Oral Contraceptives in Healthy Individuals.

BACKGROUND AND OBJECTIVE: Abrocitinib is an oral small-molecule Janus kinase (JAK)-1 inhibitor approved for the treatment of moderate-to-severe atopic dermatitis. In vitro studies indicated that abrocitinib is a weak time-dependent inhibitor of cytochrome P450 (CYP) 2C19/3A and a weak inducer of CYP1A2/2B6/2C19/3A. To assess the potential effect of abrocitinib on concomitant medications, drug-drug interaction (DDI) studies were conducted for abrocitinib with sensitive probe substrates of these CYP enzymes. The impact of abrocitinib on hormonal oral contraceptives (ethinyl estradiol and levonorgestrel), as substrates of CYP3A and important concomitant medications for female patients, was also evaluated. METHODS: Three Phase 1 DDI studies were performed to assess the impact of abrocitinib 200 mg once daily (QD) on the probe substrates of: (1) 1A2 (caffeine), 2B6 (efavirenz) and 2C19 (omeprazole) in a cocktail study; (2) 3A (midazolam); and (3) 3A (oral contraceptives). RESULTS: After multiple doses of abrocitinib 200 mg QD, there is a lack of effect on the pharmacokinetics of midazolam, efavirenz and contraceptives. Abrocitinib increased the area under the concentration time curve from 0 to infinity (AUCinf) and the maximum concentration (Cmax) of omeprazole by approximately 189 and 134%, respectively. Abrocitinib increased the AUCinf of caffeine by 40% with lack of effect on Cmax. CONCLUSIONS: Based on the study results, abrocitinib is a moderate inhibitor of CYP2C19. Caution should be exercised when using abrocitinib concomitantly with narrow therapeutic index medicines that are primarily metabolized by CYP2C19 enzyme. Abrocitinib is a mild inhibitor of CYP1A2; however, the impact is not clinically relevant, and no general dose adjustment is recommended for CYP1A2 substrates. Abrocitinib does not inhibit CYP3A or induce CYP1A2/2B6/2C19/3A and does not affect the pharmacokinetics of contraceptives. CLINICAL TRIALS REGISTRATION: ClinicalTrials.gov registration IDs: NCT03647670, NCT05067439, NCT03662516.

In vitro metabolism of the emerging contaminant 6PPD-quinone in human and rat liver microsomes: Kinetics, pathways, and mechanism.

N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine-quinone (6PPD-Q) is an ozonation product of the rubber antioxidant N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine (6PPD). 6PPD-Q has recently been detected in various environmental media, which may enter the human body via inhalation and skin contact pathways. However, the human metabolism of 6PPD-Q has remained unknown. This study investigated the in vitro Cytochrome P450-mediated metabolism of 6PPD-Q in human and rat liver microsomes (HLMs and RLMs). 6PPD-Q was significantly metabolized at lower concentrations but slowed at high concentrations. The intrinsic clearance (CLint) of 6PPD-Q was 21.10 and 18.58 µL min-1 mg-1 protein of HLMs and RLMs, respectively, suggesting low metabolic ability compared with other reported pollutants. Seven metabolites and one intermediate were identified, and metabolites were predicted immunotoxic or mutagenic toxicity. Mono- and di-oxygenation reactions were the main phase I in vitro metabolic pathways. Enzyme inhibition experiments and molecular docking techniques were further used to reveal the metabolic mechanism. CYP1A2, 3A4, and 2C19, especially CYP1A2, play critical roles in 6PPD-Q metabolism in HLMs, whereas 6PPD-Q is extensively metabolized in RLMs. Our study is the first to demonstrate the in vitro metabolic profile of 6PPD-Q in HLMs and RLMs. The results will significantly contribute to future human health management targeting the emerging pollutant 6PPD-Q.

Water Exchange from the Buried Binding Sites of Cytochrome P450 Enzymes 1A2, 2D6, and 3A4 Correlates with Conformational Fluctuations.

Human cytochrome P450 enzymes (CYPs) are critical for the metabolism of small-molecule pharmaceuticals (drugs). As such, the prediction of drug metabolism by and drug inhibition of CYP activity is an important component of the drug discovery and design process. Relative to the availability of a wide range of experimental atomic-resolution CYP structures, the development of structure-based CYP activity models has been limited. To better characterize the role of CYP conformational fluctuations in CYP activity, we perform multiple microsecond-scale all-atom explicit-solvent molecular dynamics (MD) simulations on three CYP isoforms, 1A2, 2D6, and 3A4, which together account for the majority of CYP-mediated drug metabolism. The MD simulations employ a variety of positional restraints, ranging from keeping all CYP atoms close to their experimentally determined coordinates to allowing full flexibility. We find that, with full flexibility, large fluctuations in the CYP binding sites correlate with efficient water exchange from these buried binding sites. This is especially true for 1A2, which, when restrained to its crystallographic conformation, is unable to exchange water between the binding site and bulk solvent. These findings imply that, in addition to crystal structures, a representative ensemble of conformational states ought to be included when developing structure-based CYP activity models.

Habitual coffee consumption and risk of dementia in older persons: modulation by CYP1A2 polymorphism.

Higher coffee consumption has been associated with reduced dementia risk, yet with inconsistencies across studies. CYP1A2 polymorphisms, which affects caffeine metabolism, may modulate the association between coffee and the risk of dementia and Alzheimer's disease (AD). We included 5964 participants of the Three-City Study (mean age 74 years-old), free of dementia at baseline when they reported their daily coffee consumption, with available genome-wide genotyping and followed for dementia over a median of 9.0 (range 0.8-18.7) years. In Cox proportional-hazards models, the relationship between coffee consumption and dementia risk was modified by CYP1A2 polymorphism at rs762551 (p for interaction = 0.034). In multivariable-adjusted models, coffee intake was linearly associated with a decreased risk of dementia among carriers of the C allele only ("slower caffeine metabolizers"; HR for 1-cup increased [95% CI] 0.90 [0.83-0.97]), while in non-carriers ("faster caffeine metabolizers"), there was no significant association but a J-shaped trend toward a decrease in dementia risk up to 3 cups/day and increased risk beyond. Thus, compared to null intake, drinking ≥ 4 cups of coffee daily was associated with a reduced dementia risk in slower but not faster metabolizers (HR [95% CI] for ≥ 4 vs. 0 cup/day = 0.45 [0.25-0.80] and 1.32 [0.89-1.96], respectively). Results were similar when studying AD and another CYP1A2 candidate polymorphism (rs2472304), but no interaction was found with CYP1A2 rs2472297 or rs2470893. In this cohort, a linear association of coffee intake to lower dementia risk was apparent only among carriers of CYP1A2 polymorphisms predisposing to slower caffeine metabolism.

Ciprofol is primarily glucuronidated by UGT1A9 and predicted not to cause drug-drug interactions with typical substrates of CYP1A2, CYP2B6, and CYP2C19.

Ciprofol is a novel intravenous anesthetic agent. Its major glucuronide metabolite, M4, is found in plasma and urine. However, the specific isoforms of UDP-glucuronosyltransferases (UGTs) that metabolize ciprofol to M4 remain unknown. This study systematically characterized UGTs that contribute to the formation of M4 using human liver microsomes (HLM), human intestinal microsomes (HIM), and human recombinant UGTs. The inhibitory potential of ciprofol and M4 against major human UGTs and cytochrome P450 enzymes (P450s) was also explored. In vitro-in vivo extrapolation (IVIVE) and physiologically-based pharmacokinetic (PBPK) simulations were performed to predict potential in vivo drug-drug interactions (DDIs) caused by ciprofol. Glucuronidation of ciprofol followed Michaelis-Menten kinetics in both HLM and HIM with apparent Km values of 345 and 412 µM, Vmax values of 2214 and 444 nmol min-1·mg protein-1, respectively. The in vitro intrinsic clearances (CLint = Vmax/Km) for ciprofol glucuronidation by HLM and HIM were 6.4 and 1.1 µL min-1·mg protein-1, respectively. Human recombinant UGT studies revealed that UGT1A9 is the predominant isoform mediating M4 formation, followed by UGT1A7, with UGT1A8 playing a minor role. Ciprofol competitively inhibited CYP1A2 (Ki = 12 µM) and CYP2B6 (Ki = 4.7 µM), and noncompetitively inhibited CYP2C19 (Ki = 29 µM). No time-dependent inhibition by ciprofol was noted for CYP1A2, CYP2B6, or CYP2C19. In contrast, M4 showed limited or no inhibitory effects against selected P450s. Neither ciprofol nor M4 inhibited UGTs significantly. Initial IVIVE suggested potential ciprofol-mediated inhibition of CYP1A2, CYP2B6, and CYP2C19 inhibition in vivo. However, PBPK simulations showed no significant effect on phenacetin, bupropion, and S-mephenytoin exposure or peak plasma concentration. Our findings are pertinent for future DDI studies of ciprofol as either a perpetrator or victim drug.

Effect of lemborexant on pharmacokinetics of clozapine: A potential drug-drug interaction mediated by time-dependent inhibition of CYP3A4.

Clozapine (CLZ) is extensively used for treatment-resistant schizophrenia (TRS) with caution to avoid serious adverse events such as agranulocytosis and drug-drug interactions (DDIs). In the current report, we present a case of a 35-year-old male non-smoking TRS patient whose steady-state plasma trough concentrations (Ctrough ) of CLZ and its active metabolite, N-desmethylclozapine (NDMC), were significantly increased after initiating oral administration of lemborexant (LEM), a dual orexin receptor antagonist, for the treatment of insomnia. The patient experienced oversedation with sleepiness and fatigue while maintaining high levels of Ctrough of CLZ. The increased concentrations of CLZ returned to normal ranges after the discontinuation of LEM dosing, implying a pharmacokinetic DDI between CLZ and LEM. To gain insight into possible mechanisms, we performed in vitro assays of CYP1A2- and CYP3A4-mediated CLZ metabolism by measuring the formations of NDMC and clozapine N-oxide (CNO). In accordance with previous studies, the incubation of CLZ with each enzyme resulted in the production of both metabolites. LEM had only a weak inhibitory effect on CYP1A2- and CYP3A4-mediated CLZ metabolism. However, the preincubation of LEM with CYP3A4 in the presence of NADPH showed a significant enhancement of inhibitory effects on CLZ metabolism with IC50 values for the formations of CNO and NDMC of 2.8 µM and 4.1 µM, respectively, suggesting that LEM exerts as a potent time-dependent inhibitor for CYP3A4. Taken together, the results of the current study indicate that co-medication of CLZ with LEM may lead to increase in exposure to CLZ and risks of CLZ-related adverse events.

Multi-Fusarium mycotoxin exposure activates Nrf2 and Ahr pathway in the liver of laying hens.

This study investigates gene expression changes in laying hens exposed to trichothecene mycotoxins, known to induce oxidative stress and affect xenobiotic transformation and antioxidants. A 3-day feeding trial tested low and high doses of T-2/HT-2 toxin, DON/3-AcDON/15-AcDON, and FB1 in hen feed. Results showed increased expression of AHR, AHRR, HSP90, and CYP1A2 genes on days 2 and 3, suggesting a response to mycotoxin exposure. High doses down-regulated CYP1A2, AHR, and AHRR on day 1. KEAP1 expression decreased on day 1 but increased dose-dependently on days 2 and 3. NRF2 was up-regulated by low and down-regulated by high doses on day 1, then increased on days 2 and 3. Antioxidant-related genes (GPX3, GPX4, GSS, GSR) showed dose-dependent responses. Low doses up-regulated GPX3 and GPX4 throughout, while high doses up-regulated GPX3 on days 2 and 3 and GPX4 on day 3. GSS was up-regulated on day 3. Results indicate that toxic metabolites formed by phase I biotransformation rapidly induce ROS formation at low doses through the AHR/Hsp90/CYP1A2 pathway at the gene expression level, but at high levels, ROS-induced oxidative stress manifests later. Study showed simultaneous activation of redox-sensitive pathways: aryl hydrocarbon receptor (Ahr) and nuclear factor erythroid-derived 2-like 2 (Nrf2) by multi-mycotoxin exposure.

Prenatal exposure to polycyclic aromatic hydrocarbons (PAHs) augments neonatal hyperoxic lung injury: Role of cytochrome P450 (CYP)1A1, 1A2, and 1B1.

Pregnant women exposed to polycyclic aromatic hydrocarbons (PAHs) are at increased risk for premature delivery. Premature infants often require supplemental oxygen, a known risk factor for bronchopulmonary dysplasia (BPD). Cytochrome P450 (CYP) enzymes have been implicated in hyperoxic lung injury. We hypothesize that prenatal PAH exposure exacerbates oxygen-mediated lung injury in neonatal mice, and that this effect is differentially altered in mice lacking the gene for (Cyp)1a1, 1a2, or 1b1. Timed pregnant wild type (WT) (C57BL/6J) mice were orally administered a PAH mixture of benzo[a]pyrene (BP) and benzo[b]fluoranthene (BbF) or the vehicle corn oil (CO) once daily on gestational days 16-19, and the dose response on postnatal lung injury was examined. In addition, timed pregnant mice with one of four genotypes, WT, Cyp1a1-null, Cyp1a2-null, and Cyp1b1-null, were treated orally with CO or PAH on gestational days 16-19 and exposed to hyperoxia or room air for 14 days. Lung injury was assessed on PND15 by radial alveolar count (RAC) and mean linear intercept (MLI) Gene expression of DNA repair genes in lung and liver were measured. Results showed that neonatal hyperoxic lung injury is augmented by prenatal PAH exposure in a dose-dependent manner. This effect was differentially altered in the Cyp-null mice, with Cyp1a2-null showing the greatest extent of lung injury. We concluded that newborn mice exposed to PAH in utero had more significant lung injury in response to hyperoxia than non-PAH exposed pups, and that CYP1A1 and CYP1A2 are protective against lung injury while CYP1B1 augments lung injury.

Dihydrotanshinone I-Induced CYP1 Enzyme Inhibition and Alteration of Estradiol Metabolism.

Dihydrotanshinone I (DHTI) is a pharmacologically active component occurring in the roots of the herbal medicine Salvia miltiorrhiza Bunge. This study investigated DHTI-induced inhibition of CYP1A1, CYP1A2, and CYP1B1 with the aim to determine the potential effects of DHTI on the bioactivation of estradiol (E2), possibly related to preventive/therapeutic strategy for E2-associated breast cancer. Ethoxyresorufin as a specific substrate for CYP1s was incubated with human recombinant CYP1A1, CYP1A2, or CYP1B1 in the presence of DHTI at various concentrations. Enzymatic inhibition and kinetic behaviors were examined by monitoring the formation of the corresponding product. Molecular docking was further conducted to define the interactions between DHTI and the three CYP1s. The same method and procedure were employed to examine the DHTI-induced alteration of E2 metabolism. DHTI showed significant inhibition of ethoxyresorufin O-deethylation activity catalyzed by CYP1A1, CYP1A2 and CYP1B1 in a concentration-dependent manner (IC50 = 0.56, 0.44, and 0.11 µM, respectively). Kinetic analysis showed that DHTI acted as a competitive type of inhibitor of CYP1A1 and CYP1B1, whereas it noncompetitively inhibited CYP1A2. The observed enzyme inhibition was independent of NADPH and time. Molecular docking analysis revealed hydrogen bonding interactions between DHTI and Asp-326 of CYP1B1. Moreover, DHTI displayed preferential activity to inhibit 4-hydroxylation of E2 (a genotoxic pathway) mediated by CYP1B1. Exposure to DHTI could reduce the risk of genotoxicity induced by E2. SIGNIFICANCE STATEMENT: CYP1A1, CYP1A2, and CYP1B1 enzymes are involved in the conversion of estradiol (E2) into 2-hydroxyestradiol (2-OHE2) and 4-hydroxyestradiol (4-OHE2) through oxidation. 2-OHE2 is negatively correlated with breast cancer risk, and 4-OHE2 may be a significant initiator and promoter of breast cancer. The present study revealed that dihydrotanshinone I (DHTI) competitively inhibits CYP1A1/CYP1B1 and noncompetitively inhibits CYP1A2. DHTI exhibits a preference for inhibiting the genotoxicity associated with E2 4-hydroxylation pathway mediated by CYP1B1, potentially reducing the risk of 4-OHE2-induced genotoxicity.

CYP1A inhibitors: Recent progress, current challenges, and future perspectives.

Mammalian cytochrome P450 1A (CYP1A) are key phase I xenobiotic-metabolizing enzymes that play a distinctive role in metabolic activation or metabolic clearance of a variety of procarcinogens, drugs, and endogenous substances. Human CYP1A subfamily contains two members (hCYP1A1 and hCYP1A2), which are known to catalyze the oxidative activation of some environmental procarcinogens into carcinogenic species. Increasing evidence has demonstrated that CYP1A inhibitor therapies are promising strategies for cancer chemoprevention or overcoming CYP1A-associated drug toxicity and resistance. Herein, we reviewed recent advances in the discovery and characterization of hCYP1A inhibitors, from the discovery approaches to structural features and biomedical applications of hCYP1A inhibitors. The inhibition potentials, inhibition modes, and inhibition constants of all reported hCYP1A inhibitors are comprehensively summarized. Meanwhile, the structural features and structure-activity relationships of different classes of hCYP1A1 and hCYP1A2 inhibitors are analyzed and discussed in depth. Furthermore, the major challenges and future directions for this field are presented and highlighted. Collectively, the information and knowledge presented here will strongly facilitate the researchers to discover and develop more efficacious CYP1A inhibitors for specific purposes, such as chemo-preventive agents or as tool molecules in hCYP1A-related fundamental studies.

Evaluation of Supervised Machine Learning Algorithms and Computational Structural Validation of Single Nucleotide Polymorphisms Related to Acute Liver Injury with Paracetamol.

AIMS: To identify single nucleotide polymorphisms (SNPs) of paracetamol-metabolizing enzymes that can predict acute liver injury. BACKGROUND: Paracetamol is a commonly administered analgesic/antipyretic in critically ill and chronic renal failure patients and several SNPs influence the therapeutic and toxic effects. OBJECTIVE: To evaluate the role of machine learning algorithms (MLAs) and bioinformatics tools to delineate the predictor SNPs as well as to understand their molecular dynamics. METHODS: A cross-sectional study was undertaken by recruiting critically ill patients with chronic renal failure and administering intravenous paracetamol as a standard of care. Serum concentrations of paracetamol and the principal metabolites were estimated. Following SNPs were evaluated: CYP2E1*2, CYP2E1_-1295G>C, CYP2D6*10, CYP3A4*1B, CYP3A4*2, CYP1A2*1K, CYP1A2*6, CYP3A4*3, and CYP3A5*7. MLAs were used to identify the predictor genetic variable for acute liver failure. Bioinformatics tools such as Predict SNP2 and molecular docking (MD) were undertaken to evaluate the impact of the above SNPs with binding affinity to paracetamol. RESULTS: CYP2E1*2 and CYP1A2*1C genotypes were identified by MLAs to significantly predict hepatotoxicity. The predictSNP2 revealed that CYP1A2*3 was highly deleterious in all the tools. MD revealed binding energy of -5.5 Kcal/mol, -6.9 Kcal/mol, and -6.8 Kcal/mol for CYP1A2, CYP1A2*3, and CYP1A2*6 against paracetamol. MD simulations revealed that CYP1A2*3 and CYP1A2*6 missense variants in CYP1A2 affect the binding ability with paracetamol. In-silico techniques found that CYP1A2*2 and CYP1A2*6 are highly harmful. MD simulations revealed CYP3A4*2 (A>G) had decreased binding energy with paracetamol than CYP3A4, and CYP3A4*2(A>T) and CYP3A4*3 both have greater binding energy with paracetamol. CONCLUSION: Polymorphisms in CYP2E1, CYP1A2, CYP3A4, and CYP3A5 significantly influence paracetamol's clinical outcomes or binding affinity. Robust clinical studies are needed to identify these polymorphisms' clinical impact on the pharmacokinetics or pharmacodynamics of paracetamol.

Cytochrome P450 1A2 and 2C enzymes autoinduced by omeprazole in dog hepatocytes and human HepaRG and HepaSH cells are involved in omeprazole 5-hydroxylation and sulfoxidation.

The induction assay for the cytochromes P450 (P450s) is an important tool in drug discovery and development. The inductions of dog P450 1A2 and 3A12 by omeprazole and rifampicin were functionally characterised in dog hepatocytes and were compared with induction in human HepaRG and HepaSH cells.P450 1A2-dependent ethoxyresorufin O-deethylation was induced by R,S-omeprazole and P450 3 A-dependent midazolam 1'-hydroxylation was induced by rifampicin, and both reactions were significantly enhanced in cultured dog hepatocytes and human HepaRG and HepaSH cells.Recombinant dog P450 1A2 exhibited activities towards R- and S-omeprazole 5-hydroxylation with low Km values of 23-28 µM, whereas dog P450 2C21 and 3A12 efficiently mediated S-omeprazole 5-hydroxylation and sulfoxidation, respectively, with high Vmax values of 12-17 min-1.Although omeprazole 5-hydroxylation by human P450 2C19 (and sulfoxidation by P450 3A4) in human HepaSH cells were slightly (∼2-fold) induced by R,S-omeprazole, dog P450 1A2 was autoinduced by omeprazole in dog hepatocytes and showed enhanced R-omeprazole 5-hydroxylation activity (∼5-fold).These results indicate that omeprazole can be an autoinducer of P450 1A2 in hepatocytes, and this enzyme was found to be involved in omeprazole 5-hydroxylation and sulfoxidation in dog hepatocytes and human HepaRG and HepaSH cells.

CYP1A2 expression rather than genotype is associated with olanzapine concentration in psychiatric patients.

Olanzapine is a commonly prescribed atypical antipsychotic agent for treatment of patients with schizophrenia and bipolar disorders. Previous in vitro studies using human liver microsomes identified CYP1A2 and CYP2D6 enzymes being responsible for CYP-mediated metabolism of olanzapine. The present work focused on the impact of CYP1A2 and CYP2D6 genetic polymorphisms as well as of CYP1A2 metabolizing capacity influenced by non-genetic factors (sex, age, smoking) on olanzapine blood concentration in patients with psychiatric disorders (N = 139). CYP2D6 genotype-based phenotype appeared to have negligible contribution to olanzapine metabolism, whereas a dominant role of CYP1A2 in olanzapine exposure was confirmed. However, CYP1A2 expression rather than CYP1A2 genetic variability was demonstrated to be associated with olanzapine concentration in patients. Significant contribution of - 163C > A (rs762551), the most common SNP (single nucleotide polymorphism) in CYP1A2 gene, to enhanced inducibility was confirmed by an increase in CYP1A2 mRNA expression in smokers carrying - 163A, and smoking was found to have appreciable impact on olanzapine concentration normalized by the dose/bodyweight. Furthermore, patients' olanzapine exposure was in strong association with CYP1A2 expression; therefore, assaying CYP1A2 mRNA level in leukocytes can be an appropriate tool for the estimation of patients' olanzapine metabolizing capacity and may be relevant in optimizing olanzapine dosage.

Melatonin Activation by Human Cytochrome P450 Enzymes: A Comparison between Different Isozymes.

Cytochrome P450 enzymes in the human body play a pivotal role in both the biosynthesis and the degradation of the hormone melatonin. Melatonin plays a key role in circadian rhythms in the body, but its concentration is also linked to mood fluctuations as well as emotional well-being. In the present study, we present a computational analysis of the binding and activation of melatonin by various P450 isozymes that are known to yield different products and product distributions. In particular, the P450 isozymes 1A1, 1A2, and 1B1 generally react with melatonin to provide dominant aromatic hydroxylation at the C6-position, whereas the P450 2C19 isozyme mostly provides O-demethylation products. To gain insight into the origin of these product distributions of the P450 isozymes, we performed a comprehensive computational study of P450 2C19 isozymes and compared our work with previous studies on alternative isozymes. The work covers molecular mechanics, molecular dynamics and quantum mechanics approaches. Our work highlights major differences in the size and shape of the substrate binding pocket amongst the different P450 isozymes. Consequently, substrate binding and positioning in the active site varies substantially within the P450 isozymes. Thus, in P450 2C19, the substrate is oriented with its methoxy group pointing towards the heme, and therefore reacts favorably through hydrogen atom abstraction, leading to the production of O-demethylation products. On the other hand, the substrate-binding pockets in P450 1A1, 1A2, and 1B1 are tighter, direct the methoxy group away from the heme, and consequently activate an alternative site and lead to aromatic hydroxylation instead.

Will ChatGPT3 Substitute for us as Clozapine Experts?

BACKGROUND: ChatGPT3 is a new artificial intelligence program released on February 13, 2023. METHOD: The authors tested ChatGPT3 on February 18, 2023, and repeated the test a week later. They used their expertise on the effects of ethnic ancestry in the stratification of clozapine dosing and the new idea that they published in March 2022 that African-Americans need higher clozapine doses because they have higher clozapine clearance. RESULTS: In the first interaction on February 18, ChatGPT3 provided reasonable and very up-to-date information, which included a comment that patients of African ancestry have higher clozapine metabolism. The other 4 interactions became progressively more concerning as we asked ChatGPT3 to provide references to justify the latter statement. ChatGPT3 provided non-existent "references" using articles from real journals, with real authors, false PubMed identifiers, and false titles. Moreover, ChatGPT3 said that the first author wrote in 2003 that African-Americans had higher CYP1A2 activity when that did not happen until 2022. One week later, the second author repeated the same set of questions. This time ChatGPT3 described the opposite, that African-Americans have "lower" CYP1A2 activity and "slower" metabolism. ChatGPT3 provided another set of articles to justify the information; some were real but did not comment on clozapine metabolism in African-Americans while others did not exist. CONCLUSIONS: ChatGPT3 provided a mixture of truth, twisted reality, and non-existent "facts." Within one week it defended opposite positions regarding a clinically relevant issue such as using higher or lower clozapine doses in African-Americans.

High-throughput screening of the Saccharomyces cerevisiae genome for 2-amino-3-methylimidazo [4,5-f] quinoline resistance identifies colon cancer-associated genes.

Heterocyclic aromatic amines (HAAs) are potent carcinogenic agents found in charred meats and cigarette smoke. However, few eukaryotic resistance genes have been identified. We used Saccharomyces cerevisiae (budding yeast) to identify genes that confer resistance to 2-amino-3-methylimidazo[4,5-f] quinoline (IQ). CYP1A2 and NAT2 activate IQ to become a mutagenic nitrenium compound. Deletion libraries expressing human CYP1A2 and NAT2 or no human genes were exposed to either 400 or 800 µM IQ for 5 or 10 generations. DNA barcodes were sequenced using the Illumina HiSeq 2500 platform and statistical significance was determined for exactly matched barcodes. We identified 424 ORFs, including 337 genes of known function, in duplicate screens of the "humanized" collection for IQ resistance; resistance was further validated for a select group of 51 genes by growth curves, competitive growth, or trypan blue assays. Screens of the library not expressing human genes identified 143 ORFs conferring resistance to IQ per se. Ribosomal protein and protein modification genes were identified as IQ resistance genes in both the original and "humanized" libraries, while nitrogen metabolism, DNA repair, and growth control genes were also prominent in the "humanized" library. Protein complexes identified included the casein kinase 2 (CK2) and histone chaperone (HIR) complex. Among DNA Repair and checkpoint genes, we identified those that function in postreplication repair (RAD18, UBC13, REV7), base excision repair (NTG1), and checkpoint signaling (CHK1, PSY2). These studies underscore the role of ribosomal protein genes in conferring IQ resistance, and illuminate DNA repair pathways for conferring resistance to activated IQ.

GTP1 metabolic stability assessment: A study of the tau PET tracer [18F]GTP1.

Tau PET imaging using the tau specific PET tracer [18F]GTP1 has been and is part of therapeutic trials in Alzheimer's disease to monitor the accumulation of tau aggregates in the brain. Herein, we examined the metabolic processes of GTP1 and assessed the influence of smoking on its metabolism through in vitro assays. The tracer metabolic profile was assessed by incubating GTP1 with human liver microsomes (HLM) and human hepatocytes. Since smoking strongly stimulates the CYP1A2 enzyme activity, we incubated GTP1 with recombinant CYP1A2 to evaluate the role of the enzyme in tracer metabolism. It was found that GTP1 could form up to eleven oxidative metabolites with higher polarity than the parent. Only a small amount (2.6 % at 60 min) of a defluorinated metabolite was detected in HLM and human hepatocytes incubations highlighting the stability of GTP1 with respect to enzymatic defluorination. Moreover, the major GTP1 metabolites were not the product of CYP1A2 activity suggesting that smoking may not impact in vivo tracer metabolism and subsequently GTP1 brain kinetics.

Effect of Saccharolactone on CYP-mediated Metabolism of Xenobiotics.

BACKGROUND: Saccharolactone is used as a ß-glucuronidase inhibitor in in vitro microsomal and recombinant uridine diphosphoglucuronosyl transferases (rUGTs) incubations to enhance glucuronide pathway and, thereby, formation of glucuronide metabolites. We investigated its effect on CYP mediated metabolism of drugs (compound-174, phenacetin and quinidine) using human liver microsomes (HLM) supplemented with Phase-1 and Phase-2 co-factors. METHODS: Compounds were incubated in HLM supplemented with co-factors to assess Phase-1 (NADPH) and Phase-2 (NADPH, alamethicin, saccharolactone and UDPGA) metabolism. CYP phenotype assay for compound-174 was conducted in HLM (± 1-ABT) and human recombinant CYP isoforms. CYP inhibition profile of saccharolactone was also generated in HLM. RESULTS: The metabolism of compound-174, phenacetin and quinidine in HLM significantly decreased in reactions containing additional components like alamethicin, saccharolactone and UDPGA and indicated that the addition of saccharolactone inhibited the metabolism. Phenacetin and quinidine are known substrates of CYP1A2 and CYP3A4 isoforms. The metabolism of compound- 174 was significantly inhibited in the presence of 1-ABT in HLM, and CYP3A4 and CYP2C8 isoforms were found to be the predominant isoforms responsible for its metabolism. Further evaluation of CYP inhibition in HLM indicated saccharolactone to be a strong inhibitor of CYP1A2, 2D6, 3A4 and 2C8 isoforms with IC50 values of less than 4 mM. CONCLUSION: The findings indicated that saccharolactone being a strong inhibitor of CYP1A2, 2D6, 3A4 and 2C8 isoforms (IC50 < 4 mM), resulted in significant inhibition of the metabolism of compound-174, phenacetin and quinidine in HLM and caution should be exercised in using it with proper titration of the concentrations.

Evaluation of the impact of vindoline, an active components of Catharanthus roseus, on rat hepatic cytochrome P450 enzymes by using a cocktail of probe drugs.

The objection of this study was to investigate the effects of vindoline(VDL) on the cytochrome P450 (CYP 450) isoforms (CYP1A2, 2B, 2C11, 2D1 and 3A) in rats. Firstly, the rats were randomly divided into VDL pretreatment group and blank group, each group had six rats. VDL pretreatment group was administrated VDL (20 mg·kg-1) by oral gavage for fifteen days consecutively, and the equivalent CMC-Na solution without VDL was given to the blank group by gavage. Secondly, a cocktail of caffeine, bupropion, diclofenac, dextromethorphan and midazolam was then administered on the sixteenth day. Finally, blood samples were collected at the specified time point, and the plasma concentration of the probe drug was determined by UHPLC-QTOF-MS/MS. The effects of VDL on the activity of these CYP enzymes in rats were evaluated by pharmacokinetic parameters. VDL pretreatment group compared with the blank group, accelerated the metabolism of diclofenac, and weakened the metabolism of caffeine. These results suggested that VDL could induce the activity of CYP2C11, and inhibits the activity of CYP1A2, but had no significant effects on CYP2B, CYP2D1 and CYP3A. The results in this study can provide beneficial information for the later clinical application of VDL.

Phase I study to assess the effect of adavosertib (AZD1775) on the pharmacokinetics of substrates of CYP1A2, CYP2C19, and CYP3A in patients with advanced solid tumors.

PURPOSE: Adavosertib may alter exposure to substrates of the cytochrome P450 (CYP) family of enzymes. This study assessed its effect on the pharmacokinetics of a cocktail of probe substrates for CYP3A (midazolam), CYP2C19 (omeprazole), and CYP1A2 (caffeine). METHODS: Period 1: patients with locally advanced or metastatic solid tumors received 'cocktail': caffeine 200 mg, omeprazole 20 mg, and midazolam 2 mg (single dose); period 2: after 7- to 14-day washout, patients received adavosertib 225 mg twice daily on days 1-3 (five doses), with cocktail on day 3. After cocktail alone or in combination with adavosertib administration, 24-h pharmacokinetic sampling occurred for probe substrates and their respective metabolites paraxanthine, 5-hydroxyomeprazole (5-HO), and 1'-hydroxymidazolam (1'-HM). Safety was assessed throughout. RESULTS: Of 33 patients (median age 60.0 years, range 41-83) receiving cocktail, 30 received adavosertib. Adavosertib co-administration increased caffeine, omeprazole, and midazolam exposure by 49%, 80%, and 55% (AUC0-12), respectively; AUC0-t increased by 61%, 98%, and 55%. Maximum plasma drug concentration (Cmax) increased by 4%, 46%, and 39%. Adavosertib co-administration increased 5-HO and 1'-HM exposure by 43% and 54% (AUC0-12) and 49% and 58% (AUC0-t), respectively; paraxanthine exposure was unchanged. Adavosertib co-administration decreased Cmax for paraxanthine and 5-HO by 19% and 7%; Cmax increased by 33% for 1'-HM. After receiving adavosertib, 19 (63%) patients had treatment-related adverse events (six [20%] grade ≥ 3). CONCLUSION: Adavosertib (225 mg bid) is a weak inhibitor of CYP1A2, CYP2C19, and CYP3A. CLINICALTRIALS: GOV: NCT03333824.