Common Cytochrome P450 Research Articles

Systematic engineering pinpoints a versatile strategy for the expression of functional cytochrome P450 enzymes in Escherichia coli cell factories

Production of plant secondary metabolites in engineered microorganisms provides a scalable and sustainable alternative to their sourcing from nature or through chemical synthesis. However, the biosynthesis of many valuable plant-derived products relies on cytochromes P450 – enzymes notoriously difficult to express in microbes. To improve their expression in Escherichia coli, an arsenal of engineering strategies was developed, often paired with an extensive screening of enzyme variants. Here, attempting to identify a broadly applicable strategy, we systematically evaluated six common cytochrome P450 N-terminal modifications and their effect on in vivo activity of enzymes from the CYP79 and CYP83 families. We found that transmembrane domain truncation was the only modification with a significantly positive effect for all seven tested enzymes, increasing their product titres by 2- to 170-fold. Furthermore, when comparing the changes in the protein titre and product generation, we show that higher protein expression does not directly translate to higher in vivo activity, thus making the protein titre an unreliable screening target in the context of cell factories. We propose the transmembrane domain truncation as a first-line approach that enables the expression of wide range of highly active P450 enzymes in E. coli and circumvents the time-consuming screening process. Our results challenge the notion that the engineering strategy must be tailored for each individual cytochrome P450 enzyme and have the potential to simplify and accelerate the future design of E. coli cell factories.

GENETIC VARIATION AND PREDICTED PHENOTYPES IN CYP2C9, CYP2C19, CYP3A4, CYP3A5 AND CYP2D6 GENES AMONG 552 PATIENTS WITH UNCONTROLLED TREATED HYPERTENSION

Objective: Altered drug metabolism may be a contributing factor to uncontrolled hypertension. Beta-blockers, alpha-blockers, calcium antagonists and angiotensin receptor blockers undergo hepatic metabolism by cytochrome P450 (CYP) enzymes. Genetic variation can contribute to interindividual variation, lack of treatment response or side effects with discontinuation of treatment. We aimed to determine common CYP gene variations in patients with treated but uncontrolled hypertension and to compare the distribution with the prevalence in the overall Caucasian population. Design and method: In this national multicenter study we mapped CYP2D6, CYP2C9, CYP2C19, CYP3A4 and CYP3A5 polymorphisms in 552 patients prescribed > = 2 antihypertensive agents and with systolic daytime ambulatory blood pressure (ABPM) > = 135 mmHg. On the (MALDI-TOF) MassARRAY analyzer, single nucleotide variants and small insertions/deletions (indels) were investigated with the VeriDose Core Panel and CYP2D6 copy-number variation with the VeriDose CYP2D6 CNV Panel (Agena Biosciences). Each allele is associated with either normal, decreased, absent, increased or unknown enzyme function based on activity scores defined by the The Clinical Pharmacogenetics Implementation Consortium (CPIC) and describe function and phenotypes for genes (IM = Intermediate metabolizer, PM = poor metabolizer, RM = rapid metabolizer and UM = ultra-rapid metabolizer). Results: 94% of patients were Caucasian. Mean daytime ABPM was 146/84 mmHg (135-190/55-106 mmHg), median age 63 (26-86) years and 35% were female. The distributions of the predicted phenotypes (illustrated in colors in Figure) for each enzyme did not differ from that reported in Caucasian population by The Pharmacogenomics Knowledgebase (PharmGK) (gray). 11% had normal phenotype in all five enzymes whereas 49% had > = 2 enzymes affected by gene variants. Conclusions: The phenotype distribution of five relevant CYP genes in patients with uncontrolled hypertension did not differ from the general Caucasian population. Dosing guidelines based on CYP2D6 genotype currently exist for metoprolol, but for other antihypertensive drugs further investigation is needed. The low proportion of patients with five normal enzymes and the high proportion with > = 2 enzymes affected suggest that pharmacogenetic CYP-testing may be useful for individualizing drug treatment in patients with uncontrolled hypertension.

Development of a bioprinter-based method for incorporating metabolic competence into high-throughput in vitro assays.

The acceptance and use of in vitro data for hazard identification, prioritization, and risk evaluation is partly limited by uncertainties associated with xenobiotic metabolism. The lack of biotransformation capabilities of many in vitro systems may under- or overestimate the hazard of compounds that are metabolized to more or less active metabolites in vivo. One approach to retrofitting existing bioassays with metabolic competence is the lid-based Alginate Immobilization of Metabolic Enzymes (AIME) method, which adds hepatic metabolism to conventional high-throughput screening platforms. Here, limitations of the lid-based AIME method were addressed by incorporating bioprinting, which involved depositing S9-encapsulated microspheres into standard 384-well plates with requisite cofactors for phase I and II hepatic metabolism. Objectives of this study included: 1) compare the lid-based and AIME bioprinting methods by assessing the enzymatic activity of a common cytochrome P450 (CYP) enzyme, 2) use biochemical assays with the bioprinting method to characterize additional measures of phase I and II metabolic activity, and 3) evaluate the bioprinting method by screening 25 chemicals of known metabolism-dependent bioactivity in the VM7Luc estrogen receptor transactivation (ERTA) assay. A comparison of the two methods revealed comparable precision and dynamic range. Activity of additional CYP enzymes and glucuronidation was observed using the AIME bioprinting method. The ERTA experiment identified 19/21ER-active test chemicals, 14 of which were concordant with expected biotransformation effects (73.7%). Additional refinement of the AIME bioprinting method has the potential to expand high-throughput screening capabilities in a robust, accessible manner to incorporate in vitro metabolic competence.

Pharmacogenomics in Psychiatry Practice: The Value and the Challenges.

The activity of cytochrome P450 enzymes is influenced by genetic and nongenetic factors; hence, the metabolism of exogenous psychotropic medications and potentially some endogenous neuropeptides is variably affected among different ethnic groups of psychiatric patients. The aim of this review is to highlight the most common cytochrome P450 isoenzymes associated with the metabolism of psychotropic medications (antidepressants, antipsychotics, and mood stabilizers), their variations among different populations, their impact on endogenous neurotransmitters (dopamine and serotonin), and the effect of nongenetic factors, particularly smoking, age, and pregnancy, on their metabolic activity. Furthermore, the adverse effects of psychiatric medications may be associated with certain human leukocytic antigen (HLA) genotypes. We also highlight the gene variants that may potentially increase susceptibility to obesity and metabolic syndrome, as the adverse effects of some psychiatry medications. Collectively, the literature revealed that variation of CYP450 activity is mostly investigated in relation to genetic polymorphism, and is directly correlated with individualized clinical outcomes; whereas adverse effects are associated with HLA variants, projecting the value of pharmacogenetics implementation in psychiatry clinics. Only a few previous studies have discussed the impact of such genetic variations on the metabolism of endogenous neuropeptides. In this review, we also report on the prevalence of key variants in different ethnicities, by demonstrating publicly available data from the 1000 Genomes Project and others. Finally, we highlight the future direction of further investigations to enhance the predictability of the individual gene variants to achieve precision therapies for psychiatric patients.

Multiple circulating alkaloids and saponins from intravenous Kang-Ai injection inhibit human cytochrome P450 and UDP-glucuronosyltransferase isozymes: potential drug\u2013drug interactions

BackgroundKang-Ai injection is widely used as an adjuvant therapy drug for many cancers, leukopenia, and chronic hepatitis B. Circulating alkaloids and saponins are believed to be responsible for therapeutic effects. However, their pharmacokinetics (PK) and excretion in vivo and the risk of drug–drug interactions (DDI) through inhibiting human cytochrome P450 (CYP) and UDP-glucuronosyltransferase (UGT) enzymes remain unclear.MethodsPK and excretion of circulating compounds were investigated in rats using a validated ultra-high-performance liquid chromatography tandem mass spectrometry (UHPLC–MS) method. Further, the inhibitory effects of nine major compounds against eleven CYP and UGT isozymes were assayed using well-accepted specific substrate for each enzyme.ResultsAfter dosing, 9 alkaloids were found with Cmax and t1/2 values of 0.17–422.70 μmol/L and 1.78–4.33 h, respectively. Additionally, 28 saponins exhibited considerable systemic exposure with t1/2 values of 0.63–7.22 h, whereas other trace saponins could be negligible or undetected. Besides, over 90% of alkaloids were excreted through hepatobiliary and renal excretion. Likewise, astragalosides and protopanaxatriol (PPT) type ginsenosides also involved in hepatobiliary and/or renal excretion. Protopanaxadiol (PPD) type ginsenosides were mainly excreted to urine. Furthermore, PPD-type ginsenosides were extensively bound (fu-plasma approximately 1%), whereas astragalosides and PPT-type ginsenosides displayed fu-plasma values of 12.35% and 60.23–87.36%, respectively. Moreover, matrine, oxymatrine, astragaloside IV, ginsenoside Rg1, ginsenoside Re, ginsenoside Rd, ginsenoside Rc, and ginsenoside Rb1 exhibited no inhibition or weak inhibition against several common CYP and UGT enzymes IC50 values between 8.81 and 92.21 μM. Through kinetic modeling, their inhibition mechanisms towards those CYP and UGT isozymes were explored with obtained Ki values. In vitro-in vivo extrapolation showed the inhibition of systemic clearance for CYP or UGT substrates seemed impossible due to [I]/Ki no more than 0.1.ConclusionsWe summarized the PK behaviors, excretion characteristics and protein binding rates of circulating alkaloids, astragalosides and ginsenosides after intravenous Kang-Ai injection. Furthermore, weak inhibition or no inhibition towards these CYP and UGT activities could not trigger harmful DDI when Kang-Ai injection is co-administered with clinical drugs primarily cleared by these CYP or UGT isozymes.

Functional and structural characterisation of common cytochrome P450 2D6 allelic variants-roles of Pro34 and Thr107 in catalysis and inhibition.

One major source of inter-individual variability in drug pharmacokinetics is genetic polymorphism of the cytochrome P450 (CYP) genes. This study aimed to elucidate the enzyme kinetic and molecular basis for altered activity in three major alleles of CYP2D6, namely CYP2D6*2, CYP2D6*10 and CYP2D6*17. The E. coli-expressed allelic variants were examined using substrate (venlafaxine and 3-cyano-7-ethoxycoumarin[CEC]) and inhibitor (quinidine, fluoxetine, paroxetine, terbinafine) probes in enzyme assays as well as molecular docking. The kinetics data indicated that R296C and S486T mutations in CYP2D6*2 have caused enhanced ligand binding (enhanced intrinsic clearance for venlafaxine and reduced IC50 for quinidine, paroxetine and terbinafine), suggesting morphological changes within the active site cavity that favoured ligand docking and binding. Mutations in CYP2D6*10 and CYP2D6*17 tended to cause deleterious effect on catalysis, with reduced clearance for venlafaxine and CEC. Molecular docking indicated that P34S and T107I, the unique mutations in the alleles, have negatively impacted activity by affecting ligand access and binding due to alteration of the substrate access channel and active site morphology. IC50 values however were quite variable for quinidine, fluoxetine and terbinafine, and a general decrease in IC50 was observed for paroxetine, suggesting ligand-specific altered susceptibility to inhibition in the alleles. This study indicates that CYP2D6 allele selectivity for ligands was not solely governed by changes in the active site architecture induced by the mutations, but that the intrinsic properties of the substrates and inhibitors also played vital role.

406 - Sublingual Administration of Tacrolimus for Graft-Versus-Host Disease Prevention in Pediatric Allogeneic Hematopoietic Stem Cell Transplantation, a Case Series

Background: Tacrolimus is an immunosuppressive agent that is widely used for prevention of graft-vs-host disease in patients who have undergone allogeneic hematopoietic stem cell transplantation. The drug has a narrow therapeutic window; so, it is important to monitor serum drug levels and critically evaluate the effect of interacting medications. Tacrolimus is metabolized through CYP3A4, the most common cytochrome P450 enzyme in the liver, which can lead to several drug interactions. Both CYP3A4 inhibitors and inducers can cause large fluctuations in tacrolimus levels which may risk the development of graft-vs-host disease. Tacrolimus is only FDA approved to be given orally or intravenously; however, accounts in the literature have demonstrated that sublingual administration is a unique and reliable alternative mode of drug delivery. When given sublingually, tacrolimus is absorbed directly into the blood stream and bypasses the liver's first pass metabolism. This direct systemic absorption helps avoid CYP3A4 drug interactions. To our knowledge, this is the first study looking at sublingual tacrolimus in the pediatric hematopoietic stem cell transplantation population. Methods: We conducted a retrospective chart review of all hematopoietic stem cell transplant patients treated with sublingual tacrolimus at the University of Iowa Stead Family Children's Hospital between March 2017 and September 2017. Results: A total of five patients received sublingual tacrolimus. Ages ranged from 4-20 years. Two patients were not included in the analysis because they only received one and two doses of sublingual tacrolimus respectively. One patient who was receiving rifampin had persistently subtherapeutic tacrolimus levels despite escalating oral doses; however, once the patient was switched to sublingual tacrolimus, her levels became therapeutic. In the three patients that were analyzed, sublingual doses ranged from .025-0.078 mg/kg/day divided twice daily. Two of the three patients had haploidentical transplants and the third had a matched unrelated donor. There were minimal dose changes required to keep therapeutic drug levels and no patients experienced graft-vs-host disease. Conclusions: Sublingual tacrolimus is a reliable alternative mode of drug delivery for pediatric hematopoietic stem cell transplant patients.

Transcription factor cap n collar C regulates multiple cytochrome P450 genes conferring adaptation to potato plant allelochemicals and resistance to imidacloprid in Leptinotarsa decemlineata (Say)

Colorado potato beetle (CPB), Leptinotarsa decemlineata is a notorious pest of potato. Co-evolution with Solanaceae plants containing high levels of toxins (glycoalkaloids) helped this insect to develop an efficient detoxification system and resist almost every chemical insecticide introduced for its control. Even though the cross-resistance between plant allelochemicals and insecticides is well acknowledged, the underlying molecular mechanisms are not understood. Here, we investigated the molecular mechanisms involved in detoxification of potato plant allelochemicals and imidacloprid resistance in the field-collected CPB. Our results showed that the imidacloprid-resistant beetles employ metabolic detoxification of both potato plant allelochemicals and imidacloprid by upregulation of common cytochrome P450 genes. RNAi aided knockdown identified four cytochromes P450 genes (CYP6BJa/b, CYP6BJ1v1, CYP9Z25, and CYP9Z29) that are required for defense against both natural and synthetic chemicals. These P450 genes are regulated by the xenobiotic transcription factors Cap n Collar C, CncC and muscle aponeurosis fibromatosis, Maf. Studies on the CYP9Z25 promoter using the luciferase reporter assay identified two binding sites (i.e. GCAGAAT and GTACTGA) for CncC and Maf. Overall, these data showed that CPB employs the metabolic resistance mediated through xenobiotic transcription factors CncC and Maf to regulate multiple P450 genes and detoxify both imidacloprid and potato plant allelochemicals.

Amphiphilic xanthones as a potent chemical entity of anti-mycobacterial agents with membrane-targeting properties

Tuberculosis (TB) remains a deadly disease and infects one-third of the world's population. Given the low success rates encountered in clinical development, there is an urgent need to identify structurally novel antimicrobials for tuberculosis. The present report details the anti-mycobacterial activities, structure-activity relationships (SARs) and mechanism of action of amphiphilic xanthone derivatives. The xanthones exhibited potent MIC, rapid time-kill and no cross-resistance with the current anti-TB drugs. Evidence is presented that these compounds disrupted the inner membrane and led to ATP depletion. Amphiphilic xanthone derivatives exhibited superior metabolic stability, low cytotoxicity and low activity against the common cytochrome P450. Compound 5 was selected for an in vivo pharmacokinetic study. Its bioavailability at an oral dose of 2 mg/kg was 15%. The xanthones thuse provide valuable insight for the development of a new class of membrane targeting antimycobacterial agents that may assist in overcoming the limitations of the current TB medications.

Biodegradation of 1-alkoxy-2,4-dichlorobenzenes by Yarrowia lipolytica KCTC 17618

Detailed analyses of the biodegradation of 1-alkoxy-2,4-dichlorobenzenes by Yarrowia lipolytica KCTC 17618 were conducted by gas chromatography-mass spectrometry. Eight different 1-alkoxy-2,4-dichlorobenzenes with a linear alkylgroup (butyl to tetradecyl) were subjected to liquid culture. Y. lipolytica could rapidly metabolize short-chain analogues (butyl to hexyl) and utilize them as the sole carbon source for growth, whereas the analogues with longer alkyl groups were unable to support the growth but gradually metabolized in the nutrient media. The metabolic rates decreased with an increase of alkyl chain length up to a decyl group. Comprehensive metabolite analysis indicated that 4-(2,4-dichlorophenoxy)-butyric acid was the final metabolite of substrates with even-numbered alkoxy groups. 4-(2,4-dichlorophenoxy)-butyric acid is a well-known plant growth regulator and herbicide. In case of odd-numbered alkoxybenzenes, 3-(2,4-dichlorophenoxy)-propionic acid was the most abundant metabolite and no trace of 4-(2,4-dichlorophenoxy)-butyric acid was observed. These findings suggested that alkyl chain metabolism by the yeast follows a typical β-oxidation mechanism. The metabolism was partially inhibited by piperonylbutoxide, a common cytochrome P450 inhibitor. Dose-dependent production of 4-(2,4-dichlorophenoxy)-butyric acid from 1-butoxy-2,4-dichlorobenzene was evaluated. The maximum yield was 487 mg l−1 at 1 g l−1 of substrate. In summary, Y. lipolytica could efficiently produce 4-(2,4-dichlorophenoxy)-butyric acid from 1-alkoxy-2,4-dichlorobenzenesthrough oxidative metabolism.

CYP2J2 rs890293 polymorphism is associated with susceptibility to Alzheimer’s disease in the Chinese Han population

Alzheimer’s disease (AD) is a common neurodegenerative disorder characterized by progressive cognitive dysfunction and memory loss. Increasing evidence indicates that inflammation in the brain is a powerful factor in AD progression. Epoxyeicosatrienoic acids, the biologically active derivatives of arachidonic acid, synthesized by cytochrome P450 (CYP) epoxygenases, have been proven to have powerful anti-inflammatory effects. The aim of this study was to examine whether polymorphism in CYP2J2, encoding one of the most common CYP epoxygenase isoforms, is associated with late-onset AD (LOAD). This case-control study genotyped 672 representatives of the Chinese Han population, including 321 LOAD patients and 351 healthy controls matched for age and gender, for the functional rs890293 polymorphism within CYP2J2 by means of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI–TOF MS). The CYP2J2 rs890293 T allele and GT+TT genotype were significantly associated with an increased risk of LOAD. Further data stratification according to the presence of the apolipoprotein E (APOE) e4 allele confirmed a strong association between CYP2J2 rs890293 and LOAD, and indicated that the involvement of CYP2J2 in LOAD was independent of ApoE-ϵ4. Our study demonstrated that CYP2J2 rs890293 is a possible predisposing genetic factor for progression of LOAD.

Abstract P6-12-02: Use of cytochrome P450 interacting medications in the setting of adjuvant therapy for breast cancer.

Abstract Background: In the current era of personalized medicine, oral targeted therapies are increasingly used in cancer treatment. In breast cancer, oral anti-estrogen agents have historically been part of standard treatment for hormone receptor positive disease. More recently, other targeted agents have been introduced in the metastatic setting, and are being evaluated as adjuvant therapies. Many oral medications, including anticancer therapies, are metabolized by cytochrome P450 (CYP450) enzymes raising possibility of drug-drug interactions that may affect toxicities or breast cancer outcomes. We sought to evaluate concomitant CYP450 medication use among women seeing a medical oncologist to discuss adjuvant systemic therapy for breast cancer. Methods: We performed an electronic medical record database extraction. Adult women diagnosed with breast cancer from 1/2008-7/2011 were identified from the University of Wisconsin Hospital and Clinics Cancer Registry. Medication lists were extracted from the first encounter with a medical oncologist after the initial breast cancer diagnosis. Non-systemic medications were excluded. Cytochrome P450 (CYP450) enzyme-interacting medications were categorized as inhibitors, inducers, and/or substrates of specific enzymes including CYP1A2, CYP2A6, CYP2B6, CYP2C19, CYP2C8, CYP2C9, CYP2D6, CYP2E1, and CYP3A4. CYP450 inhibitors and inducers were further characterized as strong, moderate or weak acting. Results: A total of 455 women with non-metastatic breast cancer were identified. Mean age was 56.6 years (range of 23–90) and 413 (91%) were Caucasian. Polypharmacy, defined as use of 3–5 medications, was seen in 123 (27.0%) women. A total of 236 (51.9%) women were on 0–4; 109 (24.0%) on 5–10; and 13 (2.9%) on > 10 medications at the time of first encounter with a medical oncologist after a breast cancer diagnosis. 23 (5.05%) women were on strong CYP450 enzyme inhibitors while 72 (15.8%) were on strong inducers. CYP450 enzymes most commonly affected were CYP3A4, CYP2C9, and CYP2D6. Among medications taken on a fixed schedule, levothyroxine and simvastatin were the most commonly used, while simvastatin and ranitidine were the most common CYP450 interacting medications. Further classification of potential CYP450 interactions is ongoing. Conclusions: A significant proportion of patients were on one or more CYP450 interacting medications in the setting of adjuvant therapy for breast cancer. Given the number of new oral cancer agents that are also CYP450 interacting, the potential for drug interactions should be recognized and appropriate management strategies implemented. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P6-12-02.

Abstract 1602: Time-to-recurrence is decreased in head and neck cancer patients with a common cytochrome P450 1B1 variant (N453S); epidemiological and in vitro findings

Abstract Squamous cell carcinoma of the head and neck (HNSCC) is the sixth most common type of cancer. Major risk factors for HNSCC in Western countries include tobacco and alcohol use. Carcinogenic components of tobacco smoke and ethanol can be metabolized by cytochrome P450 1B1 (CYP1B1) into mutagenic intermediates. Several common single nucleotide polymorphisms (SNPs) that introduce an amino acid change have been identified in the coding region of CYP1B1. Two of these SNPs (R48G and A119S) are linked and their function is unclear. The L432V variant has been associated with increased CYP1B1 activity while the N453S variant has been shown to accelerate the degradation of CYP1B1. The goal of this study was to evaluate the contribution of common SNPs in CYP1B1 to both the recurrence of HNSCC and overall survival of patients with HNSCC. Patients (N=155) with HNSCC, excluding oropharynx and nasopharynx, were diagnosed at FCCC between 2001-2008 (median time of follow-up - 3.3 years). DNA samples were genotyped for CYP1B1 SNPs using standard TaqMan assays. The impact of polymorphic genotypes on overall survival and recurrence was assessed using the Kaplan-Meier estimation method and the Cox proportional hazards model. Time-to-recurrence for N453S carriers was significantly greater than that of those carrying the wild-type genotype (p = 0.03). Furthermore, among patients with wild-type CYP1B1, women had a shorter time-to-recurrence than men (p = 0.02). The latter result was consistent with the known ability of CYP1B1 to metabolize estrogens to potentially carcinogenic intermediates. No difference in either survival or recurrence was observed among carriers of either R48G or L432V, as compared to wild-type carriers. In order to begin to understand the mechanism by which the N453S polymorphism may confer a shorter time-to-recurrence, cultured HNSCC cells (SCC15 and UPCI:SCC56) were transfected with plasmids encoding either wild-type CYP1B1 or variant CYP1B1 (N453S) as well as cytochrome P450 oxidoreductase (required for electron transfer) and green fluorescent protein (to measure transfection efficiency). The impact of the variant (N453S) and wild-type CYP1B1 proteins on cell proliferation and apoptosis was compared using the Fluorescent DNA Quantitation kit and the Nexin reagent, respectively. Cells with abundant levels of the N453S variant exhibited a rate of proliferation that was 5 to 8-fold lower than that of cells that overexpressed wild-type CYP1B1. In contrast, the rate of apoptosis among cells overexpressing either wild-type or variant CYP1B1 was comparable. These data provide novel insight into the mechanisms underlying carcinogenesis of the head and neck. More accurate identification of HNSCC patients with a poor prognosis could lead to new opportunities for tailored intervention with more aggressive therapeutic and surveillance strategies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1602. doi:1538-7445.AM2012-1602

Inhibition of platelet aggregation by prostaglandin E1 (PGE1) in diabetic patients during therapy with clopidogrel and aspirin

Diabetes mellitus (DM) is associated with increased platelet activation and reduced platelet inhibition by clopidogrel. Prostaglandin E1 (PGE1) stimulates adenyl cyclase activity in platelets and increases cyclic AMP concentrations, which inhibit Ca2+release and platelet aggregation induced by P2Y1 receptor activation. PGE1 is included in the VerifyNow P2Y12 assay to suppress P2Y1 induced platelet aggregation. We hypothesized that diabetes mellitus may be associated with altered response to PGE1 in subjects treated with clopidogrel. Subjects with established coronary artery disease who were taking clopidogrel 75 mg daily and aspirin for >14 days were enrolled (n = 96). Diabetic (n = 34) were compared with non-diabetic subjects (n = 62). VerifyNow P2Y12 assay and light transmittance aggregometry (LTA) were performed using ADP as agonist with and without addition of PGE1. Genomic DNA was genotyped for common cytochrome P450 (CYP) 2C19 variants using Taqman assays. Residual on-treatment platelet aggregation induced by 20 µM ADP was not significantly different between subjects with and without DM. Addition of 22 nM and 88 nM PGE1 to 20 µM ADP resulted in a significant reduction of maximal platelet aggregation (MPA). Residual LTA platelet aggregation with PGE1 and VerifyNow P2Y12 platelet reactivity were significantly higher in subjects with DM than those without DM and in carriers of CYP 2C19*2 polymorphism. We conclude that an impaired inhibitory response to PGE1 may contribute to the high platelet reactivity phenotype in subjects with DM treated with clopidogrel. Addition of PGE1 to ADP agonist platelet assays may identify subjects with blunted inhibitory response to prostaglandins and result in a higher proportion of subjects with DM being classified as non-responders.

Reporting Patterns Indicative of Adverse Drug Interactions

Adverse drug interaction surveillance in collections of Individual Case Safety Reports (ICSRs) remains underdeveloped. Most efforts to date have focused on disproportionality analysis, but the empirical support for its value is based on isolated examples. Additionally, too little attention has been given to the potential value of the detailed content of ICSRs for improved adverse drug interaction surveillance. The aim of the study was to identify reporting patterns indicative of suspected adverse drug interactions before the drug interactions are generally established. A reference set of known adverse drug interactions and drug pairs not known to interact was constructed from information added to Stockley's Drug Interactions Alerts between the first quarter of 2007 and the third quarter of 2009. The reference set was used to systematically study differences in reporting patterns between adverse drug interactions before they are generally established and adverse drug reactions (ADRs) to drug pairs that are not known to interact, in the WHO Global ICSR Database, VigiBase. The scope of the study included pharmacological properties such as common cytochrome P450 metabolism, explicit suspicions of drug interactions as noted by the reporter, clinical details such as dose and treatment overlap, and the lower limit of the 95% credibility interval of a three-way measure of disproportionality, Omega(025) (Ω(025)), based on the total number of reports on two drugs and one ADR together. Analyses were carried out including and excluding concomitant medicines. Five reporting patterns were highlighted as particularly strong indicators of adverse drug interactions before they are known: suspicion of interactions as noted by the reporter in a case narrative, the assignment of the two drugs as interacting or through an ADR term; co-reporting of effect increased with the drug pair; and, finally, an excess total number of reports on the ADR together with the two drugs, as measured by Ω(025). Overall, the inclusion of concomitant medicines led to a larger number of true adverse drug interactions being highlighted, but at a substantial decrease in the strength of most indicators. Notably, the inclusion of concomitant medicines completely eliminated the value of Ω(025) as an indicator of adverse drug interactions, in this systematic evaluation. Reported suspicion of interactions as noted by the reporter in a case narrative, the assignment of the two drugs as interacting or through an ADR term; co-reporting of effect increased with the drug pair and by the Ω(025) each provide unique information to highlight adverse drug interactions before they become known in the literature. To our knowledge, this is the first systematic analysis demonstrating the value of disproportionality analysis for adverse drug interactions using a comprehensive reference set, and the first study to consider a broader basis including clinical information for systematic drug interaction surveillance.

Expression and Characterization of Dog Cytochrome P450 2A13 and 2A25 in Baculovirus-Infected Insect Cells

Dog CYP2A13 and CYP2A25 were coexpressed with dog NADPH-cytochrome P450 reductase (OR) in baculovirus-infected Sf9 insect cells. CYP2A13 effectively catalyzed 7-ethoxycoumarin (7EC) deethylation and coumarin hydroxylation with apparent K(m) values of 4.8 and 2.1 microM, respectively, similar to those observed using dog liver microsomes (7.5 and 0.75 microM, respectively). CYP2A25 exhibited much lower affinity toward 7EC, with an apparent K(m) value of 150 microM, which indicates that CYP2A13 plays a more significant role in the metabolism of these CYP2A substrates. Similar to the dog CYP1A2 enzyme, CYP2A13 efficiently catalyzed phenacetin deethylation with a K(m) value of 3.9 microM, which suggests that phenacetin is not a selective probe for dog CYP1A2 activity. Both dog CYP2A13 and CYP2A25 exhibited little or no catalytic activity toward other common cytochrome P450 probe substrates, including bupropion, amodiaquine, diclofenac, S-mephenytoin, bufuralol, dextromethorphan, midazolam, and testosterone. These results provided additional information about the selectivity of these commonly used probe substrates.

Role of Ticagrelor in Clopidogrel Nonresponders: Resistance Is Futile?

Dual antiplatelet therapy with aspirin and a thienopyridine drug that blocks the platelet adenosine diphosphate receptor P2Y12 is the cornerstone of management in patients presenting with acute coronary syndrome (ACS). Clopidogrel is the most widely used thienopyridine, with evidence of benefit in non–ST-elevation myocardial infarction (MI),1 percutaneous coronary intervention (PCI),2 and ST-elevation MI treated with thrombolysis or primary PCI.3,4 Article see p 1188 However, clopidogrel has several suboptimal characteristics. First, despite administration of a 600 mg loading dose, clopidogrel requires at least 2 hours to achieve effective platelet inhibition, which is clearly disadvantageous during urgent PCI. Second, the irreversible P2Y12 receptor inhibition may be associated with an increased risk of hemorrhagic complications, particularly in patients requiring urgent coronary artery bypass grafting or other surgery. Furthermore, clopidogrel is a prodrug that requires 2-stage hepatic activation by cytochrome P450 (CYP) enzymes. This is susceptible to genetic polymorphisms, resulting in a variable response and an attenuated antiplatelet effect. Indeed, reduced function alleles (especially CYP2C19*2), which are present in ≈30% of the population, lead to diminished production of the active metabolite.5 This phenomenon, often called clopidogrel resistance, is associated with increased risk of atherothrombotic complications, including stent thrombosis.5,6 Clopidogrel’s limitations have stimulated the search for alternative antiplatelet agents. The development of prasugrel was a major step forward. Prasugrel is a thienopyridine with 10-fold more potent anti-P2Y12 receptor inhibitory activity than clopidogrel and a more rapid onset of action. Like clopidogrel, prasugrel also requires biotransformation to active metabolites via CYP enzymes. In contrast, CYP polymorphisms do not affect active metabolite levels, platelet inhibition, or clinical cardiovascular event rates in patients treated with prasugrel.7 In the study by Mega et al,7 the common CYP functional variants associated with clopidogrel resistance were assessed in healthy volunteers and a cohort of …

Impact of CYP3A5 and CYP3A4 gene polymorphisms on dose requirement of calcineurin inhibitors, cyclosporine and tacrolimus, in renal allograft recipients of North India

The present study investigated pharmacogenetic associations of common cytochrome P450 3A (CYP3A5 and CYP3A4) polymorphisms with dose requirements of calcineurin inhibitors, cyclosporine (CsA) and tacrolimus (Tac) in renal transplant recipients of North India. Two hundred twenty four patients on CsA and 73 patients on Tac-based immunosuppression regimen were genotyped for CYP3A5*3 (6986A>G) and CYP3A4*1B (-290A>G) and correlated with CsA/Tac dose requirement (mg/kg/day) and dose-adjusted CsA (C(2))/Tac (T (0)) blood levels (concentration/dose ratio) at 1 month and 3 months posttransplantation. The dose-adjusted levels were significantly lower in CYP3A5 expressers for CsA (p = 0.037; 3 months) and Tac (p < 0.001; 1 month and p < 0.001; 3 months) compared to the non-expressers, suggesting that for a given dose their CsA/Tac blood concentration is lower. The CYP3A5 non-expresser genotype was associated with reduced risk for allograft rejection (HR-0.18, 95% CI 0.03-0.99). No influence of CYP3A4*1B on CsA/Tac pharmacokinetics was observed. CYP3A5 expressers were associated with significantly lower dose-adjusted CsA/Tac concentrations and higher allograft rejection episodes in patients on Tac therapy.

Influence of CYP2C9 genotype on warfarin dose requirements—a systematic review and meta-analysis

To quantify the influence of common cytochrome P450 2C9 (CYP2C9) polymorphisms on warfarin dose requirements. A systematic review and a meta-analysis, calculating the warfarin dose reduction associated with the five most common variant CYP2C9 genotypes. Thirty-nine studies (7,907 patients) were included in the meta-analysis. Compared to the CYP2C9*1/*1 genotype, the CYP2C9*1/*2, CYP2C9*1/*3, CYP2C9*2/*2, CYP2C9*2/*3, and CYP2C9*3/*3 required warfarin doses that were 19.6 (95% confidence interval 17.4, 21.9), 33.7 (29.4, 38.1), 36.0 (29.9, 42.0), 56.7 (49.1, 64.3), and 78.1% (72.0, 84.3) lower, respectively. The impact of CYP2C9 genotype tended to be larger in patients without interacting drugs. Previous studies have rarely been powered to determine the quantitative influence of specific CYP2C9 genotypes on warfarin dose requirements. The results from our pooled analysis are likely to be the most accurate to date and the methodology could serve as a model for future pharmacogenetic meta-analyses.

A polymorphism of the CYP17 gene related to sex steroid metabolism is associated with female-to-male but not male-to-female transsexualism

To assess the association between transsexualism and allele and genotype frequencies of the common cytochrome P450 (CYP) 17 -34 T>C single nucleotide polymorphism (SNP). Case-control study. Academic research institution. 102 male-to-female (MtF) and 49 female-to-male (FtM) transsexuals, 756 male controls, and 915 female controls. Buccal swabs and multiplex polymerase chain reaction on a microarray system. Analysis of the CYP17 -34 T>C SNP. CYP17 -34 T>C SNP allele frequencies were statistically significantly different between FtM transsexuals and female controls (CYP17 T: 55/98 [56%] and CYP17 C: 43/98 [44%] versus CYP17 T: 1253/1826 [69%] and CYP17 C: 573/1826 [31%], respectively). In accordance, genotype distributions were also different between FtM transsexuals and female controls using a recessive genotype model (CYP17 T/T+T/C: 39/49 [80%] and C/C 10/49 [20%] vs. CYP17 T/T+T/C: 821/913 [90%] and C/C 92/913 [10%], respectively). The CYP17 -34 T>C allele and genotype distributions were not statistically significantly different between MtF transsexuals and male controls. Of note, the CYP17 -34 T>C allele distribution was gender-specific among controls (CYP17 C: males; 604 of 1512 [40%] vs. females; 573 of 1826 [31%]). The MtF transsexuals had an allele distribution equivalent to male controls, whereas FtM transsexuals did not follow the gender-specific allele distribution of female controls but rather had an allele distribution equivalent to MtF transsexuals and male controls. These data support CYP17 as a candidate gene of FtM transsexualism and indicate that loss of a female-specific CYP17 T -34C allele distribution pattern is associated with FtM transsexualism.