P450 Isozymes Research Articles

Characterization of mavacamten pharmacokinetics in patients with hypertrophic cardiomyopathy to inform dose titration.

Mavacamten is a selective, allosteric, reversible cardiac myosin inhibitor that has been developed for the treatment of adults with symptomatic obstructive hypertrophic cardiomyopathy (HCM). A population pharmacokinetic (PopPK) model was developed to characterize mavacamten pharmacokinetics (PK) and the variation in mavacamten exposure associated with intrinsic and extrinsic factors. Data from 12 clinical studies (phases 1, 2, and 3) were used. Evaluable participants were those who had at least one mavacamten concentration measurement with associated sampling time and dosing information. The base model included key covariates: body weight, cytochrome P450 isozyme 2C19 (CYP2C19) phenotype with respect to PK, and formulation. The final model was generated using stepwise covariate testing and refinement processes. Simulations were performed to evaluate PK: apparent clearance (CL/F); apparent central and peripheral volumes of distribution; and steady-state average, trough, and maximum concentrations. Overall, 9244 measurable PK observations from 497 participants were included. A two-compartment model structure was selected. After stepwise covariate model building and refinement, additional covariates included were: specified mavacamten dose, omeprazole or esomeprazole administration, health/disease status, estimated glomerular filtration rate, fed status, and sex. The final PopPK model accurately characterized mavacamten concentrations. At any given dose, CYP2C19 phenotype was the most influential covariate on exposure parameters (e.g., median CL/F was reduced by 72% in CYP2C19:poor metabolizers compared with the reference participant [CYP2C19:normal metabolizer]). CL/F was also approximately 16% higher in women than in men but lower in participants receiving concomitant omeprazole or esomeprazole (by 33% and 42%, respectively) than in participants not receiving such concomitant therapy.

Implementation of CYP2C19 and CYP2D6 genotyping to guide antidepressant use in a large rural health system.

We describe the implementation and ongoing maintenance of CYP2C19 and CYP2D6 focused pharmacogenetic (PGx) testing to guide antidepressant and antianxiety medication prescriptions in a large rural, nonprofit health system. Depression and anxiety are common psychiatric conditions. Sanford Health implemented PGx testing for metabolism of cytochrome P450 (CYP) isozymes 2C19 and 2D6 in 2014 to inform prescribing for multiple medications, including antidepressant and antianxiety therapies. As guidelines, genotype to phenotype translation, panel offerings, and other resources are updated, we adapt our approach. We make educational and informational materials available to providers and patients. Pharmacogenomic clinical pharmacists review PGx results with discrete values and provide guidance documentation in the electronic medical record. A robust clinical decision support system is in place to provide interruptive alerts, noninterruptive alerts, and genomic indicators. A referral-based interdisciplinary clinic is also available to provide in-depth education to patients regarding PGx results and implications. Additionally, partnering with our health plan has expanded access to PGx testing for patients with anxiety or depression. The implementation and maintenance of Sanford Health's PGx program to guide antidepressant and antianxiety medication use continues to evolve and requires a multipronged approach relying on both human and informatics-based resources.

Mechanism of the Oxidative Ring-Closure Reaction during Gliotoxin Biosynthesis by Cytochrome P450 GliF.

During gliotoxin biosynthesis in fungi, the cytochrome P450 GliF enzyme catalyzes an unusual C-N ring-closure step while also an aromatic ring is hydroxylated in the same reaction cycle, which may have relevance to drug synthesis reactions in biotechnology. However, as the details of the reaction mechanism are still controversial, no applications have been developed yet. To resolve the mechanism of gliotoxin biosynthesis and gain insight into the steps leading to ring-closure, we ran a combination of molecular dynamics and density functional theory calculations on the structure and reactivity of P450 GliF and tested a range of possible reaction mechanisms, pathways and models. The calculations show that, rather than hydrogen atom transfer from the substrate to Compound I, an initial proton transfer transition state is followed by a fast electron transfer en route to the radical intermediate, and hence a non-synchronous hydrogen atom abstraction takes place. The radical intermediate then reacts by OH rebound to the aromatic ring to form a biradical in the substrate that, through ring-closure between the radical centers, gives gliotoxin products. Interestingly, the structure and energetics of the reaction mechanisms appear little affected by the addition of polar groups to the model and hence we predict that the reaction can be catalyzed by other P450 isozymes that also bind the same substrate. Alternative pathways, such as a pathway starting with an electrophilic attack on the arene to form an epoxide, are high in energy and are ruled out.

Effect of CYP2D6 genetic variation on patient-reported symptom improvement and side effects among children and adolescents treated with amphetamines.

Amphetamine-based medications are recommended as a first-line pharmacotherapy for the treatment of attention-deficit/hyperactivity disorder in children and adolescents. However, the efficacy and tolerability of these medications vary across individuals, which could be related to interindividual differences in amphetamine metabolism. This study examined if genotype-predicted phenotypes of the cytochrome P450 isozyme CYP2D6 were associated with self-reported side effects and symptom improvement in youth treated with amphetamines. Two hundred fourteen participants aged 6-24 who had a history of past or current amphetamine treatment were enrolled from Western Canada. Amphetamine dose and duration information was collected from the participants along with questions regarding adherence, concomitant medications, symptom improvement and side effects. DNA was extracted from saliva samples and genotyped for CYP2D6 . Binomial logistic regression models were used to determine the effect of CYP2D6 metabolizer phenotype with and without correction for phenoconversion on self-reported symptom improvement and side effects. Genotype-predicted CYP2D6 poor metabolizers had significantly higher odds of reporting symptom improvement when compared to intermediate metabolizers (OR = 3.67, 95% CI = 1.15-11.7, P = 0.029) after correction for phenoconversion and adjusting for sex, age, dose, duration, and adherence. There was no association between CYP2D6 metabolizer phenotype and self-reported side effects. Our findings indicate that phenoconverted and genotype-predicted CYP2D6 poor metabolizer phenotype is significantly associated with higher odds of symptom improvement in children and adolescents treated with amphetamine. If replicated, these results could inform the development of future dosing guidelines for amphetamine treatment in children and adolescents.

Implementation of clopidogrel pharmacogenetic clinical decision support for a preemptive return of results program.

To describe our experiences implementing and iterating CYP2C19 genotype-guided clopidogrel pharmacogenetic clinical decision support (CDS) tools over time in the setting of a large health system-wide, preemptive pharmacogenomics program. Clopidogrel-treated patients who are genetically predicted cytochrome P450 isozyme 2C19 (CYP2C19) intermediate or poor metabolizers have an increased risk of atherothrombotic events, some of which can be life-threatening. The Clinical Pharmacogenetics Implementation Consortium provides guidance for the use of clopidogrel based on CYP2C19 genotype in patients with cardiovascular and cerebrovascular diseases. Our multidisciplinary team implemented an automated, interruptive alert that fires when clopidogrel is ordered or refilled for biobank participants with structured CYP2C19 intermediate or poor metabolizer genomic indicators in the electronic health record. The implementation began with a narrow cardiovascular indication and setting and was then scaled in 4 primary dimensions: (1) clinical indication; (2) availability across health-system locations; (3) care venue (e.g., inpatient vs outpatient); and (4) provider groups (eg, cardiology and neurology). We iterated our approach over time based on evolving clinical evidence and proactive strategies to optimize CDS maintenance and sustainability. A key facilitator of expansion was socialization of the broader pharmacogenomics initiative among our academic medical center community, accompanied by clinician acceptance of pharmacogenetic alerts in practice. A multidisciplinary collaboration is recommended to facilitate the use of CYP2C19 genotype-guided antiplatelet therapy in patients with cardiovascular and cerebrovascular diseases. Evolving clopidogrel pharmacogenetic evidence necessitates thoughtful iteration of implementation efforts and strategies to optimize long-term maintenance and sustainability.

The deleterious effects of cadmium on oxidative stress markers, drug-metabolizing, and antioxidant enzyme activities: Role of Silymarin and Garlic as Antioxidants.

Exposure to cadmium has been related to liver and kidney diseases such as polycystic and nephrotic syndrome. It is still unclear how cadmium contributes to these diseases. It is believed that the induction of oxidative stress resulting from the inhibition of antioxidant enzyme activities and changes in drug-metabolizing enzymes in the liver could explain the role of cadmium in the development of different diseases in the kidney and probably other organs. Changes in oxidative stress markers, antioxidant enzymes, and drug-metabolizing enzyme activities were assessed in the liver of male rats exposed to cadmium chloride. Additionally, the protective effects of silymarin and garlic extract against cadmium toxicosis were evaluated. Rats were randomly divided into eight groups as follows, groups 1, 2, 3, 4, and 5, received orally saline, CdCl2 (1mg/kg), garlic extract [800mg/kg], silymarin (25mg/kg) and silymarin plus garlic extract respectively for 28 consecutive days. Rats in groups 6, 7, and 8 were pretreated with the same doses of garlic, silymarin, and garlic plus silymarin, respectively for two hours before cadmium administration. The Western immunoblotting technique was used to investigate the protein expression of cytochrome P450 isozymes. Spectrophotometric methods were used to assess the activity of both antioxidant- and drug-metabolizing enzymes. Free radical levels [measured as thiobarbituric acid reactive substances (TBARS)], catalase, superoxide dismutase, and glutathione peroxidase activities increased whereas the levels of glutathione and the activities of glutathione S-transferase, glutathione reductase, and glutamyl transferase, cytochrome P450, aryl hydrocarbon dehydrogenase (AHH), dimethylnitrosamine-N-demethylase I (DMN-dI), 7-ethoxycoumarine-O-deethylase (ECOD), cytochrome b5 and NADPH-Cytochrome-c-reductase enzyme activities decreased after cadmium treatment. Furthermore, Western immunoblotting data revealed that glutathione peroxidase protein expression increased following cadmium exposure, but cytochrome P450 2E1 and 3A4 expressions were downregulated. However, pretreatment of rats with silymarin or garlic extract or both before cadmium administration was found to restore the protein expression of cytochrome P450 2E1 and 3A4, the level of free radicals, antioxidant enzymes, drug-metabolizing enzyme activities to their normal levels. Similarly, histological studies revealed that silymarin and/or garlic extract reduced the liver damage caused by cadmium. Silymarin and/or garlic extract reduced the adverse effects of cadmium on the activity of both drug-metabolizing and antioxidant enzymes activity. These antioxidants could be provided to those who work in cadmium-based sectors to help them cope with the adverse effects of cadmium on their kidneys. In addition, Inhibiting drug-metabolizing enzyme activity should be considered when administering therapeutic medications to persons exposed to cadmium because most therapeutic drugs and many endogenous substances are largely metabolized by these enzymes.

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.

Pharmacokinetics and Absorption, Distribution, Metabolism and Excretion of RGLS4326 in Mouse and Monkey, an Anti-miR-17 Oligonucleotide for the Treatment of Polycystic Kidney Disease.

RGLS4326 is a short oligonucleotide inhibitor of microRNA-17 (miR-17) that preferentially distributes to the kidney and displaces miR-17 from translationally active polysomes. Here, we present pharmacokinetics and absorption, distribution, metabolism, and excretion properties of RGLS4326 from mice and monkeys. RGLS4326 was absorbed rapidly after subcutaneous administration, distributed extensively to the kidney and liver, with preferential distribution to the kidney, and cleared rapidly from plasma by tissue uptake and renal excretion. Plasma exposure increased in a dose-proportional manner with no notable accumulation after repeat doses. Plasma protein binding of RGLS4326 across all species tested was between 79% and 96%. RGLS4326 predominantly distributed to the kidney with a long half-life (t1/2; t1/2 ranged from 8-11 days) and no marked (≤twofold) accumulation in kidney and liver after repeat doses. RGLS4326 was minimally metabolized by nucleases, not cytochrome P450 (P450) isozymes, across species and underwent sequential hydrolysis from both 3' and 5' ends to produce chain-shortened metabolites. There were no human unique metabolites observed. Renal excretion was the major route of elimination of RGLS4326, and a significant fraction (50%-79%) of the dose was recovered intact in the urine of mice and monkeys across all dose levels. RGLS4326 is not a substrate, inhibitor, or inducer of P450 isozymes, and it is not a substrate or inhibitor of uptake and most efflux transporters. Thus, RGLS4326 exhibits low potential of mediating drug-drug interactions involving P450 isozymes and drug transporters. SIGNIFICANCE STATEMENT: Pharmacokinetics (PK) and absorption, distribution, metabolism, and excretion (ADME) properties of RGLS4326 were characterized in vivo and in vitro. RGLS4326 shows similar PK and ADME properties across mice and monkeys in vivo and across human and animal matrices in vitro. Subcutaneous administration results in preferential exposure of RGLS4326 to the intended target organ (kidney) to drive maximum target engagement. These studies support the interpretation of toxicology and efficacy studies and help characterize the disposition of RGLS4326 in humans.

Reducing Hepatotoxicity Mechanism of Radix Wikstroemia Indica by Processing with “Sweat Soaking Method” Using UPLC-MS/MS and a Cocktail Probe Substrate

Background: Radix Wikstroemia indica is a traditional Chinese medicine (TCM) used as anti-inflammatory and anti-tumor drug. However, it has serious hepatotoxicity, "Sweat soaking method" processed could effectively decrease its hepatotoxicity. Objective: The objective of this study is to study the effects of Radix Wikstroemia indica on six kinds of cytochrome P450(CYP450) isozymes of rat liver microsomes before and after processing, and to study the mechanism of Radix Wikstroemia indica processed by the "Sweat soaking method" to reduce liver toxicity in rats. Methods: In this study, the effects of Radix Wikstroemia indica and processed Radix Wikstroemia indica on the six main CYP450 isoforms (2E1, 1A2, 2C6, 2D1, 2C11, and 3A1) were investigated in vitro. Using a cocktail probe of CYP450 isoform-specific substrates and their metabolites, we carried out in vitro enzymatic studies in liver microsomal incubation systems via UPLC-MS/MS. Results: The results showed that the established UPLC-MS/MS method was precise and reliable. Compared with the blank group, the activities of six enzymes in the RWI and PRWI groups were higher than those in the blank group. At the same dose, the enzyme activities of CYP2E1, CYP1A2, CYP2C6, CYP2C11, and CYP3A1 increased with the increase in dose, and the enzyme activities of the RWI group were higher than those of the PRWI group. The enzyme activities of CYP2E1 and CYP1A2 in the Radix Wikstroemia indica group were significantly increased compared with the blank group, CYP3A1 in the RWI high-dose group was higher than that in the blank group and PRWI group with statistical differences (p<0.05 or p<0.01). Conclusion: The processed Radix Wikstroemia indica could reduce liver injury, and its detoxication mechanism might be related to the decrease in enzyme activity of CYP1A2, CYP2E1 and CYP3A1.

An update on drug-drug interactions for care of the acutely ill in the era of COVID-19.

In an effort to expedite the publication of articles, AJHP is posting manuscripts online as soon as possible after acceptance. Accepted manuscripts have been peer-reviewed and copyedited, but are posted online before technical formatting and author proofing. These manuscripts are not the final version of record and will be replaced with the final article (formatted per AJHP style and proofed by the authors) at a later time. To provide key pharmacological concepts underlying drug-drug interactions (DDIs), a decision-making framework, and a list of DDIs that should be considered in the context of contemporary acutely ill patients with COVID-19. DDIs are frequently encountered in the acutely ill. The implications of DDIs include either increased risk of drug toxicity or decreased effectiveness, which may have severe consequences in the acutely ill due to lower physiological and neurocognitive reserves in these patients. In addition, an array of additional therapies and drug classes have been used for COVID-19 that were not typically used in the acute care setting. In this update on DDIs in the acutely ill, we provide key pharmacological concepts underlying DDIs, including a discussion of the gastric environment, the cytochrome P-450 (CYP) isozyme system, transporters, and pharmacodynamics in relation to DDIs. We also provide a decision-making framework that elucidates the identification of DDIs, risk assessment, selection of alternative therapies, and monitoring. Finally, important DDIs pertaining to contemporary acute care clinical practice related to COVID-19 are discussed. Interpreting and managing DDIs should follow a pharmacologically based approach and a systematic decision-making process to optimize patient outcomes.

Pharmacokinetics and Drug-Drug Interaction of Ocedurenone (KBP-5074) in vitro and in vivo.

Ocedurenone (KBP-5074) is a novel nonsteroidal mineralocorticoid receptor antagonist that has demonstrated safety and efficacy in clinical trials in patients with uncontrolled hypertension and stage 3b/4 chronic kidney disease. This study evaluated the involvement of cytochrome P450 (CYP) isozymes and drug transporters in the biotransformation of ocedurenone, and whether ocedurenone inhibited or induced CYP enzymes and transporters. Clinical pharmacokinetic drug-drug interaction (DDI) of ocedurenone with CYP3A inhibitor and inducer were investigated in healthy volunteers. In vitro tests were conducted to determine which CYP enzymes were involved in ocedurenone's metabolism and whether ocedurenone inhibited or induced these CYP enzymes; ocedurenone substrate characteristics for efflux and uptake transporters and its inhibitory potential on major drug transporters were also assessed. A clinical DDI study was conducted in healthy volunteers to evaluate the effects of a strong CYP3A inhibitor (itraconazole) and inducer (rifampin) on ocedurenone's pharmacokinetics. The in vitro study showed that ocedurenone was primarily metabolized by CYP3A4 and that it did not inhibit CYP enzymes. Ocedurenone appeared to be a substrate of BCRP and P-gp efflux transporters and inhibited BCRP, BSEP, MDR1, MATE1 and 2-K, OATP1B1/3, and OCT1. The clinical DDI study showed that itraconazole reduced ocedurenone's oral clearance by 51% and increased area under the plasma concentration-time curve extrapolated to infinity(AUC0-inf) by104%, while rifampin increased its oral clearance by 6.4-fold and decreased plasma AUC0-inf by 84%. Ocedurenone was shown to be a CYP3A substrate, with no inhibition potential on major drug metabolizing CYP enzymes and transporters at clinical efficacious doses. Ocedurenone did not induce CYP1A2 and 3A4 activity in cultured human primary hepatocytes. Clinical DDI study indicated ocedurenone was well tolerated when administered as a single 0.5-mg dose both alone and with itraconazole or rifampin, and while itraconazole had a weak effect on ocedurenone's pharmacokinetics, rifampin had a significant effect reducing systemic exposures.

Impact of transitioning to an active, noninterruptive CYP2C19/proton pump inhibitor alert on prescribing patterns.

To compare rates of prescriber acceptance of interruptive and noninterruptive clinical decision support (CDS) alerts regarding potential diminished therapeutic effectiveness and safety risks associated with proton pump inhibitor (PPI) use in carriers of gene variants affecting cytochrome P450 (CYP) isozyme 2C19 metabolism. A retrospective study was conducted at a large rural health system to examine different approaches to improving CDS alert acceptance while minimizing alert fatigue. Manual reviews were conducted to identify alerts regarding CYP2C19 metabolizer status displayed at the time of PPI ordering over 30-day periods before and after the transition from interruptive to noninterruptive CDS alert functionality. A chi-square test was conducted to analyze prescriber acceptance of CDS recommendations by alert modality and type of treatment modification. Overall, interruptive alerts had an acceptance rate of 18.6% (64/344), compared to 8.4% acceptance (30/357 alerts) for noninterruptive alerts (P ≤ 0.0001). Analysis of acceptance criteria -revealed the noninterruptive alert cohort had higher acceptance, as determined by documented medication dose adjustments, than the interruptive alert cohort (53.3% [16/30] and 4.7% [3/64], respectively). The difference in acceptance rates by CDS modality and treatment modification was statistically significant (P ≤ 0.00001). The predominant indication for PPI use was gastroesophageal reflux disease (GERD) in both cohorts. Interruptive alerts that actively influenced workflow had higher acceptance rates than noninterruptive alerts that served an informational purpose without a direct disruption of workflow. The study results suggest the utilization of noninterruptive alerts may be a beneficial tool for prompting clinicians to alter dosing regimens rather than transition to an alternative agent.

Molecular Factors and Pathways of Hepatotoxicity Associated with HIV/SARS-CoV-2 Protease Inhibitors.

Antiviral protease inhibitors are peptidomimetic molecules that block the active catalytic center of viral proteases and, thereby, prevent the cleavage of viral polyprotein precursors into maturation. They continue to be a key class of antiviral drugs that can be used either as boosters for other classes of antivirals or as major components of current regimens in therapies for the treatment of infections with human immunodeficiency virus (HIV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, sustained/lifelong treatment with the drugs or drugs combined with other substance(s) often leads to severe hepatic side effects such as lipid abnormalities, insulin resistance, and hepatotoxicity. The underlying pathogenic mechanisms are not fully known and are under continuous investigation. This review focuses on the general as well as specific molecular mechanisms of the protease inhibitor-induced hepatotoxicity involving transporter proteins, apolipoprotein B, cytochrome P450 isozymes, insulin-receptor substrate 1, Akt/PKB signaling, lipogenic factors, UDP-glucuronosyltransferase, pregnane X receptor, hepatocyte nuclear factor 4α, reactive oxygen species, inflammatory cytokines, off-target proteases, and small GTPase Rab proteins related to ER-Golgi trafficking, organelle stress, and liver injury. Potential pharmaceutical/therapeutic solutions to antiviral drug-induced hepatic side effects are also discussed.

Evaluation of the cytochrome P450 2C19 and 3A4 inhibition potential of the complement factor 5a receptor antagonist ACT-1014-6470 in vitro and in vivo.

ACT-1014-6470 is an orally available complement factor 5a receptor 1 antagonist and a novel treatment option in autoinflammatory diseases. The in vitro inhibition potential of ACT-1014-6470 on cytochrome P450 isozymes (CYPs) and its effect on the pharmacokinetics (PK) of the CYP2C19 and CYP3A4 substrates omeprazole and midazolam, respectively, in humans were assessed. In vitro assays were conducted with isoform-specific substrates in human liver microsomes. In an open-label, two-period, fixed-sequence cocktail study, single doses of 20 mg omeprazole and 2 mg midazolam were administered concomitantly to 20 healthy male subjects alone (Treatment A) and after a single dose of 100 mg ACT-1014-6470 (Treatment B) under fed conditions. Safety and PK assessments were performed. Geometric mean ratios (GMRs) and 90% confidence intervals (CIs) of noncompartmental PK parameters of Treatment B vs Treatment A were calculated. In vitro, no time-dependent inhibition was observed and the lowest inhibition constant of 4.3 μM ACT-1014-6470 was recorded for CYP2C19. In humans, GMRs (90% CI) of omeprazole PK were 1.9 (1.5-2.5) for maximum plasma concentration (Cmax ) and 1.9 (1.5-2.3) for area under the plasma concentration-time curve (AUC) from 0 to 12 h. Midazolam PK showed GMRs (90% CI) of 1.1 (1.1-1.2) for Cmax and 1.5 (1.4-1.6) for AUC from 0 to 24 h. All treatments were well tolerated. In line with in vitro results and regulatory risk factor calculation, the increased exposure to omeprazole and midazolam in humans after concomitant administration with a single dose of 100 mg ACT-1014-6470 reflected a weak inhibition of CYP2C19 and CYP3A4.

Salivary clearance of caffeine in children.

Measurement of salivary caffeine (1,3,7-trimethylpurine-2,6-dione or 1,3,7-trimethylxanthine) clearance can, in principle, be used to assess hepatic function, diagnose chronic hepatic disease and conduct investigations of substrates of hepatic cytochrome P450 (CYP) isozymes in children, without recourse to venepuncture. However, little is known about childhood sexual dimorphism of hepatic CYP isoforms. Furthermore, the association, if any, between salivary caffeine clearance and age in children has not hitherto been established. The aims of this study were to assess whether salivary caffeine clearance differs between boys and girls and whether it varies with age during childhood. Following at least 24h' abstinence from dietary caffeine, nine boys (mean (standard error) age 9.6 (1.1) y) and eight girls (mean age 11.0 (1.2) y), none of whom was a smoker or suffered from chronic hepatic disease, ingested an oral caffeine dose titrated by body mass, namely 3mgkg-1. Salivary samples collected two and 14h later underwent spectrophotometric caffeine analysis. The boys and the girls were age matched. The mean caffeine clearance in the boys was 2.47 (0.33) mL min-1 kg-1, while that in the girls was 2.20 (0.31) mL min-1 kg-1 (p=0.56). The salivary caffeine clearance was negatively correlated with age (r=-0.59, p=0.01). Stratification by sex appears to be unnecessary when considering childhood salivary caffeine clearance or when conducting investigations in children of CYP1A2 and xanthine oxidase substrates. Furthermore, childhood salivary caffeine clearance is negatively correlated with age.

Melatonin Activation by Cytochrome P450 Isozymes: How Does CYP1A2 Compare to CYP1A1?

Cytochrome P450 enzymes are versatile enzymes found in most biosystems that catalyze mono-oxygenation reactions as a means of biosynthesis and biodegradation steps. In the liver, they metabolize xenobiotics, but there are a range of isozymes with differences in three-dimensional structure and protein chain. Consequently, the various P450 isozymes react with substrates differently and give varying product distributions. To understand how melatonin is activated by the P450s in the liver, we did a thorough molecular dynamics and quantum mechanics study on cytochrome P450 1A2 activation of melatonin forming 6-hydroxymelatonin and N-acetylserotonin products through aromatic hydroxylation and O-demethylation pathways, respectively. We started from crystal structure coordinates and docked substrate into the model, and obtained ten strong binding conformations with the substrate in the active site. Subsequently, for each of the ten substrate orientations, long (up to 1 μs) molecular dynamics simulations were run. We then analyzed the orientations of the substrate with respect to the heme for all snapshots. Interestingly, the shortest distance does not correspond to the group that is expected to be activated. However, the substrate positioning gives insight into the protein residues it interacts with. Thereafter, quantum chemical cluster models were created and the substrate hydroxylation pathways calculated with density functional theory. These relative barrier heights confirm the experimental product distributions and highlight why certain products are obtained. We make a detailed comparison with previous results on CYP1A1 and identify their reactivity differences with melatonin.

Boscalid shows increased thyroxin-glucuronidation in rat but not in human hepatocytes in vitro.

The fungicide boscalid induces thyroid histopathological and hormone effects in the rat, secondary to liver enzyme induction. To assess the human relevance of liver enzyme induction presumably leading to thyroid hormone disruption, a species comparative in vitro study on T4-glucuronidation was conducted. Currently, no guidelines how to evaluate Phase II induction are in place. Therefore, we investigated the optimal conditions to evaluate Phase I and Phase II induction potential of boscalid in primary rat (PRH) and human (PHH) hepatocytes. Endpoints included mRNA gene expression and enzyme activities (cytochrome P450 isozymes [CYPs] and uridine diphosphate-glucuronosyltransferases [UGTs]), measured after 3 (D3) and 7 (D7) days of exposure to reference compounds and to 5, 10, and 20 μM boscalid, focusing on T4-glucuronidation. Basal CYP activities and T4 glucuronidation were similar or higher on D7 than D3. The highest induction responses of CYPs were on D3, whereas UGT induction and T4-glucuronidation increases were highest on D7. Boscalid induced CYP1A, CYP2B, and CYP3A mRNA and/or increased related activities in PRH and PHH. Species differences in the induction pattern of UGT genes by reference inducers (ß-naphthoflavone [BNF], 5-pregnen-3ß-ol-20-one-16α-carbonitirile [PCN], rifampicin [RIF], and phenobarbital [PB]) and boscalid were seen: UGT1A1, UGT1A3, and UGT1A9 were predominantly induced in PHH, while UGT2B1 was predominantly induced in PRH. Basal activity levels for T4-glucuronidation were very low in humans and an order of magnitude higher in rat, for this reason increases in activities were assessed as delta activity to the control. Significant increases in T4-glucuronidation occurred with boscalid in rat but not in human hepatocytes.

Biotransformation of Bisphenol by Human Cytochrome P450 2C9 Enzymes: A Density Functional Theory Study.

Bisphenol A (BPA, 2,2-bis-(4-hydroxyphenyl)propane) is used as a precursor in the synthesis of polycarbonate and epoxy plastics; however, its availability in the environment is causing toxicity as an endocrine-disrupting chemical. Metabolism of BPA and their analogues (substitutes) is generally performed by liver cytochrome P450 enzymes and often leads to a mixture of products, and some of those are toxic. To understand the product distributions of P450 activation of BPA, we have performed a computational study into the mechanisms and reactivities using large model structures of a human P450 isozyme (P450 2C9) with BPA bound. Density functional theory (DFT) calculations on mechanisms of BPA activation by a P450 compound I model were investigated, leading to a number of possible products. The substrate-binding pocket is tight, and as a consequence, aliphatic hydroxylation is not feasible as the methyl substituents of BPA cannot reach compound I well due to constraints of the substrate-binding pocket. Instead, we find low-energy pathways that are initiated with phenol hydrogen atom abstraction followed by OH rebound to the phenolic ortho- or para-position. The barriers of para-rebound are well lower in energy than those for ortho-rebound, and consequently, our P450 2C9 model predicts dominant hydroxycumyl alcohol products. The reactions proceed through two-state reactivity on competing doublet and quartet spin state surfaces. The calculations show fast and efficient substrate activation on a doublet spin state surface with a rate-determining electrophilic addition step, while the quartet spin state surface has multiple high-energy barriers that can also lead to various side products including C4-aromatic hydroxylation. This work shows that product formation is more feasible on the low spin state, while the physicochemical properties of the substrate govern barrier heights of the rate-determining step of the reaction. Finally, the importance of the second-coordination sphere is highlighted that determines the product distributions and guides the bifurcation pathways.

Mechanistic aspects of reactive metabolite formation in clomethiazole catalyzed biotransformation by cytochrome P450 enzymes.

Clomethiazole (CLM), a sedative and anticonvulsant drug, is commonly employed for the treatment of alcohol withdrawal syndromebecause it suppresses cytochrome P450 (P450) activity associated with the generation of free radicals and liver damage. The catalyzed biotransformation of thiazole-containing drugs by P450 is known to afford reactive metabolites. These metabolites can alter the biological functions of macromolecules and result in toxicity and adverse drug interactions. Multitargeted molecular modeling and quantum chemical DFT calculations were performed to explore the binding modes and molecular mechanisms underlying the mechanism-based inactivation (MBI) of P450 by CLM. The mechanistic details associated with reactive metabolite formation from further metabolic processes were extensively assessed. Seven possible routes were proposed for CLM-P450 biotransformation including CLM hydroxylation, sulfoxidation, N-oxidation, CN epoxidation (oxaziridine formation), and CC epoxidation. The results revealed a degree of preference for the C-N epoxidation pathway because of the low energy requirements of its rate-determining step (8.74 and 10.07 kcal mol-1 for LS and HS states, respectively). A kinetic competition for the CLM-methyl hydroxylation pathway was detected because the H-abstraction energy barrier was relatively comparable to the thermodynamically prevailing oxaziridine formation rate-determining step (12.58 and 14.52 kcal mol-1 for quartet and doublet states, respectively). Our studies assessed the mechanisms of covalent nucleophilic epoxide adduct formation through nucleophilic addition, hydrolysis of epoxidation products, and nonenzymatic degradation. CLM was shown to display P450-inhibitory activity by forming covalent adducts rather than further metabolization to reactive metabolites. The outcomes of molecular docking allowed assessing the binding profile of CLM with three human P450 isozymes, namely, CYP2E1, CYP3A4, and CYP2D6.

Medication with fenbendazole in feed: plasma concentrations and effects on hepatic xenobiotic metabolizing enzymes in swine.

Fenbendazole (FBZ), a benzymidazole (BZD) anthelmintic drug, is used for in-feed medication in pigs. BZD-containing drugs may induce cytochrome P450 isozymes (CYPs), particularly those members of the CYP1A subfamily. The current research evaluated the plasma and liver availability and metabolism of FBZ and its metabolites, oxfendazole (OFZ) and fenbendazole sulphone (FBZSO2), after the administration of the parent drug in feed, and characterized the effect of the sustained administration of the anthelmintic on the catalytic activities of xenobiotic metabolizing enzymes in pig liver. Five female Landrace piglets remained untreated (controls), and other six were treated with a pre-mix of FBZ, combined with feed, for 9 consecutive days as usually is recommended. Blood samples were collected from each treated animal up to day 9 and analyzed by HPLC; all animals were slaughtered for preparation of liver microsomes. Plasma concentration ratios OFZ/FBZ and FBZSO2/OFZ increased significantly (p < 0.05) from the beginning to the end of drug exposure, which may indicate an enhanced conversion of FBZ into its metabolites. FBZ represented 45.8 ± 3.4% of the total anthelmintic molecules in liver tissue. Increased CYP1A-dependent 7-ethoxy (24.5-fold, p = 0.0032) and 7-methoxyresorufin (17.2-fold, p = 0.0006) O-dealkylase activities was observed in liver microsomes from FBZ-treated animals. In addition, a 64% increase (p = 0.042) in the rate of FBZ S-oxidation was observed in pigs treated with the anthelmintic drug compared to that measured in untreated animals. Thus, the continuous FBZ administration may accelerate its own in vivo hepatic metabolism through the CYP1A pathway.