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Sample records for p450-mediated drug interaction

  1. Cytochrome P450 enzyme mediated herbal drug interactions (Part 1)

    PubMed Central

    Wanwimolruk, Sompon; Prachayasittikul, Virapong

    2014-01-01

    It is well recognized that herbal supplements or herbal medicines are now commonly used. As many patients taking prescription medications are concomitantly using herbal supplements, there is considerable risk for adverse herbal drug interactions. Such interactions can enhance the risk for an individual patient, especially with regard to drugs with a narrow therapeutic index such as warfarin, cyclosporine A and digoxin. Herbal drug interactions can alter pharmacokinetic or/and pharmacodynamic properties of administered drugs. The most common pharmacokinetic interactions usually involve either the inhibition or induction of the metabolism of drugs catalyzed by the important enzymes, cytochrome P450 (CYP). The aim of the present article is to provide an updated review of clinically relevant metabolic CYP-mediated drug interactions between selected herbal supplements and prescription drugs. The commonly used herbal supplements selected include Echinacea, Ginkgo biloba, garlic, St. John's wort, goldenseal, and milk thistle. To date, several significant herbal drug interactions have their origins in the alteration of CYP enzyme activity by various phytochemicals. Numerous herbal drug interactions have been reported. Although the significance of many interactions is uncertain but several interactions, especially those with St. John’s wort, may have critical clinical consequences. St. John’s wort is a source of hyperforin, an active ingredient that has a strong affinity for the pregnane xenobiotic receptor (PXR). As a PXR ligand, hyperforin promotes expression of CYP3A4 enzymes in the small intestine and liver. This in turn causes induction of CYP3A4 and can reduce the oral bioavailability of many drugs making them less effective. The available evidence indicates that, at commonly recommended doses, other selected herbs including Echinacea, Ginkgo biloba, garlic, goldenseal and milk thistle do not act as potent or moderate inhibitors or inducers of CYP enzymes. A good

  2. Cytochrome P450 enzyme mediated herbal drug interactions (Part 2)

    PubMed Central

    Wanwimolruk, Sompon; Phopin, Kamonrat; Prachayasittikul, Virapong

    2014-01-01

    To date, a number of significant herbal drug interactions have their origins in the alteration of cytochrome P450 (CYP) activity by various phytochemicals. Among the most noteworthy are those involving St. John's wort and drugs metabolized by human CYP3A4 enzyme. This review article is the continued work from our previous article (Part 1) published in this journal (Wanwimolruk and Prachayasittikul, 2014[ref:133]). This article extends the scope of the review to six more herbs and updates information on herbal drug interactions. These include black cohosh, ginseng, grape seed extract, green tea, kava, saw palmetto and some important Chinese medicines are also presented. Even though there have been many studies to determine the effects of herbs and herbal medicines on the activity of CYP, most of them were in vitro and in animal studies. Therefore, the studies are limited in predicting the clinical relevance of herbal drug interactions. It appeared that the majority of the herbal medicines have no clear effects on most of the CYPs examined. For example, the existing clinical trial data imply that black cohosh, ginseng and saw palmetto are unlikely to affect the pharmacokinetics of conventional drugs metabolized by human CYPs. For grape seed extract and green tea, adverse herbal drug interactions are unlikely when they are concomitantly taken with prescription drugs that are CYP substrates. Although there were few clinical studies on potential CYP-mediated interactions produced by kava, present data suggest that kava supplements have the ability to inhibit CYP1A2 and CYP2E1 significantly. Therefore, caution should be taken when patients take kava with CYP1A2 or CYP2E1 substrate drugs as it may enhance their therapeutic and adverse effects. Despite the long use of traditional Chinese herbal medicines, little is known about the potential drug interactions with these herbs. Many popularly used Chinese medicines have been shown in vitro to significantly change the

  3. Short-term fasting alters cytochrome P450-mediated drug metabolism in humans.

    PubMed

    Lammers, Laureen A; Achterbergh, Roos; de Vries, Emmely M; van Nierop, F Samuel; Klümpen, Heinz-Josef; Soeters, Maarten R; Boelen, Anita; Romijn, Johannes A; Mathôt, Ron A A

    2015-06-01

    Experimental studies indicate that short-term fasting alters drug metabolism. However, the effects of short-term fasting on drug metabolism in humans need further investigation. Therefore, the aim of this study was to evaluate the effects of short-term fasting (36 h) on P450-mediated drug metabolism. In a randomized crossover study design, nine healthy subjects ingested a cocktail consisting of five P450-specific probe drugs [caffeine (CYP1A2), S-warfarin (CYP2C9), omeprazole (CYP2C19), metoprolol (CYP2D6), and midazolam (CYP3A4)] on two occasions (control study after an overnight fast and after 36 h of fasting). Blood samples were drawn for pharmacokinetic analysis using nonlinear mixed effects modeling. In addition, we studied in Wistar rats the effects of short-term fasting on hepatic mRNA expression of P450 isoforms corresponding with the five studied P450 enzymes in humans. In the healthy subjects, short-term fasting increased oral caffeine clearance by 20% (P = 0.03) and decreased oral S-warfarin clearance by 25% (P < 0.001). In rats, short-term fasting increased mRNA expression of the orthologs of human CYP1A2, CYP2C19, CYP2D6, and CYP3A4 (P < 0.05), and decreased the mRNA expression of the ortholog of CYP2C9 (P < 0.001) compared with the postabsorptive state. These results demonstrate that short-term fasting alters cytochrome P450-mediated drug metabolism in a nonuniform pattern. Therefore, short-term fasting is another factor affecting cytochrome P450-mediated drug metabolism in humans. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

  4. Development of gold-immobilized P450 platform for exploring the effect of oligomer formation on P450-mediated metabolism for in vitro to in vivo drug metabolism predictions

    NASA Astrophysics Data System (ADS)

    Kabulski, Jarod L.

    The cytochrome P450 (P450) enzyme family is responsible for the biotransformation of a wide range of endogenous and xenobiotic compounds, as well as being the major metabolic enzyme in first pass drug metabolism. In vivo drug metabolism for P450 enzymes is predicted using in vitro data obtained from a reconstituted expressed P450 system, but these systems have not always been proven to accurately represent in vivo enzyme kinetics, due to interactions caused by oligomer formation. These in vitro systems use soluble P450 enzymes prone to oligomer formation and studies have shown that increased states of protein aggregation directly affect the P450 enzyme kinetics. We have developed an immobilized enzyme system that isolates the enzyme and can be used to elucidate the effect of P450 aggregation on metabolism kinetics. The long term goal of my research is to develop a tool that will help improve the assessment of pharmaceuticals by better predicting in vivo kinetics in an in vitro system. The central hypothesis of this research is that P450-mediated kinetics measured in vitro is dependent on oligomer formation and that the accurate prediction of in vivo P450-mediated kinetics requires elucidation of the effect of oligomer formation. The rationale is that the development of a P450 bound to a Au platform can be used to control the aggregation of enzymes and bonding to Au may also permit replacement of the natural redox partners with an electrode capable of supplying a constant flow of electrons. This dissertation explains the details of the enzyme attachment, monitoring substrate binding, and metabolism using physiological and electrochemical methods, determination of enzyme kinetics, and the development of an immobilized-P450 enzyme bioreactor. This work provides alternative approaches to studying P450-mediated kinetics, a platform for controlling enzyme aggregation, electrochemically-driven P450 metabolism, and for investigating the effect of protein

  5. High-Throughput Cytochrome P450 Cocktail Inhibition Assay for Assessing Drug-Drug and Drug-Botanical Interactions

    PubMed Central

    Li, Guannan; Huang, Ke; Nikolic, Dejan

    2015-01-01

    Detection of drug-drug interactions is essential during the early stages of drug discovery and development, and the understanding of drug-botanical interactions is important for the safe use of botanical dietary supplements. Among the different forms of drug interactions that are known, inhibition of cytochrome P450 (P450) enzymes is the most common cause of drug-drug or drug-botanical interactions. Therefore, a rapid and comprehensive mass spectrometry–based in vitro high-throughput P450 cocktail inhibition assay was developed that uses 10 substrates simultaneously against nine CYP isoforms. Including probe substrates for CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and two probes targeting different binding sites of CYP3A4/5, this cocktail simultaneously assesses at least as many P450 enzymes as previous assays while remaining among the fastest due to short incubation times and rapid analysis using ultrahigh pressure liquid chromatography–tandem mass spectrometry. The method was validated using known inhibitors of each P450 enzyme and then shown to be useful not only for single-compound testing but also for the evaluation of potential drug-botanical interactions using the botanical dietary supplement licorice (Glycyrrhiza glabra) as an example. PMID:26285764

  6. Cytochrome P450 drug interactions with statin therapy.

    PubMed

    Goh, Ivanna Xin Wei; How, Choon How; Tavintharan, Subramaniam

    2013-03-01

    Statins are commonly used in the treatment of hyperlipidaemia. Although the benefits of statins are well-documented, they have the potential to cause myopathy and rhabdomyolysis due to the complex interactions of drugs, comorbidities and genetics. The cytochrome P450 family consists of major enzymes involved in drug metabolism and bioactivation. This article aims to highlight drug interactions involving statins, as well as provide updated recommendations and approaches regarding the safe and appropriate use of statins in the primary care setting.

  7. Hepatotoxicity of Herbal Supplements Mediated by Modulation of Cytochrome P450

    PubMed Central

    Chen, Taosheng

    2017-01-01

    Herbal supplements are a significant source of drug-drug interactions (DDIs), herb-drug interactions, and hepatotoxicity. Cytochrome P450 (CYP450) enzymes metabolize a large number of FDA-approved pharmaceuticals and herbal supplements. This metabolism of pharmaceuticals and supplements can be augmented by concomitant use of either pharmaceuticals or supplements. The xenobiotic receptors constitutive androstane receptor (CAR) and the pregnane X receptor (PXR) can respond to xenobiotics by increasing the expression of a large number of genes that are involved in the metabolism of xenobiotics, including CYP450s. Conversely, but not exclusively, many xenobiotics can inhibit the activity of CYP450s. Induction of the expression or inhibition of the activity of CYP450s can result in DDIs and toxicity. Currently, the United States (US) Food and Drug Administration does not require the investigation of the interactions of herbal supplements and CYP450s. This review provides a summary of herbal supplements that inhibit CYP450s, induce the expression of CYP450s, and/or whose toxicity is mediated by CYP450s. PMID:29117101

  8. Fasting-Induced Changes in Hepatic P450 Mediated Drug Metabolism Are Largely Independent of the Constitutive Androstane Receptor CAR.

    PubMed

    de Vries, E M; Lammers, L A; Achterbergh, R; Klümpen, H-J; Mathot, R A A; Boelen, A; Romijn, J A

    2016-01-01

    Hepatic drug metabolism by cytochrome P450 enzymes is altered by the nutritional status of patients. The expression of P450 enzymes is partly regulated by the constitutive androstane receptor (CAR). Fasting regulates the expression of both P450 enzymes and CAR and affects hepatic drug clearance. We hypothesized that the fasting-induced alterations in P450 mediated drug clearance are mediated by CAR. To investigate this we used a drug cocktail validated in humans consisting of five widely prescribed drugs as probes for specific P450 enzymes: caffeine (CYP1A2), metoprolol (CYP2D6), omeprazole (CYP2C19), midazolam (CYP3A4) and s-warfarin (CYP2C9). This cocktail was administered to wild type (WT, C57Bl/6) mice or mice deficient for CAR (CAR-/-) that were either fed ad libitum or fasted for 24 hours. Blood was sampled at predefined intervals and drug concentrations were measured as well as hepatic mRNA expression of homologous/orthologous P450 enzymes (Cyp1a2, Cyp2d22, Cyp3a11, Cyp2c37, Cyp2c38 and Cyp2c65). Fasting decreased Cyp1a2 and Cyp2d22 expression and increased Cyp3a11 and Cyp2c38 expression in both WT and CAR-/- mice. The decrease in Cyp1a2 was diminished in CAR-/- in comparison with WT mice. Basal Cyp2c37 expression was lower in CAR-/- compared to WT mice. Fasting decreased the clearance of all drugs tested in both WT and CAR-/- mice. The absence of CAR was associated with an decrease in the clearance of omeprazole, metoprolol and midazolam in fed mice. The fasting-induced reduction in clearance of s-warfarin was greater in WT than in CAR-/-. The changes in drug clearance correlated with the expression pattern of the specific P450 enzymes in case of Cyp1a2-caffeine and Cyp2c37-omeprazole. We conclude that CAR is important for hepatic clearance of several widely prescribed drugs metabolized by P450 enzymes. However the fasting-induced alterations in P450 mediated drug clearance are largely independent of CAR.

  9. Importance of multi-P450 inhibition in drug-drug interactions: evaluation of incidence, inhibition magnitude and prediction from in vitro data

    PubMed Central

    Isoherranen, Nina; Lutz, Justin D; Chung, Sophie P; Hachad, Houda; Levy, Rene H; Ragueneau-Majlessi, Isabelle

    2012-01-01

    Drugs that are mainly cleared by a single enzyme are considered more sensitive to drug-drug interactions (DDIs) than drugs cleared by multiple pathways. However, whether this is true when a drug cleared by multiple pathways is co-administered with an inhibitor of multiple P450 enzymes (multi-P450 inhibition) is not known. Mathematically, simultaneous equipotent inhibition of two elimination pathways that each contributes half of the drug clearance is equal to equipotent inhibition of a single pathway that clears the drug. However, simultaneous strong or moderate inhibition of two pathways by a single inhibitor is perceived as an unlikely scenario. The aim of this study was (i) to identify P450 inhibitors currently in clinical use that can inhibit more than one clearance pathway of an object drug in vivo, and (ii) to evaluate the magnitude and predictability of DDIs caused by these multi-P450 inhibitors. Multi-P450 inhibitors were identified using the Metabolism and Transport Drug Interaction Database™. A total of 38 multi-P450 inhibitors, defined as inhibitors that increased the AUC or decreased the clearance of probes of two or more P450’s, were identified. Seventeen (45 %) multi-P450 inhibitors were strong inhibitors of at least one P450 and an additional 12 (32 %) were moderate inhibitors of one or more P450s. Only one inhibitor (fluvoxamine) was a strong inhibitor of more than one enzyme. Fifteen of the multi-P450 inhibitors also inhibit drug transporters in vivo, but such data are lacking on many of the inhibitors. Inhibition of multiple P450 enzymes by a single inhibitor resulted in significant (>2-fold) clinical DDIs with drugs that are cleared by multiple pathways such as imipramine and diazepam while strong P450 inhibitors resulted in only weak DDIs with these object drugs. The magnitude of the DDIs between multi-P450 inhibitors and diazepam, imipramine and omeprazole could be predicted using in vitro data with similar accuracy as probe substrate

  10. Electronic and structural aspects of p450-mediated drug metabolism.

    PubMed

    Lewis, David F V; Ito, Yuko; Lake, Brian G

    2009-04-01

    From a consideration of first principles for enzymes kinetics, we have employed theoretical methods which enable one to analyse the kinetics of cytochrome P450-mediated reactions which have been based on the known physicochemical principles underlying the majority of chemical or enzymatic reactions. A comparison is made between the correlation equations produced from the QSAR analysis of experimental P450 reaction rate data and those obtained from first principles, where there appears to be a generally satisfactory concordance between the two procedures. In this respect, we have developed expressions based on standard reaction kinetics theory which incorporate the Eyring and Marcus relationships. The analysis of P450-catalyzed reaction rates is elaborated to encompass a treatment of metabolic clearance, and satisfactory correlations are obtained with literature values for both intrinsic clearance and whole body clearance in terms of compound lipophilicity derived from log P data, where P is the octanol/water partition coefficient. The importance of ionization potential as a factor in the overall catalytic turnover of P450-mediated reactions is noted, especially in combination with the lipophilicity parameter, log P.

  11. In silico prediction of cytochrome P450-mediated drug metabolism.

    PubMed

    Zhang, Tao; Chen, Qi; Li, Li; Liu, Limin Angela; Wei, Dong-Qing

    2011-06-01

    The application of combinatorial chemistry and high-throughput screening technique enables the large number of chemicals to be generated and tested simultaneously, which will facilitate the drug development and discovery. At the same time, it brings about a challenge of how to efficiently identify the potential drug candidates from thousands of compounds. A way used to deal with the challenge is to consider the drug pharmacokinetic properties, such as absorption, distribution, metabolism and excretion (ADME), in the early stage of drug development. Among ADME properties, metabolism is of importance due to the strong association with efficacy and safety of drug. The review will focus on in silico approaches for prediction of Cytochrome P450-mediated drug metabolism. We will describe these predictive methods from two aspects, structure-based and data-based. Moreover, the applications and limitations of various methods will be discussed. Finally, we provide further direction toward improving the predictive accuracy of these in silico methods.

  12. Endotoxin administration to humans inhibits hepatic cytochrome P450-mediated drug metabolism.

    PubMed Central

    Shedlofsky, S I; Israel, B C; McClain, C J; Hill, D B; Blouin, R A

    1994-01-01

    In experimental animals, injection of gram-negative endotoxin (LPS) decreases hepatic cytochrome P450-mediated drug metabolism. To evaluate this phenomenon in a human model of gram-negative sepsis, LPS was administered on two consecutive days to healthy male volunteers during which time a cocktail of antipyrine (AP-250 mg), hexobarbital (HB-500 mg), and theophylline (TH-150 mg) was ingested and the apparent oral clearance of each drug determined. Each subject had a control drug clearance study with saline injections. In the first experiment, six subjects received the drug cocktail 0.5 h after the first dose of LPS. In the second experiment, another six subjects received the drug cocktail 0.5 h after the second dose of LPS. In both experiments, LPS caused the expected physiologic responses of inflammation including fever with increases in serum concentrations of TNF alpha, IL-1 beta, IL-6, and acute phase reactants. In the first experiment, only minor decreases in clearances of the probe drugs were observed (7-12%). However in the second experiment, marked decreases in the clearances of AP (35, 95% CI 18-48%), HB (27, 95% CI 14-34%), and TH (22, 95% CI 12-32%) were seen. The decreases in AP clearance correlated with initial peak values of TNF alpha (r = 0.82) and IL-6 (r = 0.86). These data show that in humans the inflammatory response to even a very low dose of LPS significantly decreases hepatic cytochrome P450-mediated drug metabolism and this effect evolves over a 24-h period. It is likely that septic patients with much higher exposures to LPS have more profound inhibition of drug metabolism. PMID:7989576

  13. A current review of cytochrome P450 interactions of psychotropic drugs.

    PubMed

    Madhusoodanan, Subramoniam; Velama, Umamaheswararao; Parmar, Jeniel; Goia, Diana; Brenner, Ronald

    2014-05-01

    The number of psychotropic drugs has expanded tremendously over the past few decades with a proportional increase in drug-drug interactions. The majority of psychotropic agents are biotransformed by hepatic enzymes, which can lead to significant drug-drug interactions. Most drug-drug interactions of psychotropics occur at metabolic level involving the hepatic cytochrome P450 enzyme system. We searched the National Library of Medicine, PsycINFO, and Cochrane reviews from 1981 to 2012 for original studies including clinical trials, double-blind, placebo-controlled studies, and randomized controlled trials. In addition, case reports, books, review articles, and hand-selected journals were utilized to supplement this review. Based on the clinical intensity of outcome, cytochrome interactions can be classified as severe, moderate, and mild. Severe interactions include effects that might be acutely life threatening. They are mainly inhibitory interactions with cardiovascular drugs. Moderate interactions include efficacy issues. Mild interactions include nonserious side effects, such as somnolence. Psychotropic drugs may interact with other prescribed medications used to treat concomitant medical illnesses. A thorough understanding of the most prescribed medications and patient education will help reduce the likelihood of potentially fatal drug-drug interactions.

  14. Physical Studies of P450P450 Interactions: Predicting Quaternary Structures of P450 Complexes in Membranes from Their X-ray Crystal Structures

    PubMed Central

    Reed, James R.; Backes, Wayne L.

    2017-01-01

    Cytochrome P450 enzymes, which catalyze oxygenation reactions of both exogenous and endogenous chemicals, are membrane bound proteins that require interaction with their redox partners in order to function. Those responsible for drug and foreign compound metabolism are localized primarily in the endoplasmic reticulum of liver, lung, intestine, and other tissues. More recently, the potential for P450 enzymes to exist as supramolecular complexes has been shown by the demonstration of both homomeric and heteromeric complexes. The P450 units in these complexes are heterogeneous with respect to their distribution and function, and the interaction of different P450s can influence P450-specific metabolism. The goal of this review is to examine the evidence supporting the existence of physical complexes among P450 enzymes. Additionally, the review examines the crystal lattices of different P450 enzymes derived from X-ray diffraction data to make assumptions regarding possible quaternary structures in membranes and in turn, to predict how the quaternary structures could influence metabolism and explain the functional effects of specific P450P450 interactions. PMID:28194112

  15. Current cytochrome P450 phenotyping methods applied to metabolic drug-drug interaction prediction in dogs.

    PubMed

    Mills, Beth Miskimins; Zaya, Matthew J; Walters, Rodney R; Feenstra, Kenneth L; White, Julie A; Gagne, Jason; Locuson, Charles W

    2010-03-01

    Recombinant cytochrome P450 (P450) phenotyping, different approaches for estimating fraction metabolized (f(m)), and multiple measures of in vivo inhibitor exposure were tested for their ability to predict drug interaction magnitude in dogs. In previous reports, midazolam-ketoconazole interaction studies in dogs have been attributed to inhibition of CYP3A pathways. However, in vitro phenotyping studies demonstrated higher apparent intrinsic clearances (CL(int,app)) of midazolam with canine CYP2B11 and CYP2C21. Application of activity correction factors and isoform hepatic abundance to liver microsome CL(int,app) values further implicated CYP2B11 (f(m) >or= 0.89) as the dog enzyme responsible for midazolam- and temazepam-ketoconazole interactions in vivo. Mean area under the curve (AUC) in the presence of the inhibitor/AUC ratios from intravenous and oral midazolam interaction studies were predicted well with unbound K(i) and estimates of unbound hepatic inlet inhibitor concentrations and intestinal metabolism using the AUC-competitive inhibitor relationship. No interactions were observed in vivo with bufuralol, although significant interactions with bufuralol were predicted with fluoxetine via CYP2D and CYP2C pathways (>2.45-fold) but not with clomipramine (<2-fold). The minor caffeine-fluvoxamine interaction (1.78-fold) was slightly higher than predicted values based on determination of a moderate f(m) value for CYP1A1, although CYP1A2 may also be involved in caffeine metabolism. The findings suggest promise for in vitro approaches to drug interaction assessment in dogs, but they also highlight the need to identify improved substrate and inhibitor probes for canine P450s.

  16. Cytochrome P450 and P-Glycoprotein-Mediated Interactions Involving African Herbs Indicated for Common Noncommunicable Diseases

    PubMed Central

    Kikete, Siambi; Liang, Rongjia; Wang, Lili

    2017-01-01

    Herbal remedies are regularly used to complement conventional therapies in the treatment of various illnesses in Africa. This may be because they are relatively cheap and easily accessible and are believed by many to be safe, cause fewer side effects, and are less likely to cause dependency. On the contrary, many herbs have been shown to alter the pharmacokinetics of coadministered allopathic medicines and can either synergize or antagonize therapeutic effects as well as altering the toxicity profiles of these drugs. Current disease burden data point towards epidemiological transitions characterised by increasing urbanization and changing lifestyles, risk factors for chronic diseases like hypertension, diabetes, and cancer which often present as multimorbidities. As a result, we highlight African herb-drug interactions (HDIs) modulated via cytochrome P450 enzyme family (CYP) and P-glycoprotein (P-gp) and the consequences thereof in relation to antihypertensive, antidiabetic, and anticancer drugs. CYPs are enzymes which account for to up to 70% of drug metabolism while P-gp is an efflux pump that extrudes drug substrates out of cells. Consequently, regulation of the relative activity of both CYP and P-gp by African herbs influences the effective drug concentration at the site of action and modifies therapeutic outcomes. PMID:28250793

  17. Inhibitory effects of spirulina platensis on carcinogen-activating cytochrome P450 isozymes and potential for drug interactions.

    PubMed

    Savranoglu, Seda; Tumer, Tugba Boyunegmez

    2013-01-01

    Spirulina platensis (SP) has been considered as potential food source of 21st century due to its remarkable nutrient profile and therapeutic benefits. However, the cytochrome P450 (CYP)-mediated drug/chemical interaction potential of SP has not yet been pursued. We investigated the effects of SP on the expressions and enzymatic activities of main CYP isozymes. After the rats were orally administered with SP daily for 5 consecutive weeks, there were significant downregulations in hepatic expression levels and inhibition in enzymatic activities of CYP1A2 and CYP2E1 compared to controls. In addition, a significant decrease was observed in CYP2C6-associated enzyme activity with no remarkable changes in messenger RNA (mRNA)/protein levels. The SP application resulted in significant increases in mRNA/protein levels of both CYP2B1 and CYP3A1 without a significant change in enzyme activities. These findings partly explain the chemopreventive properties of SP toward various organ toxicities, mutagenesis, and carcinogenesis; however, its coadministration with some CYP substrates may lead to undesirable drug interactions.

  18. Inactivation of Cytochrome P450 (P450) 3A4 but not P450 3A5 by OSI-930, a Thiophene-Containing Anticancer DrugS⃞

    PubMed Central

    Lin, Hsia-lien; Zhang, Haoming; Medower, Christine; Johnson, William W.

    2011-01-01

    An investigational anticancer agent that contains a thiophene moiety, 3-[(quinolin-4-ylmethyl)-amino]-N-[4-trifluoromethox)phenyl] thiophene-2-carboxamide (OSI-930), was tested to investigate its ability to modulate the activities of several cytochrome P450 enzymes. Results showed that OSI-930 inactivated purified, recombinant cytochrome P450 (P450) 3A4 in the reconstituted system in a mechanism-based manner. The inactivation was dependent on cytochrome b5 and required NADPH. Catalase did not protect against the inactivation. No inactivation was observed in studies with human 2B6, 2D6, or 3A5 either in the presence or in the absence of b5. The inactivation of 3A4 by OSI-930 was time- and concentration-dependent. The inactivation of the 7-benzyloxy-4-(trifluoromethyl)coumarin catalytic activity of 3A4 was characterized by a KI of 24 μM and a kinact of 0.04 min−1. This KI is significantly greater than the clinical OSI-930 Cmax of 1.7 μM at the maximum tolerated dose, indicating that clinical drug interactions of OSI-930 via this pathway are not likely. Spectral analysis of the inactivated protein indicated that the decrease in the reduced CO spectrum at 450 nm was comparable to the amount of inactivation, thereby suggesting that the inactivation was primarily due to modification of the heme. High-pressure liquid chromatography (HPLC) analysis with detection at 400 nm showed a loss of heme comparable to the activity loss, but a modified heme was not detected. This result suggests either that the heme must have been modified enough so as not to be observed in a HPLC chromatograph or, possibly, that it was destroyed. The partition ratio for the inactivation of P450 3A4 was approximately 23, suggesting that this P450 3A4-mediated pathway occurs with approximately 4% frequency during the metabolism of OSI-930. Modeling studies on the binding of OSI-930 to the active site of the P450 3A4 indicated that OSI-930 would be oriented properly in the active site for oxidation

  19. Mechanism-based inactivation of human cytochrome P450 enzymes: strategies for diagnosis and drug-drug interaction risk assessment.

    PubMed

    Venkatakrishnan, K; Obach, R S; Rostami-Hodjegan, A

    2007-01-01

    Among drugs that cause pharmacokinetic drug-drug interactions, mechanism-based inactivators of cytochrome P450 represent several of those agents that cause interactions of the greatest magnitude. In vitro inactivation kinetic data can be used to predict the potential for new drugs to cause drug interactions in the clinic. However, several factors exist, each with its own uncertainty, that must be taken into account in order to predict the magnitude of interactions reliably. These include aspects of in vitro experimental design, an understanding of relevant in vivo concentrations of the inactivator, and the extent to which the inactivated enzyme is involved in the clearance of the affected drug. Additionally, the rate of enzyme degradation in vivo is also an important factor that needs to be considered in the prediction of the drug interaction magnitudes. To address mechanism-based inactivation for new drugs, various in vitro experimental approaches have been employed. The selection of approaches for in vitro kinetic characterization of inactivation as well as in vitro-in vivo extrapolation should be guided by the purpose of the exercise and the stage of drug discovery and development, with an increase in the level of sophistication throughout the research and development process.

  20. FTIR studies of the redox partner interaction in cytochrome P450: the Pdx-P450cam couple.

    PubMed

    Karyakin, Andrey; Motiejunas, Domantas; Wade, Rebecca C; Jung, Christiane

    2007-03-01

    Recently we have developed a new approach to study protein-protein interactions using Fourier transform infrared spectroscopy in combination with titration experiments and principal component analysis (FTIR-TPCA). In the present paper we review the FTIR-TPCA results obtained for the interaction between cytochrome P450 and the redox partner protein in two P450 systems, the Pseudomonas putida P450cam (CYP101) with putidaredoxin (P450cam-Pdx), and the Bacillus megaterium P450BM-3 (CYP102) heme domain with the FMN domain (P450BMP-FMND). Both P450 systems reveal similarities in the structural changes that occur upon redox partner complex formation. These involve an increase in beta-sheets and alpha-helix content, a decrease in the population of random coil/3(10)-helix structure, a redistribution of turn structures within the interacting proteins and changes in the protonation states or hydrogen-bonding of amino acid carboxylic side chains. We discuss in detail the P450cam-Pdx interaction in comparison with literature data and conclusions drawn from experiments obtained by other spectroscopic techniques. The results are also interpreted in the context of a 3D structural model of the Pdx-P450cam complex.

  1. Effect of zolpidem on human cytochrome P450 activity, and on transport mediated by P-glycoprotein.

    PubMed

    von Moltke, Lisa L; Weemhoff, James L; Perloff, Michael D; Hesse, Leah M; Harmatz, Jerold S; Roth-Schechter, Barbara F; Greenblatt, David J

    2002-12-01

    The influence of high concentrations of zolpidem (100 microM, corresponding to approximately 200 times maximum therapeutic concentrations) on the activity of six human Cytochrome P450 (CYP) enzymes was evaluated in a model system using human liver microsomes. Zolpidem produced negligible or weak inhibition of human CYP1A2, 2B6, 2C9, 2C19, 2D6, and 3A. Transport of rhodamine 123, presumed to be mediated mainly by the energy-dependent efflux transport protein P-glycoprotein, was studied in a cell culture system using a human intestinal cell line. High concentrations of zolpidem (100 microM), exceeding the usual therapeutic range by more than 100-fold, produced only modest impairment of rhodamine 123 transport. The findings indicate that zolpidem is very unlikely to cause clinical drug interactions attributable to impairment of CYP activity or P-gp mediated transport. Copyright 2002 John Wiley & Sons, Ltd.

  2. Structural features of cytochromes P450 and ligands that affect drug metabolism as revealed by X-ray crystallography and NMR.

    PubMed

    Gay, Sean C; Roberts, Arthur G; Halpert, James R

    2010-09-01

    Cytochromes P450 (P450s) play a major role in the clearance of drugs, toxins, and environmental pollutants. Additionally, metabolism by P450s can result in toxic or carcinogenic products. The metabolism of pharmaceuticals by P450s is a major concern during the design of new drug candidates. Determining the interactions between P450s and compounds of very diverse structures is complicated by the variability in P450-ligand interactions. Understanding the protein structural elements and the chemical attributes of ligands that dictate their orientation in the P450 active site will aid in the development of effective and safe therapeutic agents. The goal of this review is to describe P450-ligand interactions from two perspectives. The first is the various structural elements that microsomal P450s have at their disposal to assume the different conformations observed in X-ray crystal structures. The second is P450-ligand dynamics analyzed by NMR relaxation studies.

  3. Interaction potential of Trigonella foenum graceum through cytochrome P450 mediated inhibition

    PubMed Central

    Ahmmed, Sk Milan; Mukherjee, Pulok K.; Bahadur, Shiv; Kar, Amit; Mukherjee, Kakali; Karmakar, Sanmoy; Bandyopadhyay, Arun

    2015-01-01

    Objective: The seeds of Trigonella foenum-graecum (TFG) (family: Leguminosae) are widely consumed both as a spice in food and Traditional Medicine in India. The present study was undertaken to evaluate the inhibitory effect of standardized extract of TFG and its major constituent trigonelline (TG) on rat liver microsome (RLM) and cytochrome P450 (CYP450) drug metabolizing isozymes (CYP3A4 and CYP2D6), which may indicate the possibility of a probable unwanted interaction. Materials and Methods: Reverse phase-high performance liquid chromatography method was developed to standardize the hydroalcoholic seed extract with standard TG. The inhibitory potential of the extract and TG was evaluated on RLM and CYP isozymes using CYP450-carbon monoxide (CYP450-CO) complex assay and fluorescence assay, respectively. Results: The content of TG in TFG was found to be 3.38% (w/w). The CYP-CO complex assay showed 23.32% inhibition on RLM. Fluorescence study revealed that the extract and the biomarker had some inhibition on CYP450 isozymes e.g. CYP3A4 and CYP2D6 (IC50 values of the extract: 102.65 ± 2.63–142.23 ± 2.61 µg/ml and TG: 168.73 ± 4.03–180.90 ± 2.49 µg/ml) which was very less compared to positive controls ketoconazole and quinidine. Inhibition potential of TFG was little higher than TG but very less compared to positive controls. Conclusions: From the present study, we may conclude that the TFG or TG has very less potential to inhibit the CYP isozymes (CYP3A4, CYP2D6), so administration of this plant extract or its biomarker TG may be safe. PMID:26600643

  4. Cytochrome P450s--Their expression, regulation, and role in insecticide resistance.

    PubMed

    Liu, Nannan; Li, Ming; Gong, Youhui; Liu, Feng; Li, Ting

    2015-05-01

    P450s are known to be critical for the detoxification and/or activation of xenobiotics such as drugs and pesticides and overexpression of P450 genes can significantly affect the disposition of xenobiotics in the tissues of organisms, altering their pharmacological/toxicological effects. In insects, P450s play an important role in detoxifying exogenous compounds such as insecticides and plant toxins and their overexpression can result in increased levels of P450 proteins and P450 activities. This has been associated with enhanced metabolic detoxification of insecticides and has been implicated in the development of insecticide resistance in insects. Multiple P450 genes have been found to be co-overexpressed in individual insect species via several constitutive overexpression and induction mechanisms, which in turn are co-responsible for high levels of insecticide resistance. Many studies have also demonstrated that the transcriptional overexpression of P450 genes in resistant insects is regulated by trans and/or cis regulatory genes/factors. Taken together, these earlier findings suggest not only that insecticide resistance is conferred via multi-resistance P450 genes, but also that it is mediated through the interaction of regulatory genes/factors and resistance genes. This chapter reviews our current understanding of how the molecular mechanisms of P450 interaction/gene regulation govern the development of insecticide resistance in insects and our progress along the road to a comprehensive characterization of P450 detoxification-mediated insecticide resistance. Copyright © 2015 Elsevier Inc. All rights reserved.

  5. Cytochrome P450 (CYP450) Tests

    MedlinePlus

    ... P450 (CYP450) tests Overview Your doctor may use cytochrome P450 (CYP450) tests to help determine how your body processes (metabolizes) a drug. The human body contains P450 enzymes to process medications. Because of inherited (genetic) traits ...

  6. Cytochrome p450 architecture and cysteine nucleophile placement impact raloxifene-mediated mechanism-based inactivation.

    PubMed

    VandenBrink, Brooke M; Davis, John A; Pearson, Josh T; Foti, Robert S; Wienkers, Larry C; Rock, Dan A

    2012-11-01

    The propensity for cytochrome P450 (P450) enzymes to bioactivate xenobiotics is governed by the inherent chemistry of the xenobiotic itself and the active site architecture of the P450 enzyme(s). Accessible nucleophiles in the active site or egress channels of the P450 enzyme have the potential of sequestering reactive metabolites through covalent modification, thereby limiting their exposure to other proteins. Raloxifene, a drug known to undergo CYP3A-mediated reactive metabolite formation and time-dependent inhibition in vitro, was used to explore the potential for bioactivation and enzyme inactivation of additional P450 enzymes (CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A5). Every P450 tested except CYP2E1 was capable of raloxifene bioactivation, based on glutathione adduct formation. However, raloxifene-mediated time-dependent inhibition only occurred in CYP2C8 and CYP3A4. Comparable inactivation kinetics were achieved with K(I) and k(inact) values of 0.26 μM and 0.10 min(-1) and 0.81 μM and 0.20 min(-1) for CYP2C8 and CYP3A4, respectively. Proteolytic digests of CYP2C8 and CYP3A4 Supersomes revealed adducts to Cys225 and Cys239 for CYP2C8 and CYP3A4, respectively. For each P450 enzyme, proposed substrate/metabolite access channels were mapped and active site cysteines were identified, which revealed that only CYP2C8 and CYP3A4 possess accessible cysteine residues near the active site cavities, a result consistent with the observed kinetics. The combined data suggest that the extent of bioactivation across P450 enzymes does not correlate with P450 inactivation. In addition, multiple factors contribute to the ability of reactive metabolites to form apo-adducts with P450 enzymes.

  7. Cytochrome P450-mediated metabolism of vitamin D

    PubMed Central

    Jones, Glenville; Prosser, David E.; Kaufmann, Martin

    2014-01-01

    The vitamin D signal transduction system involves a series of cytochrome P450-containing sterol hydroxylases to generate and degrade the active hormone, 1α,25-dihydroxyvitamin D3, which serves as a ligand for the vitamin D receptor-mediated transcriptional gene expression described in companion articles in this review series. This review updates our current knowledge of the specific anabolic cytochrome P450s involved in 25- and 1α-hydroxylation, as well as the catabolic cytochrome P450 involved in 24- and 23-hydroxylation steps, which are believed to initiate inactivation of the vitamin D molecule. We focus on the biochemical properties of these enzymes; key residues in their active sites derived from crystal structures and mutagenesis studies; the physiological roles of these enzymes as determined by animal knockout studies and human genetic diseases; and the regulation of these different cytochrome P450s by extracellular ions and peptide modulators. We highlight the importance of these cytochrome P450s in the pathogenesis of kidney disease, metabolic bone disease, and hyperproliferative diseases, such as psoriasis and cancer; as well as explore potential future developments in the field. PMID:23564710

  8. DrugMetZ DB: an anthology of human drug metabolizing Chytochrome P450 enzymes.

    PubMed

    Antony, Tresa Remya Thomas; Nagarajan, Shanthi

    2006-11-14

    Understandings the basics of Cytochrome P450 (P450 or CYP) will help to discern drug metabolism. CYP, a super-family of heme-thiolate proteins, are found in almost all living organisms and is involved in the biotransformation of a diverse range of xenobiotics, therapeutic drugs and toxins. Here, we describe DrugMetZ DB, a database for CYP metabolizing drugs. The DB is implemented in MySQL, PHP and HTML. www.bicpu.edu.in/DrugMetZDB/

  9. Metabolism of 4-Aminopiperidine Drugs by Cytochrome P450s: Molecular and Quantum Mechanical Insights into Drug Design

    PubMed Central

    2011-01-01

    4-Aminopiperidines are a variety of therapeutic agents that are extensively metabolized by cytochrome P450s with CYP3A4 as a major isoform catalyzing their N-dealkylation reaction. However, its catalytic mechanism has not been fully elucidated in a molecular interaction level. Here, we applied theoretical approaches including the molecular mechanics-based docking to study the binding patterns and quantum mechanics-based reactivity calculations. They were supported by the experimental human liver microsomal clearance and P450 isoform phenotyping data. Our results herein suggested that the molecular interactions between substrates and CYP3A4 active site residues are essential for the N-dealkylation of 4-aminopiperidines. We also found that the serine 119 residue of CYP3A4 may serve as a key hydrogen-bonding partner to interact with the 4-amino groups of the studied drugs. The reactivity of the side chain α-carbon hydrogens drives the direction of catalysis as well. As a result, structure-based drug design approaches look promising to guide drug discovery programs into the optimized drug metabolism space. PMID:21841964

  10. Binding Specificity Determines the Cytochrome P450 3A4 Mediated Enantioselective Metabolism of Metconazole.

    PubMed

    Zhuang, Shulin; Zhang, Leili; Zhan, Tingjie; Lu, Liping; Zhao, Lu; Wang, Haifei; Morrone, Joseph A; Liu, Weiping; Zhou, Ruhong

    2018-01-25

    Cytochrome P450 3A4 (CYP3A4) is a promiscuous enzyme, mediating the biotransformations of ∼50% of clinically used drugs, many of which are chiral molecules. Probing the interactions between CYP3A4 and chiral chemicals is thus essential for the elucidation of molecular mechanisms of enantioselective metabolism. We developed a stepwise-restrained-molecular-dynamics (MD) method to model human CYP3A4 in a complex with cis-metconazole (MEZ) isomers and performed conventional MD simulations with a total simulation time of 2.2 μs to probe the molecular interactions. Our current study, which employs a combined experimental and theoretical approach, reports for the first time on the distinct conformational changes of CYP3A4 that are induced by the enantioselective binding of cis-MEZ enantiomers. CYP3A4 preferably metabolizes cis-RS MEZ over the cis-SR isomer, with the resultant enantiomer fraction for cis-MEZ increasing rapidly from 0.5 to 0.82. cis-RS MEZ adopts a more extended structure in the active pocket with its Cl atom exposed to the solvent, whereas cis-SR MEZ sits within the hydrophobic core of the active pocket. Free-energy-perturbation calculations indicate that unfavorable van der Waals interactions between the cis-MEZ isomers and the CYP3A4 binding pocket predominantly contribute to their binding-affinity differences. These results demonstrate that binding specificity determines the cytochrome P450 3A4 mediated enantioselective metabolism of cis-MEZ.

  11. A Multiscale Approach to Modelling Drug Metabolism by Membrane-Bound Cytochrome P450 Enzymes

    PubMed Central

    Sansom, Mark S. P.; Mulholland, Adrian J.

    2014-01-01

    Cytochrome P450 enzymes are found in all life forms. P450s play an important role in drug metabolism, and have potential uses as biocatalysts. Human P450s are membrane-bound proteins. However, the interactions between P450s and their membrane environment are not well-understood. To date, all P450 crystal structures have been obtained from engineered proteins, from which the transmembrane helix was absent. A significant number of computational studies have been performed on P450s, but the majority of these have been performed on the solubilised forms of P450s. Here we present a multiscale approach for modelling P450s, spanning from coarse-grained and atomistic molecular dynamics simulations to reaction modelling using hybrid quantum mechanics/molecular mechanics (QM/MM) methods. To our knowledge, this is the first application of such an integrated multiscale approach to modelling of a membrane-bound enzyme. We have applied this protocol to a key human P450 involved in drug metabolism: CYP3A4. A biologically realistic model of CYP3A4, complete with its transmembrane helix and a membrane, has been constructed and characterised. The dynamics of this complex have been studied, and the oxidation of the anticoagulant R-warfarin has been modelled in the active site. Calculations have also been performed on the soluble form of the enzyme in aqueous solution. Important differences are observed between the membrane and solution systems, most notably for the gating residues and channels that control access to the active site. The protocol that we describe here is applicable to other membrane-bound enzymes. PMID:25033460

  12. A multiscale approach to modelling drug metabolism by membrane-bound cytochrome P450 enzymes.

    PubMed

    Lonsdale, Richard; Rouse, Sarah L; Sansom, Mark S P; Mulholland, Adrian J

    2014-07-01

    Cytochrome P450 enzymes are found in all life forms. P450s play an important role in drug metabolism, and have potential uses as biocatalysts. Human P450s are membrane-bound proteins. However, the interactions between P450s and their membrane environment are not well-understood. To date, all P450 crystal structures have been obtained from engineered proteins, from which the transmembrane helix was absent. A significant number of computational studies have been performed on P450s, but the majority of these have been performed on the solubilised forms of P450s. Here we present a multiscale approach for modelling P450s, spanning from coarse-grained and atomistic molecular dynamics simulations to reaction modelling using hybrid quantum mechanics/molecular mechanics (QM/MM) methods. To our knowledge, this is the first application of such an integrated multiscale approach to modelling of a membrane-bound enzyme. We have applied this protocol to a key human P450 involved in drug metabolism: CYP3A4. A biologically realistic model of CYP3A4, complete with its transmembrane helix and a membrane, has been constructed and characterised. The dynamics of this complex have been studied, and the oxidation of the anticoagulant R-warfarin has been modelled in the active site. Calculations have also been performed on the soluble form of the enzyme in aqueous solution. Important differences are observed between the membrane and solution systems, most notably for the gating residues and channels that control access to the active site. The protocol that we describe here is applicable to other membrane-bound enzymes.

  13. Defective Cytochrome P450-Catalysed Drug Metabolism in Niemann-Pick Type C Disease

    PubMed Central

    Wassif, Christopher A.; Gray, James; Burkert, Kathryn R.; Smith, David A.; Morris, Lauren; Cologna, Stephanie M.; Peer, Cody J.; Sissung, Tristan M.; Uscatu, Constantin-Daniel; Figg, William D.; Pavan, William J.; Vite, Charles H.; Porter, Forbes D.; Platt, Frances M.

    2016-01-01

    Niemann-Pick type C (NPC) disease is a neurodegenerative lysosomal storage disease caused by mutations in either the NPC1 or NPC2 gene. NPC is characterised by storage of multiple lipids in the late endosomal/lysosomal compartment, resulting in cellular and organ system dysfunction. The underlying molecular mechanisms that lead to the range of clinical presentations in NPC are not fully understood. While evaluating potential small molecule therapies in Npc1-/- mice, we observed a consistent pattern of toxicity associated with drugs metabolised by the cytochrome P450 system, suggesting a potential drug metabolism defect in NPC1 disease. Investigation of the P450 system in the context of NPC1 dysfunction revealed significant changes in the gene expression of many P450 associated genes across the full lifespan of Npc1-/- mice, decreased activity of cytochrome P450 reductase, and a global decrease of multiple cytochrome P450 catalysed dealkylation reactions. In vivo drug metabolism studies using a prototypic P450 metabolised drug, midazolam, confirmed dysfunction in drug clearance in the Npc1-/- mouse. Expression of the Phase II enzyme uridinediphosphate-glucuronosyltransferase (UGT) was also significantly reduced in Npc1-/- mice. Interestingly, reduced activity within the P450 system was also observed in heterozygous Npc1+/- mice. The reduced activity of P450 enzymes may be the result of bile acid deficiency/imbalance in Npc1-/- mice, as bile acid treatment significantly rescued P450 enzyme activity in Npc1-/- mice and has the potential to be an adjunctive therapy for NPC disease patients. The dysfunction in the cytochrome P450 system were recapitulated in the NPC1 feline model. Additionally, we present the first evidence that there are alterations in the P450 system in NPC1 patients. PMID:27019000

  14. WhichP450: a multi-class categorical model to predict the major metabolising CYP450 isoform for a compound

    NASA Astrophysics Data System (ADS)

    Hunt, Peter A.; Segall, Matthew D.; Tyzack, Jonathan D.

    2018-02-01

    In the development of novel pharmaceuticals, the knowledge of how many, and which, Cytochrome P450 isoforms are involved in the phase I metabolism of a compound is important. Potential problems can arise if a compound is metabolised predominantly by a single isoform in terms of drug-drug interactions or genetic polymorphisms that would lead to variations in exposure in the general population. Combined with models of regioselectivities of metabolism by each isoform, such a model would also aid in the prediction of the metabolites likely to be formed by P450-mediated metabolism. We describe the generation of a multi-class random forest model to predict which, out of a list of the seven leading Cytochrome P450 isoforms, would be the major metabolising isoforms for a novel compound. The model has a 76% success rate with a top-1 criterion and an 88% success rate for a top-2 criterion and shows significant enrichment over randomised models.

  15. [Cytochrome P-450 and the response to antimalarial drugs].

    PubMed

    Guzmán, Valentina; Carmona-Fonseca, Jaime

    2006-01-01

    To assess the relationship between the genetic and phenotypic factors linked to the cytochrome P-450 enzyme system and the response to the antimalarial drugs chloroquine, amodiaquine, mefloquine, and proguanil, as well as to determine how certain biological and social factors of the host influence the behavior of this enzymatic complex. We performed a systematic review of the medical bibliographic databases PubMed, Excerpta Medica, LILACS, and SciELO by using the following Spanish and English descriptors: "CYP-450" and "citocromo P-450" in combination with "proguanil" (and with "mefloquina," "cloroquina," and "amodiaquina"), "farmacocinética de proguanil" (and the same using "mefloquina," "cloroquina," and "amodiaquina"), "resistencia a proguanil" (and the same using "mefloquina," "cloroquina," and "amodiaquina"), "metabolismo," "farmacogenética," "enfermedad," "inflamación," "infección," "enfermedad hepática," "malaria," "nutrición," and "desnutrición." The same terms were used in English. The search included only articles published in Spanish, English, and Portuguese on or before 30 June 2005 that dealt with only four antimalarial drugs: amodiaquine, chloroquine, mefloquine, and proguanil. Some genetic factors linked to human cytochrome P-450 (mainly its polymorphism), as well as other biological and social factors (the presence of disease itself, or of inflammation and infection, the use of antimalarials in their various combinations, and the patient's nutritional status) influence the behavior of this complex enzymatic system. It has only been in the last decade that the genetics of the cytochromes has been explored and that the mechanisms underlying some therapeutic interactions and aspects of drug metabolism have been uncovered, making it possible to characterize the biotransformation pathway of amodiaquine and chloroquine. Hopefully new research will help answer the questions that still remain, some of which pertain to the metabolism of other

  16. Drug-enhanced carbon monoxide production from heme by cytochrome P450 reductase.

    PubMed

    Vukomanovic, Dragic; Rahman, Mona N; Jia, Zongchao; Nakatsu, Kanji

    2017-01-01

    Carbon monoxide (CO) formed endogenously is considered to be cytoprotective, and the vast majority of CO formation is attributed to the degradation of heme by heme oxygenases-1 and -2 (HO-1, HO-2). Previously, we observed that brain microsomes containing HO-2 produced many-fold more CO in the presence of menadione and its congeners; herein we explored these observations further. We determined the effects of various drugs on CO production of rat brain microsomes and recombinant human cytochrome P450 reductase (CPR); CO was measured by gas chromatography with reductive detection. Brain microsomes of Sprague-Dawley rats or recombinant human cytochrome P450 reductase (CPR) were incubated with NADPH and various drugs in closed vials in phosphate buffer at pH 7.4 and 37°C. After 15 minutes, the reaction was stopped by cooling in dry ice, and the headspace gas was analyzed for CO production using gas chromatography with reductive (mercuric oxide) detection. We observed drug-enhanced CO production in the presence of both microsomes and recombinant CPR alone; the presence of HO was not required. A range of structurally diverse drugs were capable of amplifying this CO formation; these molecules had structures consistent with redox cycling capability. The addition of catalase to a reaction mixture, that contained activating drugs, inhibited the production of CO. Drug-enhanced CO formation can be catalyzed by CPR. The mechanism of CPR activation was not through classical drug-receptor mediation. Redox cycling may be involved in the drug-induced amplification of CO production by CPR through the production of reactive oxygen species.

  17. Microbial P450 Enzymes in Bioremediation and Drug Discovery: Emerging Potentials and Challenges.

    PubMed

    Bhattacharya, Sukanta S; Yadav, Jagjit S

    2018-01-01

    Cytochrome P450 enzymes are a structurally conserved but functionally diverse group of heme-containing mixed function oxidases found across both prokaryotic and eukaryotic forms of the microbial world. Microbial P450s are known to perform diverse functions ranging from the synthesis of cell wall components to xenobiotic/drug metabolism to biodegradation of environmental chemicals. Conventionally, many microbial systems have been reported to mimic mammalian P450-like activation of drugs and were proposed as the in-vitro models of mammalian drug metabolism. Recent reports suggest that native or engineered forms of specific microbial P450s from these and other microbial systems could be employed for desired specific biotransformation reactions toward natural and synthetic (drug) compounds underscoring their emerging potential in drug improvement and discovery. On the other hand, microorganisms particularly fungi and actinomycetes have been shown to possess catabolic P450s with unusual potential to degrade toxic environmental chemicals including persistent organic pollutants (POPs). Wood-rotting basidiomycete fungi in particular have revealed the presence of exceptionally large P450 repertoire (P450ome) in their genomes, majority of which are however orphan (with no known function). Our pre- and post-genomic studies have led to functional characterization of several fungal P450s inducible in response to exposure to several environmental toxicants and demonstration of their potential in bioremediation of these chemicals. This review is an attempt to summarize the postgenomic unveiling of this versatile enzyme superfamily in microbial systems and investigation of their potential to synthesize new drugs and degrade persistent pollutants, among other biotechnological applications. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  18. Effect of gamma-oryzanol on cytochrome P450 activities in human liver microsomes.

    PubMed

    Umehara, Ken; Shimokawa, Yoshihiko; Miyamoto, Gohachiro

    2004-07-01

    The effects of gamma-oryzanol, a drug mainly used for the treatment of hyperlipidaemia, on several cytochrome P450 (CYP) specific reactions in human liver microsomes were investigated to predict drug interactions with gamma-oryzanol in vivo from in vitro data. The following eight CYP catalytic reactions were used in this study: CYP1A1/2-mediated 7-ethoxyresorufin O-deethylation, CYP2A6-mediated coumarin 7-hydroxylation, CYP2B6-mediated 7-benzyloxyresorufin O-debenzylation, CYP2C8/9-mediated tolbutamide methylhydroxylation, CYP2C19-mediated S-mephenytoin 4'-hydroxylation, CYP2D6-mediated bufuralol 1'-hydroxylation, CYP2E1-mediated chlorzoxazone 6-hydroxylation, and CYP3A4-mediated testosterone 6beta-hydroxylation. gamma-Oryzanol had little inhibitory effects on CYP activities, indicating that this compound would not be expected to cause clinically significant interactions with other CYP-metabolized drugs at expected therapeutic concentrations.

  19. Interactions between Cytochromes P450 2B4 (CYP2B4) and 1A2 (CYP1A2) Lead to Alterations in Toluene Disposition and P450 Uncoupling

    PubMed Central

    Reed, James R.; Cawley, George F.; Backes, Wayne L.

    2013-01-01

    The goal of this study was to characterize the effects of CYP1A2•CYP2B4 complex formation on the rates and efficiency of toluene metabolism by comparing the results from simple reconstituted systems containing P450 reductase (CPR) and a single P450 to those using a mixed system containing CPR and both P450s. In the mixed system, the rates of formation of CYP2B4-specific benzyl alcohol and p-cresol were inhibited, whereas that of CYP1A2-specific o-cresol was increased, results consistent with the formation of a CYP1A2•CYP2B4 complex where the CYP1A2 moiety has higher affinity for CPR binding. Comparison of the rates of NADPH oxidation and production of hydrogen peroxide and excess water by the simple and mixed systems indicated that excess water formed at a much lower rate in the mixed system. The commensurate increase in the rate of CYP1A2-specific product formation suggested the P450P450 interaction increased the putative rate-limiting step of CYP1A2 catalysis, abstraction of a hydrogen radical from the substrate. Cumene hydroperoxide-supported metabolism was measured to determine whether the effects of the P450P450 interaction required the presence of CPR. Peroxidative metabolism was not affected by the interaction of the two P450s, even with CPR present. However, CPR did stimulate peroxidative metabolism by the simple system containing CYP1A2. These results suggest the major functional effects of the P450P450 interaction are mediated by changes in the relative abilities of the P450s to receive electrons from CPR. Furthermore, CPR may play an effector role by causing a conformation change in CYP1A2 that makes its metabolism more efficient. PMID:23675771

  20. VX-509 (Decernotinib)-Mediated CYP3A Time-Dependent Inhibition: An Aldehyde Oxidase Metabolite as a Perpetrator of Drug-Drug Interactions.

    PubMed

    Zetterberg, Craig; Maltais, Francois; Laitinen, Leena; Liao, Shengkai; Tsao, Hong; Chakilam, Ananthsrinivas; Hariparsad, Niresh

    2016-08-01

    (R)-2-((2-(1H-pyrrolo[2,3-b]pyridin-3-yl)pyrimidin-4-yl)amino)-2-methyl-N-(2,2,2-trifluoroethyl)butanamide (VX-509, decernotinib) is an oral Janus kinase 3 inhibitor that has been studied in patients with rheumatoid arthritis. Patients with rheumatoid arthritis often receive multiple medications, such as statins and steroids, to manage the signs and symptoms of comorbidities, which increases the chances of drug-drug interactions (DDIs). Mechanism-based inhibition is a subset of time-dependent inhibition (TDI) and occurs when a molecule forms a reactive metabolite which irreversibly binds and inactivates drug-metabolizing enzymes, potentially increasing the systemic load to toxic concentrations. Traditionally, perpetrating compounds are screened using human liver microsomes (HLMs); however, this system may be inadequate when the precipitant is activated by a non-cytochrome P450 (P450)-mediated pathway. Even though studies assessing competitive inhibition and TDI using HLM suggested a low risk for CYP3A4-mediated DDI in the clinic, VX-509 increased the area under the curve of midazolam, atorvastatin, and methyl-prednisolone by approximately 12.0-, 2.7-, and 4.3-fold, respectively. Metabolite identification studies using human liver cytosol indicated that VX-509 is converted to an oxidative metabolite, which is the perpetrator of the DDIs observed in the clinic. As opposed to HLM, hepatocytes contain the full complement of drug-metabolizing enzymes and transporters and can be used to assess TDI arising from non-P450-mediated metabolic pathways. In the current study, we highlight the role of aldehyde oxidase in the formation of the hydroxyl-metabolite of VX-509, which is involved in clinically significant TDI-based DDIs and represents an additional example in which a system-dependent prediction of TDI would be evident. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

  1. Regio- and Stereo-Selective Oxidation of a Cardiovascular Drug, Metoprolol, Mediated by Cytochrome P450 2D and 3A Enzymes in Marmoset Livers.

    PubMed

    Uehara, Shotaro; Ishii, Sakura; Uno, Yasuhiro; Inoue, Takashi; Sasaki, Erika; Yamazaki, Hiroshi

    2017-08-01

    A β -blocker, metoprolol, is one of the in vivo probes for human cytochrome P450 (P450) 2D6. Investigation of nonhuman primate P450 enzymes helps to improve the accuracy of the extrapolation of pharmacokinetic data from animals into humans. Common marmosets ( Callithrix jacchus ) are a potential primate model for preclinical research, but the detailed roles of marmoset P450 enzymes in metoprolol oxidation remain unknown. In this study, regio- and stereo-selectivity of metoprolol oxidations by a variety of P450 enzymes in marmoset and human livers were investigated in vitro. Although liver microsomes from cynomolgus monkeys and rats preferentially mediated S -metoprolol O -demethylation and R -metoprolol α -hydroxylation, respectively, those from humans, marmosets, minipigs, and dogs preferentially mediated R -metoprolol O -demethylation, in contrast to the slow rates of R - and S -metoprolol oxidation in mouse liver microsomes. R - and S -metoprolol O -demethylation activities in marmoset livers were strongly inhibited by quinidine and ketoconazole, and were significantly correlated with bufuralol 1'-hydroxylation and midazolam 1'-hydroxylation activities and also with P450 2D and 3A4 contents, which is different from the case in human livers that did not have any correlations with P450 3A-mediated midazolam 1'-hydroxylation. Recombinant human P450 2D6 enzyme and marmoset P450 2D6/3A4 enzymes effectively catalyzed R -metoprolol O -demethylation, comparable to the activities of human and marmoset liver microsomes, respectively. These results indicated that the major roles of P450 2D enzymes for the regio- and stereo-selectivity of metoprolol oxidation were similar between human and marmoset livers, but the minor roles of P450 3A enzymes were unique to marmosets. Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.

  2. An in vitro evaluation of cytochrome P450 inhibition and P-glycoprotein interaction with goldenseal, Ginkgo biloba, grape seed, milk thistle, and ginseng extracts and their constituents.

    PubMed

    Etheridge, Amy S; Black, Sherry R; Patel, Purvi R; So, James; Mathews, James M

    2007-07-01

    Drug-herb interactions can result from the modulation of the activities of cytochrome P450 (P450) and/or drug transporters. The effect of extracts and individual constituents of goldenseal, Ginkgo biloba (and its hydrolyzate), grape seed, milk thistle, and ginseng on the activities of cytochrome P450 enzymes CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4 in human liver microsomes were determined using enzyme-selective probe substrates, and their effect on human P-glycoprotein (Pgp) was determined using a baculovirus expression system by measuring the verapamil-stimulated, vanadate-sensitive ATPase activity. Extracts were analyzed by HPLC to standardize their concentration(s) of constituents associated with the pharmacological activity, and to allow comparison of their effects on P450 and Pgp with literature values. Many of the extracts/constituents exerted > or = 50 % inhibition of P450 activity. These include those from goldenseal (normalized to alkaloid content) inhibiting CYP2C8, CYP2D6, and CYP3A4 at 20 microM, ginkgo inhibiting CYP2C8 at 10 microM, grape seed inhibiting CYP2C9 and CYP3A4 at 10 microM, milk thistle inhibiting CYP2C8 at 10 microM, and ginsenosides F1 and Rh1 (but not ginseng extract) inhibiting CYP3A4 at 10 microM. Goldenseal extracts/constituents (20 microM, particularly hydrastine) and ginsenoside Rh1 stimulated ATPase at about half of the activity of the model substrate, verapamil (20 microM). The data suggest that the clearance of a variety of drugs may be diminished by concomitant use of these herbs via inhibition of P450 enzymes, but less so by Pgp-mediated effects.

  3. Identification of human cytochrome P450s as autoantigens.

    PubMed

    Manns, M P; Johnson, E F

    1991-01-01

    Antimicrosomal antibodies in inflammatory liver diseases all seem to be directed against members of the cytochrome P450 family of proteins. These autoantigens seem to be genetically polymorphic, the autoantibodies are inhibitory, and the autoepitopes are generally conserved among species. Anti-P450 autoantibodies share these characteristics with other autoantibodies, for example, antinuclear antibodies in systemic lupus erythematosus. The identification of P450s as human autoantigens is clinically important. Diagnostic tests will be developed on the basis of cloned antigen, facilitating a better diagnosis of drug-induced and idiopathic autoimmune hepatitis. It is unknown what triggers autoantibody production against cytochrome P450 proteins. Furthermore, their pathogenetic role and thus their involvement in tissue destruction is unclear. In this context LKM1 autoantibodies may serve as a model. Although LKM1 antibodies are inhibitory, all LKM1 antibody-positive patients tested so far are extensive metabolizers for drug metabolism mediated by P450IID6 and express this protein in their livers. Thus, the inhibitory LKM1 autoantibody does not sufficiently penetrate through the intact liver cell membrane to inhibit enzyme function in vivo. Presumably, tissue destruction in autoimmune hepatitis is mediated by liver-infiltrating T lymphocytes. T lymphocytes have been cloned from liver tissue that specifically proliferate in the presence of recombinant cytochrome P450IID6. The construction of overlapping cDNA subclones is also valuable to identify immunodominant B cell as well as relevant T cell epitopes.

  4. Inactivation of the hepatic cytochrome P450 system by conditional deletion of hepatic cytochrome P450 reductase.

    PubMed

    Henderson, Colin J; Otto, Diana M E; Carrie, Dianne; Magnuson, Mark A; McLaren, Aileen W; Rosewell, Ian; Wolf, C Roland

    2003-04-11

    Cytochrome P450 (CYP) monooxygenases catalyze the oxidation of a large number of endogenous compounds and the majority of ingested environmental chemicals, leading to their elimination and often to their metabolic activation to toxic products. This enzyme system therefore provides our primary defense against xenobiotics and is a major determinant in the therapeutic efficacy of pharmacological agents. To evaluate the importance of hepatic P450s in normal homeostasis, drug pharmacology, and chemical toxicity, we have conditionally deleted the essential electron transfer protein, NADH:ferrihemoprotein reductase (EC, cytochrome P450 reductase, CPR) in the liver, resulting in essentially complete ablation of hepatic microsomal P450 activity. Hepatic CPR-null mice could no longer break down cholesterol because of their inability to produce bile acids, and whereas hepatic lipid levels were significantly increased, circulating levels of cholesterol and triglycerides were severely reduced. Loss of hepatic P450 activity resulted in a 5-fold increase in P450 protein, indicating the existence of a negative feedback pathway regulating P450 expression. Profound changes in the in vivo metabolism of pentobarbital and acetaminophen indicated that extrahepatic metabolism does not play a major role in the disposition of these compounds. Hepatic CPR-null mice developed normally and were able to breed, indicating that hepatic microsomal P450-mediated steroid hormone metabolism is not essential for fertility, demonstrating that a major evolutionary role for hepatic P450s is to protect mammals from their environment.

  5. QSAR Modeling and Prediction of Drug-Drug Interactions.

    PubMed

    Zakharov, Alexey V; Varlamova, Ekaterina V; Lagunin, Alexey A; Dmitriev, Alexander V; Muratov, Eugene N; Fourches, Denis; Kuz'min, Victor E; Poroikov, Vladimir V; Tropsha, Alexander; Nicklaus, Marc C

    2016-02-01

    Severe adverse drug reactions (ADRs) are the fourth leading cause of fatality in the U.S. with more than 100,000 deaths per year. As up to 30% of all ADRs are believed to be caused by drug-drug interactions (DDIs), typically mediated by cytochrome P450s, possibilities to predict DDIs from existing knowledge are important. We collected data from public sources on 1485, 2628, 4371, and 27,966 possible DDIs mediated by four cytochrome P450 isoforms 1A2, 2C9, 2D6, and 3A4 for 55, 73, 94, and 237 drugs, respectively. For each of these data sets, we developed and validated QSAR models for the prediction of DDIs. As a unique feature of our approach, the interacting drug pairs were represented as binary chemical mixtures in a 1:1 ratio. We used two types of chemical descriptors: quantitative neighborhoods of atoms (QNA) and simplex descriptors. Radial basis functions with self-consistent regression (RBF-SCR) and random forest (RF) were utilized to build QSAR models predicting the likelihood of DDIs for any pair of drug molecules. Our models showed balanced accuracy of 72-79% for the external test sets with a coverage of 81.36-100% when a conservative threshold for the model's applicability domain was applied. We generated virtually all possible binary combinations of marketed drugs and employed our models to identify drug pairs predicted to be instances of DDI. More than 4500 of these predicted DDIs that were not found in our training sets were confirmed by data from the DrugBank database.

  6. Antiepileptic drugs affect neuronal androgen signaling via a cytochrome P450-dependent pathway.

    PubMed

    Gehlhaus, Marcel; Schmitt, Nina; Volk, Benedikt; Meyer, Ralf P

    2007-08-01

    Recent data imply an important role for brain cytochrome P450 (P450) in endocrine signaling. In epileptic patients, treatment with P450 inducers led to reproductive disorders; in mouse hippocampus, phenytoin treatment caused concomitant up-regulation of CYP3A11 and androgen receptor (AR). In the present study, we established specific in vitro models to examine whether CYP3A isoforms cause enhanced AR expression and activation. Murine Hepa1c1c7 cells and neuronal-type rat PC-12 cells were used to investigate P450 regulation and its effects on AR after phenytoin and phenobarbital administration. In both cell lines, treatment with antiepileptic drugs (AEDs) led to concomitant up-regulation of CYP3A (CYP3A11 in Hepa1c1c7 and CYP3A2 in PC-12) and AR mRNA and protein. Inhibition of CYP3A expression and activity by the CYP3A inhibitor ketoconazole or by CYP3A11-specific short interfering RNA molecules reduced AR expression to basal levels. The initial up-regulation of AR signal transduction, measured by an androgen-responsive element chloramphenicol-acetyltransferase reporter gene assay, was completely reversed after specific inhibition of CYP3A11. Withdrawal of the CYP3A11 substrate testosterone prevented AR activation, whereas AR mRNA expression remained up-regulated. In addition, recombinant CYP3A11 was expressed heterologously in PC-12 cells, thereby eliminating any direct drug influence on the AR. Again, the initial up-regulation of AR mRNA and activity was reduced to basal levels after silencing of CYP3A11. In conclusion, we show here that CYP3A2 and CYP3A11 are crucial mediators of AR expression and signaling after AED application. These findings point to an important and novel function of P450 in regulation of steroid hormones and their receptors in endocrine tissues such as liver and brain.

  7. Cytochrome P450-Mediated Phytoremediation using Transgenic Plants: A Need for Engineered Cytochrome P450 Enzymes

    PubMed Central

    Kumar, Santosh; Jin, Mengyao; Weemhoff, James L

    2013-01-01

    There is an increasing demand for versatile and ubiquitous Cytochrome P450 (CYP) biocatalysts for biotechnology, medicine, and bioremediation. In the last decade there has been an increase in realization of the power of CYP biocatalysts for detoxification of soil and water contaminants using transgenic plants. However, the major limitations of mammalian CYP enzymes are that they require CYP reductase (CPR) for their activity, and they show relatively low activity, stability, and expression. On the other hand, bacterial CYP enzymes show limited substrate diversity and usually do not metabolize herbicides and industrial contaminants. Therefore, there has been a considerable interest for biotechnological industries and the scientific community to design CYP enzymes to improve their catalytic efficiency, stability, expression, substrate diversity, and the suitability of P450-CPR fusion enzymes. Engineered CYP enzymes have potential for transgenic plants-mediated phytoremediation of herbicides and environmental contaminants. In this review we discuss: 1) the role of CYP enzymes in phytoremediation using transgenic plants, 2) problems associated with wild-type CYP enzymes in phytoremediation, and 3) examples of engineered CYP enzymes and their potential role in transgenic plant-mediated phytoremediation. PMID:25298920

  8. The Evaluation of CP-001 (a Standardized Herbal Mixture of Houttuynia cordata, Rehmannia glutinosa, Betula platyphylla, and Rubus coreanus) for Cytochrome P450-Related Herb-Drug Interactions

    PubMed Central

    Yoo, Hye Hyun; Kim, Sun-A; Kim, In Sook; Ko, Seong-Gyu

    2013-01-01

    In the present study, the effect of CP-001, a standardized herbal mixture of Houttuynia cordata, Rehmannia glutinosa, Betula platyphylla, and Rubus coreanus, on cytochrome P450 (CYP) enzyme-mediated drug metabolism was investigated in vitro to evaluate the potential for herb-drug interactions. CP-001 was tested at concentrations of 1, 3, 10, 30, and 100 μg/mL. A CYP-specific substrate mixture was incubated with CP-001 in human liver microsomes, and the metabolites generated by each CYP-specific metabolic reaction were measured by liquid chromatography-tandem mass spectrometry. CP-001 seemed to slightly inhibit some CYP isozymes, but the IC50 values for all CYP isozymes were greater than 100 μg/mL. Furthermore, CP-001 did not exhibit time-dependent CYP inhibitory activities, indicating that it does not act as a mechanism-based inactivator of CYP enzymes. In conclusion, the effects of CP-001 on CYP isozyme activities were negligible at the concentrations tested. Therefore, the likelihood of herbal mixture-drug interaction is considered minimal. PMID:23935684

  9. Cytochrome P450-mediated metabolic engineering: current progress and future challenges.

    PubMed

    Renault, Hugues; Bassard, Jean-Etienne; Hamberger, Björn; Werck-Reichhart, Danièle

    2014-06-01

    Cytochromes P450 catalyze a broad range of regiospecific, stereospecific and irreversible steps in the biosynthetic routes of plant natural metabolites with important applications in pharmaceutical, cosmetic, fragrance and flavour, or polymer industries. They are consequently essential drivers for the engineered bioproduction of such compounds. Two ground-breaking developments of commercial products driven by the engineering of P450s are the antimalarial drug precursor artemisinic acid and blue roses or carnations. Tedious optimizations were required to generate marketable products. Hurdles encountered in P450 engineering and their potential solutions are summarized here. Together with recent technical developments and novel approaches to metabolic engineering, the lessons from this pioneering work should considerably boost exploitation of the amazing P450 toolkit emerging from accelerated sequencing of plant genomes. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.

  10. The Use of Human Liver Cell Model and Cytochrome P450 Substrate-Inhibitor Panel for Studies of Dasatinib and Warfarin Interactions.

    PubMed

    Zakharyants, A A; Burmistrova, O A; Poloznikov, A A

    2017-02-01

    The possibility of interactions between warfarin and dasatinib and their interactions with other drugs metabolized by cytochrome P450 isoform CYP3A4 was demonstrated using a previously created cytochrome P450 substrate-inhibitor panel for preclinical in vitro studies of drug biotransformation on a 3D histotypical microfluidic cell model of human liver (liver-on-a-chip technology). Dasatinib and warfarin are inhibitors of CYP2C19 isoform and hence, can interfere the drugs metabolized by this isoform. Our findings are in line with the data obtained on primary culture of human hepatocytes and suggest that the model can be used in preclinical in vitro studies of drugs.

  11. New reactions and products resulting from alternative interactions between the P450 enzyme and redox partners.

    PubMed

    Zhang, Wei; Liu, Yi; Yan, Jinyong; Cao, Shaona; Bai, Fali; Yang, Ying; Huang, Shaohua; Yao, Lishan; Anzai, Yojiro; Kato, Fumio; Podust, Larissa M; Sherman, David H; Li, Shengying

    2014-03-05

    Cytochrome P450 enzymes are capable of catalyzing a great variety of synthetically useful reactions such as selective C-H functionalization. Surrogate redox partners are widely used for reconstitution of P450 activity based on the assumption that the choice of these auxiliary proteins or their mode of action does not affect the type and selectivity of reactions catalyzed by P450s. Herein, we present an exceptional example to challenge this postulate. MycG, a multifunctional biosynthetic P450 monooxygenase responsible for hydroxylation and epoxidation of 16-membered ring macrolide mycinamicins, is shown to catalyze the unnatural N-demethylation(s) of a range of mycinamicin substrates when partnered with the free Rhodococcus reductase domain RhFRED or the engineered Rhodococcus-spinach hybrid reductase RhFRED-Fdx. By contrast, MycG fused with the RhFRED or RhFRED-Fdx reductase domain mediates only physiological oxidations. This finding highlights the larger potential role of variant redox partner protein-protein interactions in modulating the catalytic activity of P450 enzymes.

  12. Immobilized Cytochrome P450 2C9 (CYP2C9): Applications for Metabolite Generation, Monitoring Protein-Protein Interactions, and Improving In-vivo Predictions Using Enhanced In-vitro Models

    NASA Astrophysics Data System (ADS)

    Wollenberg, Lance A.

    Cytochrome P450 (P450) enzymes are a family of oxoferroreductase enzymes containing a heme moiety and are well known to be involved in the metabolism of a wide variety of endogenous and xenobiotic materials. It is estimated that roughly 75% of all pharmaceutical compounds are metabolized by these enzymes. Traditional reconstituted in-vitro incubation studies using recombinant P450 enzymes are often used to predict in-vivo kinetic parameters of a drug early in development. However, in many cases, these reconstituted incubations are prone to aggregation which has been shown to affect the catalytic activity of an enzyme. Moreover, the presence of other isoforms of P450 enzymes present in a metabolic incubation, as is the case with microsomal systems, may affect the catalytic activity of an enzyme through isoform-specific protein-protein interactions. Both of these effects may result in inaccurate prediction of in-vivo drug metabolism using in-vitro experiments. Here we described the development of immobilized P450 constructs designed to elucidate the effects of aggregation and protein-protein interactions between P450 isoforms on catalytic activities. The long term objective of this project is to develop a system to control the oligomeric state of Cytochrome P450 enzymes to accurately elucidate discrepancies between in vitro reconstituted systems and actual in vivo drug metabolism for the precise prediction of metabolic activity. This approach will serve as a system to better draw correlations between in-vivo and in-vitro drug metabolism data. The central hypothesis is that Cytochrome P450 enzymes catalytic activity can be altered by protein-protein interactions occurring between Cytochrome P450 enzymes involved in drug metabolism, and is dependent on varying states of protein aggregation. This dissertation explains the details of the construction and characterization of a nanostructure device designed to control the state of aggregation of a P450 enzyme. Moreover

  13. Genetic determinants of drug responsiveness and drug interactions.

    PubMed

    Caraco, Y

    1998-10-01

    Six cytochrome P450 enzymes mediate the oxidative metabolism of most drugs in common use: CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4. These enzymes have selective substrate specificity, and their activity is characterized by marked interindividual variation. Some of these systems (CYP2C19, CYP2D6) are polymorphically distributed; thus, a subset of the population may be genetically deficient in enzyme activity. Phenotyping procedures designed to identify subjects with impaired metabolism who may be at increased risk for drug toxicity have been developed and validated. This has been supplemented in recent years by the availability of genetic analysis and the identification of specific alleles that are associated with altered (i.e., reduced, deficient, or increased) enzyme activity. The potential of genotyping to predict pharmacodynamics holds great promise for the future because it does not involve the administration of exogenous compound and is not confounded by drug therapy. Drug interactions caused by the inhibition or induction of oxidative drug metabolism may be of great clinical importance because they may result in drug toxicity or therapeutic failure. Further understanding of cytochrome P450 complexity may allow, through a combined in vitro-in vivo approach, the reliable prediction and possible prevention of deleterious drug interactions.

  14. Novel approaches to mitigating parathion toxicity: targeting cytochrome P450-mediated metabolism with menadione.

    PubMed

    Jan, Yi-Hua; Richardson, Jason R; Baker, Angela A; Mishin, Vladimir; Heck, Diane E; Laskin, Debra L; Laskin, Jeffrey D

    2016-08-01

    Accidental or intentional exposures to parathion, an organophosphorus (OP) pesticide, can cause severe poisoning in humans. Parathion toxicity is dependent on its metabolism by the cytochrome P450 (CYP) system to paraoxon (diethyl 4-nitrophenyl phosphate), a highly poisonous nerve agent and potent inhibitor of acetylcholinesterase. We have been investigating inhibitors of CYP-mediated bioactivation of OPs as a method of preventing or reversing progressive parathion toxicity. It is well recognized that NADPH-cytochrome P450 reductase, an enzyme required for the transfer of electrons to CYPs, mediates chemical redox cycling. In this process, the enzyme diverts electrons from CYPs to support chemical redox cycling, which results in inhibition of CYP-mediated biotransformation. Using menadione as the redox-cycling chemical, we discovered that this enzymatic reaction blocks metabolic activation of parathion in rat and human liver microsomes and in recombinant CYPs important to parathion metabolism, including CYP1A2, CYP2B6, and CYP3A4. Administration of menadione to rats reduces metabolism of parathion, as well as parathion-induced inhibition of brain cholinesterase activity. This resulted in inhibition of parathion neurotoxicity. Menadione has relatively low toxicity and is approved by the Food and Drug Administration for other indications. Its ability to block parathion metabolism makes it an attractive therapeutic candidate to mitigate parathion-induced neurotoxicity. © 2016 New York Academy of Sciences.

  15. The Role of Protein-Protein and Protein-Membrane Interactions on P450 Function

    PubMed Central

    Scott, Emily E.; Wolf, C. Roland; Otyepka, Michal; Humphreys, Sara C.; Reed, James R.; Henderson, Colin J.; McLaughlin, Lesley A.; Paloncýová, Markéta; Navrátilová, Veronika; Berka, Karel; Anzenbacher, Pavel; Dahal, Upendra P.; Barnaba, Carlo; Brozik, James A.; Jones, Jeffrey P.; Estrada, D. Fernando; Laurence, Jennifer S.; Park, Ji Won

    2016-01-01

    This symposium summary, sponsored by the ASPET, was held at Experimental Biology 2015 on March 29, 2015, in Boston, Massachusetts. The symposium focused on: 1) the interactions of cytochrome P450s (P450s) with their redox partners; and 2) the role of the lipid membrane in their orientation and stabilization. Two presentations discussed the interactions of P450s with NADPH-P450 reductase (CPR) and cytochrome b5. First, solution nuclear magnetic resonance was used to compare the protein interactions that facilitated either the hydroxylase or lyase activities of CYP17A1. The lyase interaction was stimulated by the presence of b5 and 17α-hydroxypregnenolone, whereas the hydroxylase reaction was predominant in the absence of b5. The role of b5 was also shown in vivo by selective hepatic knockout of b5 from mice expressing CYP3A4 and CYP2D6; the lack of b5 caused a decrease in the clearance of several substrates. The role of the membrane on P450 orientation was examined using computational methods, showing that the proximal region of the P450 molecule faced the aqueous phase. The distal region, containing the substrate-access channel, was associated with the membrane. The interaction of NADPH-P450 reductase (CPR) with the membrane was also described, showing the ability of CPR to “helicopter” above the membrane. Finally, the endoplasmic reticulum (ER) was shown to be heterogeneous, having ordered membrane regions containing cholesterol and more disordered regions. Interestingly, two closely related P450s, CYP1A1 and CYP1A2, resided in different regions of the ER. The structural characteristics of their localization were examined. These studies emphasize the importance of P450 protein organization to their function. PMID:26851242

  16. Investigation of drug-drug interactions caused by human pregnane X receptor-mediated induction of CYP3A4 and CYP2C subfamilies in chimeric mice with a humanized liver.

    PubMed

    Hasegawa, Maki; Tahara, Harunobu; Inoue, Ryo; Kakuni, Masakazu; Tateno, Chise; Ushiki, Junko

    2012-03-01

    The induction of cytochrome P450 (P450) enzymes is one of the risk factors for drug-drug interactions (DDIs). To date, the human pregnane X receptor (PXR)-mediated CYP3A4 induction has been well studied. In addition to CYP3A4, the expression of CYP2C subfamily is also regulated by PXR, and the DDIs caused by the induction of CYP2C enzymes have been reported to have a major clinical impact. The purpose of the present study was to investigate whether chimeric mice with a humanized liver (PXB mice) can be a suitable animal model for investigating the PXR-mediated induction of CYP2C subfamily, together with CYP3A4. We evaluated the inductive effect of rifampicin (RIF), a typical human PXR ligand, on the plasma exposure to the four P450 substrate drugs (triazolam/CYP3A4, pioglitazone/CYP2C8, (S)-warfarin/CYP2C9, and (S)-(-)-mephenytoin/CYP2C19) by cassette dosing in PXB mice. The induction of several drug-metabolizing enzymes and transporters in the liver was also examined by measuring the enzyme activity and mRNA expression levels. Significant reductions in the exposure to triazolam, pioglitazone, and (S)-(-)-mephenytoin, but not to (S)-warfarin, were observed. In contrast to the in vivo results, all the four P450 isoforms, including CYP2C9, were elevated by RIF treatment. The discrepancy in the (S)-warfarin results between in vivo and in vitro studies may be attributed to the relatively small contribution of CYP2C9 to (S)-warfarin elimination in the PXB mice used in this study. In summary, PXB mice are a useful animal model to examine DDIs caused by PXR-mediated induction of CYP2C and CYP3A4.

  17. Vitamin K3 (menadione) redox cycling inhibits cytochrome P450-mediated metabolism and inhibits parathion intoxication

    SciTech Connect

    Jan, Yi-Hua; Richardson, Jason R., E-mail: jricha3@eohsi.rutgers.edu; Baker, Angela A.

    Parathion, a widely used organophosphate insecticide, is considered a high priority chemical threat. Parathion toxicity is dependent on its metabolism by the cytochrome P450 system to paraoxon (diethyl 4-nitrophenyl phosphate), a cytotoxic metabolite. As an effective inhibitor of cholinesterases, paraoxon causes the accumulation of acetylcholine in synapses and overstimulation of nicotinic and muscarinic cholinergic receptors, leading to characteristic signs of organophosphate poisoning. Inhibition of parathion metabolism to paraoxon represents a potential approach to counter parathion toxicity. Herein, we demonstrate that menadione (methyl-1,4-naphthoquinone, vitamin K3) is a potent inhibitor of cytochrome P450-mediated metabolism of parathion. Menadione is active in redox cycling,more » a reaction mediated by NADPH-cytochrome P450 reductase that preferentially uses electrons from NADPH at the expense of their supply to the P450s. Using human recombinant CYP 1A2, 2B6, 3A4 and human liver microsomes, menadione was found to inhibit the formation of paraoxon from parathion. Administration of menadione bisulfite (40 mg/kg, ip) to rats also reduced parathion-induced inhibition of brain cholinesterase activity, as well as parathion-induced tremors and the progression of other signs and symptoms of parathion poisoning. These data suggest that redox cycling compounds, such as menadione, have the potential to effectively mitigate the toxicity of organophosphorus pesticides including parathion which require cytochrome P450-mediated activation. - Highlights: • Menadione redox cycles with cytochrome P450 reductase and generates reactive oxygen species. • Redox cycling inhibits cytochrome P450-mediated parathion metabolism. • Short term administration of menadione inhibits parathion toxicity by inhibiting paraoxon formation.« less

  18. Feed-drug interaction of orally applied butyrate and phenobarbital on hepatic cytochrome P450 activity in chickens.

    PubMed

    Mátis, G; Kulcsár, A; Petrilla, J; Hermándy-Berencz, K; Neogrády, Zs

    2016-08-01

    The expression of hepatic drug-metabolizing cytochrome P450 (CYP) enzymes may be affected by several nutrition-derived compounds, such as by the commonly applied feed additive butyrate, possibly leading to feed-drug interactions. The aim of this study was to provide some evidence if butyrate can alter the activity of hepatic CYPs in chickens exposed to CYP-inducing xenobiotics, monitoring for the first time the possibility of such interaction. Ross 308 chickens in the grower phase were treated with daily intracoelomal phenobarbital (PB) injection (80 mg/kg BW), applied as a non-specific CYP-inducer, simultaneously with two different doses of intra-ingluvial sodium butyrate boluses (0.25 and 1.25 g/kg BW) for 5 days. Activity of CYP2H and CYP3A subfamilies was assessed by specific enzyme assays from isolated liver microsomes. According to our results, the lower dose of orally administered butyrate significantly attenuated the PB-triggered elevation of both hepatic CYP2H and CYP3A activities, which might be in association with the partly common signalling pathways of butyrate and CYP-inducing drugs, such as that of PB. Based on these data, butyrate may take part in pharmacoepigenetic interactions with simultaneously applied drugs or other CYP-inducing xenobiotics, with possible consequences for food safety and pharmacotherapy. Butyrate was found to be capable to maintain physiological CYP activity by attenuating CYP induction, underlining the safety of butyrate application in poultry nutrition. Journal of Animal Physiology and Animal Nutrition © 2015 Blackwell Verlag GmbH.

  19. Comparative study of hop-containing products on human cytochrome p450-mediated metabolism.

    PubMed

    Foster, Brian C; Kearns, Nikia; Arnason, John T; Saleem, Ammar; Ogrodowczyk, Carolina; Desjardins, Suzanne

    2009-06-10

    Thirty-five national and international brands of beer were examined for their potential to affect human cytochrome P450 (CYP)-mediated metabolism. They represented the two main categories of beer, ales and lagers, and included a number of specialty products including bitter (porter, stout), coffee, ice, wheat, Pilsner, and hemp seed. Aliquots were examined for nonvolatile soluble solids, effect on CYP metabolism and P-glycoprotein (Pgp) transport, and major alpha- and beta-hop acids. Wide variance was detected in contents of alcohol, nonvolatile suspended solids, and hop acids and in the potential to affect CYP-mediated metabolism and Pgp-mediated efflux transport. Many of the products affected CYP2C9-mediated metabolism, and only two (NRP 306 and 307) markedly affected CYP3A4; hence, some products have the capacity to affect drug safety. CYP3A4, CYP3A5, CYP3A7, and CYP19 (aromatase) inhibition to the log concentration of beta-acid content was significant with r(2) > 0.37, suggesting that these components can account for some of the variation in inhibition of CYP metabolism.

  20. NADPH-cytochrome P450 reductase-mediated denitration reaction of 2,4,6-trinitrotoluene to yield nitrite in mammals.

    PubMed

    Shinkai, Yasuhiro; Nishihara, Yuya; Amamiya, Masahiro; Wakayama, Toshihiko; Li, Song; Kikuchi, Tomohiro; Nakai, Yumi; Shimojo, Nobuhiro; Kumagai, Yoshito

    2016-02-01

    While the biodegradation of 2,4,6-trinitrotoluene (TNT) via the release of nitrite is well established, mechanistic details of the reaction in mammals are unknown. To address this issue, we attempted to identify the enzyme from rat liver responsible for the production of nitrite from TNT. A NADPH-cytochrome P450 reductase (P450R) was isolated and identified from rat liver microsomes as the enzyme responsible for not only the release of nitrite from TNT but also formation of superoxide and 4-hydroxyamino-2,6-dinitrotoluene (4-HADNT) under aerobic conditions. In this context, reactive oxygen species generated during P450R-catalyzed TNT reduction were found to be, at least in part, a mediator for the production of 4-HADNT from TNT via formation of 4-nitroso-2,6-dinitrotoluene. P450R did not catalyze the formation of the hydride-Meisenheimer complex (H(-)-TNT) that is thought to be an intermediate for nitrite release from TNT. Furthermore, in a time-course experiment, 4-HADNT formation reached a plateau level and then declined during the reaction between TNT and P450R with NADPH, while the release of nitrite was subjected to a lag period. Notably, the produced 4-HADNT can react with the parent compound TNT to produce nitrite and dimerized products via formation of a Janovsky complex. Our results demonstrate for the first time that P450R-mediated release of nitrite from TNT results from the process of chemical interaction of TNT and its 4-electron reduction metabolite 4-HADNT. Copyright © 2015 Elsevier Inc. All rights reserved.

  1. Antiretroviral Drug Interactions: Overview of Interactions Involving New and Investigational Agents and the Role of Therapeutic Drug Monitoring for Management

    PubMed Central

    Rathbun, R. Chris; Liedtke, Michelle D.

    2011-01-01

    Antiretrovirals are prone to drug-drug and drug-food interactions that can result in subtherapeutic or supratherapeutic concentrations. Interactions between antiretrovirals and medications for other diseases are common due to shared metabolism through cytochrome P450 (CYP450) and uridine diphosphate glucuronosyltransferase (UGT) enzymes and transport by membrane proteins (e.g., p-glycoprotein, organic anion-transporting polypeptide). The clinical significance of antiretroviral drug interactions is reviewed, with a focus on new and investigational agents. An overview of the mechanistic basis for drug interactions and the effect of individual antiretrovirals on CYP450 and UGT isoforms are provided. Interactions between antiretrovirals and medications for other co-morbidities are summarized. The role of therapeutic drug monitoring in the detection and management of antiretroviral drug interactions is also briefly discussed. PMID:24309307

  2. Food Polyphenol Apigenin Inhibits the Cytochrome P450 Monoxygenase Branch of the Arachidonic Acid Cascade.

    PubMed

    Steuck, Maryvonne; Hellhake, Stefan; Schebb, Nils Helge

    2016-11-30

    The product of cytochrome P450 monooxygenase (P450) ω-hydroxylation of arachidonic acid (AA), 20- hydroxyeicosatetraenoic acid (HETE), is a potent vasoconstrictor. Utilizing microsomes as well as individual CYP4 isoforms we demonstrate here that flavonoids can block 20-HETE formation. Apigenin inhibits CYP4F2 with an IC 50 value of 4.6 μM and 20-HETE formation in human liver and kidney microsomes at 2.4-9.8 μM. Interestingly, the structurally similar naringenin shows no relevant effect on the formation of 20-HETE. Based on these in vitro data, it is impossible to evaluate if a relevant blockade of 20-HETE formation can result in humans from intake of polyphenols with the diet. However, the potency of apigenin is comparable to those of P450 inhibitors such as ketoconazole. Moreover, an IC 50 value in the micromolar range is also described for the inhibition of CYP-mediated drug metabolism leading to food-drug interactions. The modulation of the arachidonic acid cascade by food polyphenols therefore warrants further investigation.

  3. Pharmacokinetics and Differential Regulation of Cytochrome P450 Enzymes in Type 1 Allergic Mice.

    PubMed

    Tanino, Tadatoshi; Komada, Akira; Ueda, Koji; Bando, Toru; Nojiri, Yukie; Ueda, Yukari; Sakurai, Eiichi

    2016-12-01

    Type 1 allergic diseases are characterized by elevated production of specific immunoglobulin E (IgE) for each antigen and have become a significant health problem worldwide. This study investigated the effect of IgE-mediated allergy on drug pharmacokinetics. To further understand differential suppression of hepatic cytochrome P450 (P450) activity, we examined the inhibitory effect of nitric oxide (NO), a marker of allergic conditions. Seven days after primary sensitization (PS7) or secondary sensitization (SS7), hepatic CYP1A2, CYP2C, CYP2E1, and CYP3A activities were decreased to 45%-75% of the corresponding control; however, CYP2D activity was not downregulated. PS7 and SS7 did not change the expression levels of five P450 proteins. Disappearance of CYP1A2 and CYP2D substrates from the plasma was not significantly different between allergic mice and control mice. In contrast, the area under the curve of a CYP1A2-mediated metabolite in PS7 and SS7 mice was reduced by 50% of control values. Total clearances of a CYP2E1 substrate in PS7 and SS7 mice were significantly decreased to 70% and 50% respectively, of the control without altering plasma protein binding. Hepatic amounts of CYP1A2 and CYP2E1 substrates were enhanced by allergic induction, being responsible for each downregulated activity. NO scavenger treatment completely improved the downregulated P450 activities. Therefore, our data suggest that the onset of IgE-mediated allergy alters the pharmacokinetics of major P450-metabolic capacity-limited drugs except for CYP2D drugs. NO is highly expected to participate in regulatory mechanisms of the four P450 isoforms. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

  4. Cytochrome P450 humanised mice

    PubMed Central

    2004-01-01

    Humans are exposed to countless foreign compounds, typically referred to as xenobiotics. These can include clinically used drugs, environmental pollutants, food additives, pesticides, herbicides and even natural plant compounds. Xenobiotics are metabolised primarily in the liver, but also in the gut and other organs, to derivatives that are more easily eliminated from the body. In some cases, however, a compound is converted to an electrophile that can cause cell toxicity and transformation leading to cancer. Among the most important xenobiotic-metabolising enzymes are the cytochromes P450 (P450s). These enzymes represent a superfamily of multiple forms that exhibit marked species differences in their expression and catalytic activities. To predict how humans will metabolise xenobiotics, including drugs, human liver extracts and recombinant P450s have been used. New humanised mouse models are being developed which will be of great value in the study of drug metabolism, pharmacokinetics and pharmacodynamics in vivo, and in carrying out human risk assessment of xenobiotics. Humanised mice expressing CYP2D6 and CYP3A4, two major drug-metabolising P450s, have revealed the feasibility of this approach. PMID:15588489

  5. Optimization of the Bacterial Cytochrome P450 BM3 System for the Production of Human Drug Metabolites

    PubMed Central

    Di Nardo, Giovanna; Gilardi, Gianfranco

    2012-01-01

    Drug metabolism in human liver is a process involving many different enzymes. Among them, a number of cytochromes P450 isoforms catalyze the oxidation of most of the drugs commercially available. Each P450 isoform acts on more than one drug, and one drug may be oxidized by more than one enzyme. As a result, multiple products may be obtained from the same drug, and as the metabolites can be biologically active and may cause adverse drug reactions (ADRs), the metabolic profile of a new drug has to be known before this can be commercialized. Therefore, the metabolites of a certain drug must be identified, synthesized and tested for toxicity. Their synthesis must be in sufficient quantities to be used for metabolic tests. This review focuses on the progresses done in the field of the optimization of a bacterial self-sufficient and efficient cytochrome P450, P450 BM3 from Bacillus megaterium, used for the production of metabolites of human enzymes. The progress made in the improvement of its catalytic performance towards drugs, the substitution of the costly NADPH cofactor and its immobilization and scale-up of the process for industrial application are reported. PMID:23443101

  6. Interaction of rocuronium with human liver cytochromes P450.

    PubMed

    Anzenbacherova, Eva; Spicakova, Alena; Jourova, Lenka; Ulrichova, Jitka; Adamus, Milan; Bachleda, Petr; Anzenbacher, Pavel

    2015-02-01

    Rocuronium is a neuromuscular blocking agent acting as a competitive antagonist of acetylcholine. Results of an inhibition of eight individual liver microsomal cytochromes P450 (CYP) are presented. As the patients are routinely premedicated with diazepam, possible interaction of diazepam with rocuronium has been also studied. Results indicated that rocuronium interacts with human liver microsomal CYPs by binding to the substrate site. Next, concentration dependent inhibition of liver microsomal CYP3A4 down to 42% (at rocuronium concentration 189 μM) was found. This effect has been confirmed with two CYP3A4 substrates, testosterone (formation of 6β-hydroxytestosterone) and diazepam (temazepam formation). CYP2C9 and CYP2C19 activities were inhibited down to 75-80% (at the same rocuronium concentration). Activities of other microsomal CYPs have not been inhibited by rocuronium. To prove the possibility of rocuronium interaction with other drugs (diazepam), the effect of rocuronium on formation of main diazepam metabolites, temazepam (by CYP3A4) and desmethyldiazepam, (also known as nordiazepam; formed by CYP2C19) in primary culture of human hepatocytes has been examined. Rocuronium has caused inhibition of both reactions by 20 and 15%, respectively. The results open a possibility that interactions of rocuronium with drugs metabolized by CYP3A4 (and possibly also CYP2C19) may be observed. Copyright © 2014 Japanese Pharmacological Society. Production and hosting by Elsevier B.V. All rights reserved.

  7. Cytochrome P450 monooxygenase CYP53 family in fungi: comparative structural and evolutionary analysis and its role as a common alternative anti-fungal drug target.

    PubMed

    Jawallapersand, Poojah; Mashele, Samson Sitheni; Kovačič, Lidija; Stojan, Jure; Komel, Radovan; Pakala, Suresh Babu; Kraševec, Nada; Syed, Khajamohiddin

    2014-01-01

    Cytochrome P450 monooxygenases (CYPs/P450s) are heme-thiolate proteins whose role as a drug target against pathogenic microbes has been explored because of their stereo- and regio-specific oxidation activity. We aimed to assess the CYP53 family's role as a common alternative drug target against animal (including human) and plant pathogenic fungi and its role in fungal-mediated wood degradation. Genome-wide analysis of fungal species revealed the presence of CYP53 members in ascomycetes and basidiomycetes. Basidiomycetes had a higher number of CYP53 members in their genomes than ascomycetes. Only two CYP53 subfamilies were found in ascomycetes and six subfamilies in basidiomycetes, suggesting that during the divergence of phyla ascomycetes lost CYP53 P450s. According to phylogenetic and gene-structure analysis, enrichment of CYP53 P450s in basidiomycetes occurred due to the extensive duplication of CYP53 P450s in their genomes. Numerous amino acids (103) were found to be conserved in the ascomycetes CYP53 P450s, against only seven in basidiomycetes CYP53 P450s. 3D-modelling and active-site cavity mapping data revealed that the ascomycetes CYP53 P450s have a highly conserved protein structure whereby 78% amino acids in the active-site cavity were found to be conserved. Because of this rigid nature of ascomycetes CYP53 P450s' active site cavity, any inhibitor directed against this P450 family can serve as a common anti-fungal drug target, particularly toward pathogenic ascomycetes. The dynamic nature of basidiomycetes CYP53 P450s at a gene and protein level indicates that these P450s are destined to acquire novel functions. Functional analysis of CYP53 P450s strongly supported our hypothesis that the ascomycetes CYP53 P450s ability is limited for detoxification of toxic molecules, whereas basidiomycetes CYP53 P450s play an additional role, i.e. involvement in degradation of wood and its derived components. This study is the first report on genome-wide comparative

  8. Acaricide, Fungicide and Drug Interactions in Honey Bees (Apis mellifera)

    PubMed Central

    Johnson, Reed M.; Dahlgren, Lizette; Siegfried, Blair D.; Ellis, Marion D.

    2013-01-01

    Background Chemical analysis shows that honey bees (Apis mellifera) and hive products contain many pesticides derived from various sources. The most abundant pesticides are acaricides applied by beekeepers to control Varroa destructor. Beekeepers also apply antimicrobial drugs to control bacterial and microsporidial diseases. Fungicides may enter the hive when applied to nearby flowering crops. Acaricides, antimicrobial drugs and fungicides are not highly toxic to bees alone, but in combination there is potential for heightened toxicity due to interactive effects. Methodology/Principal Findings Laboratory bioassays based on mortality rates in adult worker bees demonstrated interactive effects among acaricides, as well as between acaricides and antimicrobial drugs and between acaricides and fungicides. Toxicity of the acaricide tau-fluvalinate increased in combination with other acaricides and most other compounds tested (15 of 17) while amitraz toxicity was mostly unchanged (1 of 15). The sterol biosynthesis inhibiting (SBI) fungicide prochloraz elevated the toxicity of the acaricides tau-fluvalinate, coumaphos and fenpyroximate, likely through inhibition of detoxicative cytochrome P450 monooxygenase activity. Four other SBI fungicides increased the toxicity of tau-fluvalinate in a dose-dependent manner, although possible evidence of P450 induction was observed at the lowest fungicide doses. Non-transitive interactions between some acaricides were observed. Sublethal amitraz pre-treatment increased the toxicity of the three P450-detoxified acaricides, but amitraz toxicity was not changed by sublethal treatment with the same three acaricides. A two-fold change in the toxicity of tau-fluvalinate was observed between years, suggesting a possible change in the genetic composition of the bees tested. Conclusions/Significance Interactions with acaricides in honey bees are similar to drug interactions in other animals in that P450-mediated detoxication appears to play an

  9. Vitamin K3 (menadione) redox cycling inhibits cytochrome P450-mediated metabolism and inhibits parathion intoxication.

    PubMed

    Jan, Yi-Hua; Richardson, Jason R; Baker, Angela A; Mishin, Vladimir; Heck, Diane E; Laskin, Debra L; Laskin, Jeffrey D

    2015-10-01

    Parathion, a widely used organophosphate insecticide, is considered a high priority chemical threat. Parathion toxicity is dependent on its metabolism by the cytochrome P450 system to paraoxon (diethyl 4-nitrophenyl phosphate), a cytotoxic metabolite. As an effective inhibitor of cholinesterases, paraoxon causes the accumulation of acetylcholine in synapses and overstimulation of nicotinic and muscarinic cholinergic receptors, leading to characteristic signs of organophosphate poisoning. Inhibition of parathion metabolism to paraoxon represents a potential approach to counter parathion toxicity. Herein, we demonstrate that menadione (methyl-1,4-naphthoquinone, vitamin K3) is a potent inhibitor of cytochrome P450-mediated metabolism of parathion. Menadione is active in redox cycling, a reaction mediated by NADPH-cytochrome P450 reductase that preferentially uses electrons from NADPH at the expense of their supply to the P450s. Using human recombinant CYP 1A2, 2B6, 3A4 and human liver microsomes, menadione was found to inhibit the formation of paraoxon from parathion. Administration of menadione bisulfite (40mg/kg, ip) to rats also reduced parathion-induced inhibition of brain cholinesterase activity, as well as parathion-induced tremors and the progression of other signs and symptoms of parathion poisoning. These data suggest that redox cycling compounds, such as menadione, have the potential to effectively mitigate the toxicity of organophosphorus pesticides including parathion which require cytochrome P450-mediated activation. Copyright © 2015 Elsevier Inc. All rights reserved.

  10. Engineering Macaca fascicularis cytochrome P450 2C20 to reduce animal testing for new drugs.

    PubMed

    Rua, Francesco; Sadeghi, Sheila J; Castrignanò, Silvia; Di Nardo, Giovanna; Gilardi, Gianfranco

    2012-12-01

    In order to develop in vitro methods as an alternative to P450 animal testing in the drug discovery process, two main requisites are necessary: 1) gathering of data on animal homologues of the human P450 enzymes, currently very limited, and 2) bypassing the requirement for both the P450 reductase and the expensive cofactor NADPH. In this work, P450 2C20 from Macaca fascicularis, homologue of the human P450 2C8 has been taken as a model system to develop such an alternative in vitro method by two different approaches. In the first approach called "molecular Lego", a soluble self-sufficient chimera was generated by fusing the P450 2C20 domain with the reductase domain of cytochrome P450 BM3 from Bacillus megaterium (P450 2C20/BMR). In the second approach, the need for the redox partner and also NADPH were both obviated by the direct immobilization of the P450 2C20 on glassy carbon and gold electrodes. Both systems were then compared to those obtained from the reconstituted P450 2C20 monooxygenase in presence of the human P450 reductase and NADPH using paclitaxel and amodiaquine, two typical drug substrates of the human P450 2C8. The K(M) values calculated for the 2C20 and 2C20/BMR in solution and for 2C20 immobilized on electrodes modified with gold nanoparticles were 1.9 ± 0.2, 5.9 ± 2.3, 3.0 ± 0.5 μM for paclitaxel and 1.2 ± 0.2, 1.6±0.2 and 1.4 ± 0.2 μM for amodiaquine, respectively. The data obtained not only show that the engineering of M. fascicularis did not affect its catalytic properties but also are consistent with K(M) values measured for the microsomal human P450 2C8 and therefore show the feasibility of developing alternative in vitro animal tests. Copyright © 2012 Elsevier Inc. All rights reserved.

  11. Effect of Short-Term Fasting on Systemic Cytochrome P450-Mediated Drug Metabolism in Healthy Subjects: A Randomized, Controlled, Crossover Study Using a Cocktail Approach.

    PubMed

    Lammers, Laureen A; Achterbergh, Roos; van Schaik, Ron H N; Romijn, Johannes A; Mathôt, Ron A A

    2017-10-01

    Short-term fasting can alter drug exposure but it is unknown whether this is an effect of altered oral bioavailability and/or systemic clearance. Therefore, the aim of our study was to assess the effect of short-term fasting on oral bioavailability and systemic clearance of different drugs. In a randomized, controlled, crossover trial, 12 healthy subjects received a single administration of a cytochrome P450 (CYP) probe cocktail, consisting of caffeine (CYP1A2), metoprolol (CYP2D6), midazolam (CYP3A4), omeprazole (CYP2C19) and warfarin (CYP2C9), on four occasions: an oral (1) and intravenous (2) administration after an overnight fast (control) and an oral (3) and intravenous (4) administration after 36 h of fasting. Pharmacokinetic parameters of the probe drugs were analyzed using the nonlinear mixed-effects modeling software NONMEM. Short-term fasting increased systemic caffeine clearance by 17% (p = 0.04) and metoprolol clearance by 13% (p < 0.01), whereas S-warfarin clearance decreased by 19% (p < 0.01). Fasting did not affect bioavailability. The study demonstrates that short-term fasting alters CYP-mediated drug metabolism in a non-uniform pattern without affecting oral bioavailability.

  12. Cytochrome P450 Organization and Function Are Modulated by Endoplasmic Reticulum Phospholipid Heterogeneity.

    PubMed

    Brignac-Huber, Lauren M; Park, Ji Won; Reed, James R; Backes, Wayne L

    2016-12-01

    Cytochrome P450s (P450s) comprise a superfamily of proteins that catalyze numerous monooxygenase reactions in animals, plants, and bacteria. In eukaryotic organisms, these proteins not only carry out reactions necessary for the metabolism of endogenous compounds, but they are also important in the oxidation of exogenous drugs and other foreign compounds. Eukaryotic P450 system proteins generally reside in membranes, primarily the endoplasmic reticulum or the mitochondrial membrane. These membranes provide a scaffold for the P450 system proteins that facilitate interactions with their redox partners as well as other P450s. This review focuses on the ability of specific lipid components to influence P450 activities, as well as the role of the membrane in P450 function. These studies have shown that P450s and NADPH-cytochrome P450 reductase appear to selectively associate with specific phospholipids and that these lipid-protein interactions influence P450 activities. Finally, because of the heterogeneous nature of the endoplasmic reticulum as well as other biologic membranes, the phospholipids are not arranged randomly but associate to generate lipid microdomains. Together, these characteristics can affect P450 function by 1) altering the conformation of the proteins, 2) influencing the P450 interactions with their redox partners, and 3) affecting the localization of the proteins into specific membrane microdomains. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

  13. Expansion of chemical space for natural products by uncommon P450 reactions.

    PubMed

    Zhang, Xingwang; Li, Shengying

    2017-08-30

    Covering: 2000 to 2017Cytochrome P450 enzymes (P450s) are the most versatile biocatalysts in nature. The catalytic competence of these extraordinary hemoproteins is broadly harnessed by numerous chemical defenders such as bacteria, fungi, and plants for the generation of diverse and complex natural products. Rather than the common tailoring reactions (e.g. hydroxylation and epoxidation) mediated by the majority of biosynthetic P450s, in this review, we will focus on the unusual P450 enzymes in relation to new chemistry, skeleton construction, and structure re-shaping via their own unique catalytic power or the intriguing protein-protein interactions between P450s and other proteins. These uncommon P450 reactions lead to a higher level of chemical space expansion for natural products, through which a broader spectrum of bioactivities can be gained by the host organisms.

  14. Autoantibodies against cytochrome P450s in sera of children treated with immunosuppressive drugs

    PubMed Central

    LYTTON, S D; BERG, U; NEMETH, A; INGELMAN-SUNDBERG, M

    2002-01-01

    Treatment with the immunosuppressive drugs cyclosporin and tacrolimus, the mainstays of anti-graft rejection and autoimmune disease therapy, is limited by their hepato-and nephrotoxicity. The metabolic conversion of these compounds to more easily excretable products is catalysed mainly by hepatic cytochrome P4503A4 (CYP3A4) but also involves extrahepatic CYP3A5 and other P450 forms. We set out to study whether or not exposure to cyclosporin and FK506 in children undergoing organ transplantation leads to formation of autoantibodies against P450s. Immunoblotting analysis revealed anti-CYP reactivity in 16% of children on CyA for anti-graft rejection or treatment of nephrosis (n = 67), 31% of kidney transplant patients switched from CyA to FK506 (n = 16), and 21% of kidney and or liver transplant patients on FK506 (n = 14). In contrast, the frequency of reactive immunoblots was only 8·5% among the normal paediatric controls (n = 25) and 7% among adult kidney transplant patients on CyA or FK506 (n = 30). The CYP2C9+ sera were able to immunoprecipitate in vitro translated CYP2C9 and the immunoblot reactivity showed striking correlation to peaks in the age at onset of drug exposure. Sera were isoform selective as evidenced from Western blotting using human liver microsomes and heterologously expressed human P450s. These findings suggest that anti-cytochrome P450 autoantibodies, identified on the basis of their specific binding in immunoblots, are significantly increased among children on immunosuppressive drugs and in some cases are associated with drug toxicity and organ rejection. PMID:11876753

  15. Autoantibodies against cytochrome P450s in sera of children treated with immunosuppressive drugs.

    PubMed

    Lytton, S D; Berg, U; Nemeth, A; Ingelman-Sundberg, M

    2002-02-01

    Treatment with the immunosuppressive drugs cyclosporin and tacrolimus, the mainstays of anti-graft rejection and autoimmune disease therapy, is limited by their hepato- and nephrotoxicity. The metabolic conversion of these compounds to more easily excretable products is catalysed mainly by hepatic cytochrome P4503A4 (CYP3A4) but also involves extrahepatic CYP3A5 and other P450 forms. We set out to study whether or not exposure to cyclosporin and FK506 in children undergoing organ transplantation leads to formation of autoantibodies against P450s. Immunoblotting analysis revealed anti-CYP reactivity in 16% of children on CyA for anti-graft rejection or treatment of nephrosis (n = 67), 31% of kidney transplant patients switched from CyA to FK506 (n = 16), and 21% of kidney and or liver transplant patients on FK506 (n = 14). In contrast, the frequency of reactive immunoblots was only 8.5% among the normal paediatric controls (n = 25) and 7% among adult kidney transplant patients on CyA or FK506 (n = 30). The CYP2C9+ sera were able to immunoprecipitate in vitro translated CYP2C9 and the immunoblot reactivity showed striking correlation to peaks in the age at onset of drug exposure. Sera were isoform selective as evidenced from Western blotting using human liver microsomes and heterologously expressed human P450s. These findings suggest that anti-cytochrome P450 autoantibodies, identified on the basis of their specific binding in immunoblots, are significantly increased among children on immunosuppressive drugs and in some cases are associated with drug toxicity and organ rejection.

  16. The Consequence of Drug-Drug Interactions Influencing the Interplay between P-Glycoprotein and Cytochrome P450 3a: An Ex Vivo Study with Rat Precision-Cut Intestinal Slices.

    PubMed

    Li, Ming; de Graaf, Inge A M; Siissalo, Sanna; de Jager, Marina H; van Dam, Annie; Groothuis, Geny M M

    2016-05-01

    P-glycoprotein (P-gp) and cytochrome P450 3A (CYP3A) are differentially expressed along the intestine and work coordinately to reduce the intracellular concentration of xenobiotics and the absorption of orally taken drugs. Drug-drug interactions (DDIs) based on P-gp/CYP3A interplay are of clinical importance and require preclinical investigation. We investigated the P-gp/Cyp3a interplay and related DDIs with different P-gp inhibitors in the various regions of the rat intestine ex vivo using precision-cut intestinal slices (PCIS) with quinidine (Qi), a dual substrate of P-gp and Cyp3a, as the probe. The results showed that P-gp efflux was the main factor limiting the intracellular Qi content at concentrations below 5µM, whereas both efflux and metabolism were saturated at [Qi] > 50µM. The selective P-gp inhibitors CP100356 [N-(3,4-dimethoxyphenethyl)-4-(6,7-dimethoxy-3,4-dihydroisoquinolin-2[1H]-yl)-6,7-dimethoxyquinazolin-2-amine] and PSC833 [valspodar, 6-[(2S,4R,6E)-4-methyl-2-(methylamino)-3-oxo-6-octenoic acid]-7-l-valine-cyclosporin A] enhanced the Qi accumulation in slices in line with the different P-gp expression in the intestinal regions and, as a result, also enhanced metabolism in the jejunum and ileum. Dual inhibitors of both P-gp and Cyp3a (verapamil and ketoconazole) increased the concentration of Qi in the jejunum and ileum, but less 3-hydroxy-quinidine was produced due to inhibition of Cyp3a. The results indicate that the P-gp/Cyp3a interplay depends on the concentration of the drug and on the intestinal region under study. Furthermore, due to the P-gp/Cyp3a interplay, DDIs can lead to remarkable changes in the intracellular concentration of both the parent drug and the metabolite, which varies among the intestinal regions and depends on the selectivity of the inhibitors, with potentially important implications for disposition and toxicity of drugs and their metabolites. Copyright © 2016 by The American Society for Pharmacology and Experimental

  17. Clinical Drug-Drug Interactions Through Cytochrome P450 3A (CYP3A) for the Selective ALK Inhibitor Alectinib.

    PubMed

    Morcos, Peter N; Cleary, Yumi; Guerini, Elena; Dall, Georgina; Bogman, Katrijn; De Petris, Luigi; Viteri, Santiago; Bordogna, Walter; Yu, Li; Martin-Facklam, Meret; Phipps, Alex

    2017-05-01

    The efficacy and safety of alectinib, a central nervous system-active and selective anaplastic lymphoma kinase (ALK) inhibitor, has been demonstrated in patients with ALK-positive (ALK+) non-small cell lung cancer (NSCLC) progressing on crizotinib. Alectinib is mainly metabolized by cytochrome P450 3A (CYP3A) to a major similarly active metabolite, M4. Alectinib and M4 show evidence of weak time-dependent inhibition and small induction of CYP3A in vitro. We present results from 3 fixed-sequence studies evaluating drug-drug interactions for alectinib through CYP3A. Studies NP28990 and NP29042 enrolled 17 and 24 healthy subjects, respectively, and investigated potent CYP3A inhibition with posaconazole and potent CYP3A induction through rifampin, respectively, on the single oral dose pharmacokinetics (PK) of alectinib. A substudy of the global phase 2 NP28673 study enrolled 15 patients with ALK+ NSCLC to determine the effect of multiple doses of alectinib on the single oral dose PK of midazolam, a sensitive substrate of CYP3A. Potent CYP3A inhibition or induction resulted in only minor effects on the combined exposure of alectinib and M4. Multiple doses of alectinib did not influence midazolam exposure. These results suggest that dose adjustments may not be needed when alectinib is coadministered with CYP3A inhibitors or inducers or for coadministered CYP3A substrates. © 2016, The American College of Clinical Pharmacology.

  18. Characterization of the Pharmacokinetics of Vilaprisan: Bioavailability, Excretion, Biotransformation, and Drug-Drug Interaction Potential.

    PubMed

    Schultze-Mosgau, Marcus-Hillert; Höchel, Joachim; Prien, Olaf; Zimmermann, Torsten; Brooks, Ashley; Bush, Jim; Rottmann, Antje

    2018-01-12

    In-vitro data suggest that clearance of vilaprisan is mediated by cytochrome P450 3A4 (oxidation) and aldoketoreductases (reduction). To fully understand the elimination and biotransformation pathways of vilaprisan, a selective progesterone receptor modulator, and to quantify the impact of cytochrome P450 3A4 inhibition on the pharmacokinetics of vilaprisan, two clinical studies in healthy postmenopausal women were conducted. In study 1, pharmacokinetics, mass balance, and metabolite patterns were determined after single oral administration of 5 mg of [ 14 C]-labeled vilaprisan in six subjects. In study 2, pharmacokinetics were determined after single oral administration of 4 mg of vilaprisan without and with concomitant administration of the strong cytochrome P450 3A4 inhibitor itraconazole (200 mg/day) in 14 subjects. In addition, a microtracer dose of vilaprisan was given intravenously to determine absolute bioavailability, clearance, and volume of distribution. The dominant single compound in plasma was vilaprisan. No plasma metabolites exceeding 10% of total drug-related area under the concentration-time curve were detected. The absolute oral bioavailability of vilaprisan was ~ 60%. The mean clearance was ~ 7 L/h and the volume of distribution at steady state was ~ 360 L. Excretion occurred primarily via feces (73.5 ± 3.70% of dose; urine: 13.1 ± 1.71%; total recovery: 86.6 ± 2.81%), mostly in a metabolized form. Only small amounts of the parent drug were found in excreta. When vilaprisan was administered together with itraconazole, exposure to vilaprisan was increased 6.2-fold (90% confidence interval 5.4-7.2). Vilaprisan is predominantly metabolized in the liver to a complex variety of metabolites, which are mainly excreted with feces. The pivotal role of cytochrome P450 3A4 in the metabolism of vilaprisan was confirmed. EudraCT numbers 2013-000707-16 (mass balance study) and 2014-004929-41 (drug-drug interaction/microtracer study); NCT

  19. Pyrethroid activity-based probes for profiling cytochrome P450 activities associated with insecticide interactions.

    PubMed

    Ismail, Hanafy M; O'Neill, Paul M; Hong, David W; Finn, Robert D; Henderson, Colin J; Wright, Aaron T; Cravatt, Benjamin F; Hemingway, Janet; Paine, Mark J I

    2013-12-03

    Pyrethroid insecticides are used to control diseases spread by arthropods. We have developed a suite of pyrethroid mimetic activity-based probes (PyABPs) to selectively label and identify P450s associated with pyrethroid metabolism. The probes were screened against pyrethroid-metabolizing and nonmetabolizing mosquito P450s, as well as rodent microsomes, to measure labeling specificity, plus cytochrome P450 oxidoreductase and b5 knockout mouse livers to validate P450 activation and establish the role for b5 in probe activation. Using PyABPs, we were able to profile active enzymes in rat liver microsomes and identify pyrethroid-metabolizing enzymes in the target tissue. These included P450s as well as related detoxification enzymes, notably UDP-glucuronosyltransferases, suggesting a network of associated pyrethroid-metabolizing enzymes, or "pyrethrome." Considering the central role P450s play in metabolizing insecticides, we anticipate that PyABPs will aid in the identification and profiling of P450s associated with insecticide pharmacology in a wide range of species, improving understanding of P450-insecticide interactions and aiding the development of unique tools for disease control.

  20. Pyrethroid activity-based probes for profiling cytochrome P450 activities associated with insecticide interactions

    PubMed Central

    Ismail, Hanafy M.; O’Neill, Paul M.; Hong, David W.; Finn, Robert D.; Henderson, Colin J.; Wright, Aaron T.; Cravatt, Benjamin F.; Hemingway, Janet; Paine, Mark J. I.

    2013-01-01

    Pyrethroid insecticides are used to control diseases spread by arthropods. We have developed a suite of pyrethroid mimetic activity-based probes (PyABPs) to selectively label and identify P450s associated with pyrethroid metabolism. The probes were screened against pyrethroid-metabolizing and nonmetabolizing mosquito P450s, as well as rodent microsomes, to measure labeling specificity, plus cytochrome P450 oxidoreductase and b5 knockout mouse livers to validate P450 activation and establish the role for b5 in probe activation. Using PyABPs, we were able to profile active enzymes in rat liver microsomes and identify pyrethroid-metabolizing enzymes in the target tissue. These included P450s as well as related detoxification enzymes, notably UDP-glucuronosyltransferases, suggesting a network of associated pyrethroid-metabolizing enzymes, or “pyrethrome.” Considering the central role P450s play in metabolizing insecticides, we anticipate that PyABPs will aid in the identification and profiling of P450s associated with insecticide pharmacology in a wide range of species, improving understanding of P450–insecticide interactions and aiding the development of unique tools for disease control. PMID:24248381

  1. Macrolide drug interactions: an update.

    PubMed

    Pai, M P; Graci, D M; Amsden, G W

    2000-04-01

    To describe the current drug interaction profiles for the commonly used macrolides in the US and Europe, and to comment on the clinical impact of these interactions. A MEDLINE search (1975-1998) was performed to identify all pertinent studies, review articles, and case reports. When appropriate information was not available in the literature, data were obtained from the product manufacturers. All available data were reviewed to provide an unbiased account of possible drug interactions. Data for some of the interactions were not available from the literature, but were available from abstracts or company-supplied materials. Although the data were not always explicit, the best attempt was made to deliver pertinent information that clinical practitioners would need to formulate practice opinions. When more in-depth information was supplied in the form of a review or study report, a thorough explanation of pertinent methodology was supplied. Several clinically significant drug interactions have been identified since the approval of erythromycin. These interactions usually were related to the inhibition of the cytochrome P450 enzyme systems, which are responsible for the metabolism of many drugs. The decreased metabolism by the macrolides has in some instances resulted in potentially severe adverse events. The development and marketing of newer macrolides are hoped to improve the drug interaction profile associated with this class. However, this has produced variable success. Some of the newer macrolides demonstrated an interaction profile similar to that of erythromycin; others have improved profiles. The most success in avoiding drug interactions related to the inhibition of cytochrome P450 has been through the development of the azalide subclass, of which azithromycin is the first and only to be marketed. Azithromycin has not been demonstrated to inhibit the cytochrome P450 system in studies using a human liver microsome model, and to date has produced none of the

  2. Drug-drug interactions via mechanism-based cytochrome P450 inactivation: points to consider for risk assessment from in vitro data and clinical pharmacologic evaluation.

    PubMed

    Venkatakrishnan, Karthik; Obach, R Scott

    2007-06-01

    This commentary discusses the approaches to, and key considerations in the in vitro-in vivo extrapolation of drug-drug interactions (DDI) resulting from mechanism-based inactivation (MBI) of cytochrome P450 (CYP) enzymes and clinical pharmacologic implications. In vitro kinetic assessment and prediction of DDI produced via reversible inhibition and MBI rely on operationally and conceptually distinct approaches. DDI risk assessment for inactivators requires estimation of maximal inactivation rate (k(inact)) and inactivator potency (KI) in vitro, that need to be considered in context of the biological turnover rate of the enzyme (kdeg) and clinical exposures of the inactivator (I), respectively, to predict interaction magnitude. Risk assessment cannot be performed by a simple comparison of inactivator potency against in vivo exposure since inactivation is both concentration and time-dependent. MBI contour plots tracking combinations of I:KI and k(inact):k(deg) resulting in identical fold-reductions in intrinsic clearance are proposed as a useful framework for DDI risk assessment. Additionally, substrate-specific factors like fraction of the total clearance of the object drug via the enzyme being inactivated (f(m(CYP) )) and the bioavailability fraction across the intestine for CYP3A substrates (F(G)) are important determinants of interaction magnitude. Sensitivity analysis of predicted DDI magnitude to uncertainty in input parameters is recommended to inform confidence in predictions. The time course of reversal of DDI resulting from CYP inactivation is determined by the half-life of the enzyme which is an important consideration in the design and interpretation of clinical DDI studies with inactivators.

  3. ABC multidrug transporters: target for modulation of drug pharmacokinetics and drug-drug interactions.

    PubMed

    Marquez, Béatrice; Van Bambeke, Françoise

    2011-05-01

    Nine proteins of the ABC superfamily (P-glycoprotein, 7 MRPs and BCRP) are involved in multidrug transport. Being localised at the surface of endothelial or epithelial cells, they expel drugs back to the external medium (if located at the apical side [P-glycoprotein, BCRP, MRP2, MRP4 in the kidney]) or to the blood (if located at the basolateral side [MRP1, MRP3, MRP4, MRP5]), modulating thereby their absorption, distribution, and elimination. In the CNS, most transporters are oriented to expel drugs to the blood. Transporters also cooperate with Phase I/Phase II metabolism enzymes by eliminating drug metabolites. Their major features are (i) their capacity to recognize drugs belonging to unrelated pharmacological classes, and (ii) their redundancy, a single molecule being possibly substrate for different transporters. This ensures an efficient protection of the body against invasion by xenobiotics. Competition for transport is now characterized as a mechanism of interaction between co-administered drugs, one molecule limiting the transport of the other, potentially affecting bioavailability, distribution, and/or elimination. Again, this mechanism reinforces drug interactions mediated by cytochrome P450 inhibition, as many substrates of P-glycoprotein and CYP3A4 are common. Induction of the expression of genes coding for MDR transporters is another mechanism of drug interaction, which could affect all drug substrates of the up-regulated transporter. Overexpression of MDR transporters confers resistance to anticancer agents and other therapies. All together, these data justify why studying drug active transport should be part of the evaluation of new drugs, as recently recommended by the FDA.

  4. Monkey liver cytochrome P450 2C19 is involved in R- and S-warfarin 7-hydroxylation.

    PubMed

    Hosoi, Yoshio; Uno, Yasuhiro; Murayama, Norie; Fujino, Hideki; Shukuya, Mitsunori; Iwasaki, Kazuhide; Shimizu, Makiko; Utoh, Masahiro; Yamazaki, Hiroshi

    2012-12-15

    Cynomolgus monkeys are widely used as primate models in preclinical studies. However, some differences are occasionally seen between monkeys and humans in the activities of cytochrome P450 enzymes. R- and S-warfarin are model substrates for stereoselective oxidation in humans. In this current research, the activities of monkey liver microsomes and 14 recombinantly expressed monkey cytochrome P450 enzymes were analyzed with respect to R- and S-warfarin 6- and 7-hydroxylation. Monkey liver microsomes efficiently mediated both R- and S-warfarin 7-hydroxylation, in contrast to human liver microsomes, which preferentially catalyzed S-warfarin 7-hydroxylation. R-Warfarin 7-hydroxylation activities in monkey liver microsomes were not inhibited by α-naphthoflavone or ketoconazole, and were roughly correlated with P450 2C19 levels and flurbiprofen 4-hydroxylation activities in microsomes from 20 monkey livers. In contrast, S-warfarin 7-hydroxylation activities were not correlated with the four marker drug oxidation activities used. Among the 14 recombinantly expressed monkey P450 enzymes tested, P450 2C19 had the highest activities for R- and S-warfarin 7-hydroxylations. Monkey P450 3A4 and 3A5 slowly mediated R- and S-warfarin 6-hydroxylations. Kinetic analysis revealed that monkey P450 2C19 had high V(max) and low K(m) values for R-warfarin 7-hydroxylation, comparable to those for monkey liver microsomes. Monkey P450 2C19 also mediated S-warfarin 7-hydroxylation with V(max) and V(max)/K(m) values comparable to those for recombinant human P450 2C9. R-warfarin could dock favorably into monkey P450 2C19 modeled. These results collectively suggest high activities for monkey liver P450 2C19 toward R- and S-warfarin 6- and 7-hydroxylation in contrast to the saturation kinetics of human P450 2C9-mediated S-warfarin 7-hydroxylation. Copyright © 2012 Elsevier Inc. All rights reserved.

  5. CYTOCHROME P450 17A1 STRUCTURES WITH PROSTATE CANCER DRUGS ABIRATERONE AND TOK-001

    PubMed Central

    DeVore, Natasha M.; Scott, Emily E.

    2011-01-01

    Cytochrome P450 17A1 (P450c17) catalyzes the biosynthesis of androgens in humans1. Since prostate cancer cells proliferate in response to androgen steroids2,3, CYP17A1 inhibition is a new strategy to prevent androgen synthesis and treat lethal metastatic castration-resistant prostate cancer4, but drug development has been hampered by the lack of a CYP17A1 structure. Here we report the only known structures of CYP17A1, which contain either abiraterone, a first-in-class steroidal inhibitor recently approved by the FDA for late-stage prostate cancer5, or TOK-001, another inhibitor in clinical trials4,6. Both bind the heme iron forming a 60° angle above the heme plane, packing against the central I helix with the 3β-OH interacting with N202 in the F helix. Importantly, this binding mode differs substantially from those predicted by homology models or from steroids in other cytochrome P450 enzymes with known structures, with some features more similar to steroid receptors. While the overall CYP17A1 structure provides a rationale for understanding many mutations found in patients with steroidogenic diseases, the active site reveals multiple steric and hydrogen bonding features that will facilitate better understanding of the enzyme’s dual hydroxylase and lyase catalytic capabilities and assist in rational drug design. Specifically, structure-based design is expected to aid development of inhibitors that bind only CYP17A1 and solely inhibit its androgen-generating lyase activity to improve treatment of prostate and other hormone-responsive cancers. PMID:22266943

  6. N-Heterocyclic Carbene Capture by Cytochrome P450 3A4

    PubMed Central

    Jennings, Gareth K.; Ritchie, Caroline M.; Shock, Lisa S.; Lyons, Charles E.

    2016-01-01

    Cytochrome P450 3A4 (CYP3A4) is the dominant P450 enzyme involved in human drug metabolism, and its inhibition may result in adverse interactions or, conversely, favorably reduce the systemic elimination rates of poorly bioavailable drugs. Herein we describe a spectroscopic investigation of the interaction of CYP3A4 with N-methylritonavir, an analog of ritonavir, widely used as a pharmacoenhancer. In contrast to ritonavir, the binding affinity of N-methylritonavir for CYP3A4 is pH-dependent. At pH <7.4, the spectra are definitively type I, whereas at pH ≥7.4 the spectra have split Soret bands, including a red-shifted component characteristic of a P450-carbene complex. Variable-pH UV-visible spectroscopy binding studies with molecular fragments narrows the source of this pH dependence to its N-methylthiazolium fragment. The C2 proton of this group is acidic, and variable-pH resonance Raman spectroscopy tentatively assigns it a pKa of 7.4. Hence, this fragment of N-methylritonavir is expected to be readily deprotonated under physiologic conditions to yield a thiazol-2-ylidene, which is an N-heterocyclic carbene that has high-affinity for and is presumed to be subsequently captured by the heme iron. This mechanism is supported by time-dependent density functional theory with an active site model that accurately reproduces distinguishing features of the experimental UV-visible spectra of N-methylritonavir bound to CYP3A4. Finally, density functional theory calculations support that this novel interaction is as strong as the tightest-binding azaheterocycles found in P450 inhibitors and could offer new avenues for inhibitor development. PMID:27126611

  7. Multisite Phosphorylation of Human Liver Cytochrome P450 3A4 Enhances Its gp78- and CHIP-mediated Ubiquitination

    PubMed Central

    Wang, YongQiang; Guan, Shenheng; Acharya, Poulomi; Liu, Yi; Thirumaran, Ranjit K.; Brandman, Relly; Schuetz, Erin G.; Burlingame, Alma L.; Correia, Maria Almira

    2012-01-01

    CYP3A4, an integral endoplasmic reticulum (ER)-anchored protein, is the major human liver cytochrome P450 enzyme responsible for the disposition of over 50% of clinically relevant drugs. Alterations of its protein turnover can influence drug metabolism, drug-drug interactions, and the bioavailability of chemotherapeutic drugs. Such CYP3A4 turnover occurs via a classical ER-associated degradation (ERAD) process involving ubiquitination by both UBC7/gp78 and UbcH5a/CHIP E2-E3 complexes for 26 S proteasomal targeting. These E3 ligases act sequentially and cooperatively in CYP3A4 ERAD because RNA interference knockdown of each in cultured hepatocytes results in the stabilization of a functionally active enzyme. We have documented that UBC7/gp78-mediated CYP3A4 ubiquitination requires protein phosphorylation by protein kinase (PK) A and PKC and identified three residues (Ser-478, Thr-264, and Ser-420) whose phosphorylation is required for intracellular CYP3A4 ERAD. We document herein that of these, Ser-478 plays a pivotal role in UBC7/gp78-mediated CYP3A4 ubiquitination, which is accelerated and enhanced on its mutation to the phosphomimetic Asp residue but attenuated on its Ala mutation. Intriguingly, CYP3A5, a polymorphically expressed human liver CYP3A4 isoform (containing Asp-478) is ubiquitinated but not degraded to a greater extent than CYP3A4 in HepG2 cells. This suggests that although Ser-478 phosphorylation is essential for UBC7/gp78-mediated CYP3A4 ubiquitination, it is not sufficient for its ERAD. Additionally, we now report that CYP3A4 protein phosphorylation by PKA and/or PKC at sites other than Ser-478, Thr-264, and Ser-420 also enhances UbcH5a/CHIP-mediated ubiquitination. Through proteomic analyses, we identify (i) 12 additional phosphorylation sites that may be involved in CHIP-CYP3A4 interactions and (ii) 8 previously unidentified CYP3A4 ubiquitination sites within spatially associated clusters of Asp/Glu and phosphorylatable Ser/Thr residues that may

  8. Molecular modeling of cytochrome P450 3A4

    NASA Astrophysics Data System (ADS)

    Szklarz, Grazyna D.; Halpert, James R.

    1997-05-01

    The three-dimensional structure of human cytochrome P450 3A4 was modeled based on crystallographic coordinates of four bacterial P450s: P450 BM-3, P450cam, P450terp, and P450eryF. The P450 3A4 sequence was aligned to those of the known proteins using a structure-based alignment of P450 BM-3, P450cam, P450terp, and P450eryF. The coordinates of the model were then calculated using a consensus strategy, and the final structure was optimized in the presence of water. The P450 3A4 model resembles P450 BM-3 the most, but the B' helix is similar to that of P450eryF, which leads to an enlarged active site when compared with P450 BM-3, P450cam, and P450terp. The 3A4 residues equivalent to known substrate contact residues of the bacterial proteins and key residues of rat P450 2B1 are located in the active site or the substrate access channel. Docking of progesterone into the P450 3A4 model demonstrated that the substrate bound in a 6β-orientation can interact with a number of active site residues, such as 114, 119, 301, 304, 305, 309, 370, 373, and 479, through hydrophobic interactions. The active site of the enzyme can also accommodate erythromycin, which, in addition to the residues listed for progesterone, also contacts residues 101, 104, 105, 214, 215, 217, 218, 374, and 478. The majority of 3A4 residues which interact with progesterone and/or erythromycin possess their equivalents in key residues of P450 2B enzymes, except for residues 297, 480 and 482, which do not contact either substrate in P450 3A4. The results from docking of progesterone and erythromycin into the enzyme model make it possible to pinpoint residues which may be important for 3A4 function and to target them for site-directed mutagenesis.

  9. The regulation of cytochrome P450 2E1 during LPS-induced inflammation in the rat

    SciTech Connect

    Abdulla, Dalya; Goralski, Kerry B.; College of Pharmacy, Burbidge Building, Dalhousie University, Halifax, Nova Scotia, B3H 3J5

    2006-10-01

    It is well known that inflammatory and infectious conditions differentially regulate cytochrome P450 (P450)-mediated drug metabolism in the liver. We have previously outlined a potential pathway for the downregulation in hepatic cytochrome P450 following LPS-mediated inflammation in the CNS (Abdulla, D., Goralski, K.B., Garcia Del Busto Cano, E., Renton, K.W., 2005. The signal transduction pathways involved in hepatic cytochrome P450 regulation in the rat during an LPS-induced model of CNS inflammation. Drug Metab. Dispos). The purpose of this study was to outline the effects of LPS-induced peripheral and central nervous system inflammation on hepatic cytochrome P450 2E1 (CYP2E1) in vivo,more » an enzyme that plays an important role in various physiological and pathological states. We report an increase in hepatic mRNA expression of CYP2E1 that occurred as early as 2-3 h following either the intraperitoneal (i.p.) injection of 5 mg/kg LPS or i.c.v. administration of 25 {mu}g of LPS. This increase in CYP2E1 mRNA expression was sustained for 24 h. In sharp contrast to the increase in hepatic CYP2E1 mRNA, we observed a significant reduction in the catalytic activity of this enzyme 24 h following either the i.c.v. or i.p. administration of LPS. Cycloheximide or actinomycin-D did not change the LPS-mediated downregulation in hepatic CYP2E1 catalytic activity. Our results support the idea that LPS acts at two different levels to regulate hepatic CYP2E1: a transcriptional level to increase CYP2E1 mRNA expression and a post-transcriptional level to regulate CYP2E1 protein and activity.« less

  10. Grapefruit and drug interactions.

    PubMed

    2012-12-01

    Since the late 1980s, grapefruit juice has been known to affect the metabolism of certain drugs. Several serious adverse effects involving drug interactions with grapefruit juice have been published in detail. The components of grapefruit juice vary considerably depending on the variety, maturity and origin of the fruit, local climatic conditions, and the manufacturing process. No single component accounts for all observed interactions. Other grapefruit products are also occasionally implicated, including preserves, lyophylised grapefruit juice, powdered whole grapefruit, grapefruit seed extract, and zest. Clinical reports of drug interactions with grapefruit juice are supported by pharmacokinetic studies, each usually involving about 10 healthy volunteers, in which the probable clinical consequences were extrapolated from the observed plasma concentrations. Grapefruit juice inhibits CYP3A4, the cytochrome P450 isoenzyme most often involved in drug metabolism. This increases plasma concentrations of the drugs concerned, creating a risk of overdose and dose-dependent adverse effects. Grapefruit juice also inhibits several other cytochrome P450 isoenzymes, but they are less frequently implicated in interactions with clinical consequences. Drugs interacting with grapefruit and inducing serious clinical consequences (confirmed or very probable) include: immunosuppressants, some statins, benzodiazepines, most calcium channel blockers, indinavir and carbamazepine. There are large inter-individual differences in enzyme efficiency. Along with the variable composition of grapefruit juice, this makes it difficult to predict the magnitude and clinical consequences of drug interactions with grapefruit juice in a given patient. There is increasing evidence that transporter proteins such as organic anion transporters and P-glycoprotein are involved in interactions between drugs and grapefruit juice. In practice, numerous drugs interact with grapefruit juice. Although only a few

  11. Induction of P450 3A1/2 and 2C6 by gemfibrozil in Sprague-Dawley rats.

    PubMed

    Liu, Aiming; Yang, Julin; Zhao, Xin; Jiao, Xiaolan; Zhao, Weihong; Ma, Qing; Tang, Zhiyuan; Dai, Renke

    2011-01-01

    Fibrates are a group of peroxisome proliferator-activated receptor α agonists used in the treatment of dyslipidemia; however, they have been reported to cause species-related hepatocarcinogenesis and clinical myotoxicity. Gemfibrozil is one of the most commonly used fibrates, and it shows the highest risk for myotoxicity among the fibrates. The inhibitory drug-drug interaction mechanism associated with gemfibrozil has been explored recently, and the induction of human P450 3A4 and 2C8 has been reported. In this study, in vivo induction of rat P450 by gemfibrozil was studied in Sprague-Dawley rats. After the rats were dosed with gemfibrozil by oral gavage, microsomes were prepared. The metabolic activities of P450 3A1/2, 2C6, and 2D2 were assayed using probe substrates, and the systemic concentration of gemfibrozil during its administration was determined. P450 3A1/2 and 2C6 activities were induced 32-77% in the rats by gemfibrozil when the exposure concentration was in the clinical range. These data indicate that the inducibility of homologous P450 isoforms by gemfibrozil is similar in Sprague-Dawley rats and in humans. Inductive drug-drug interactions and inhibitory actions are involved in the co-administration of gemfibrozil with other drugs, which suggests the relevance for a fibrate-toxicology investigation.

  12. Regulation of P450-mediated permethrin resistance in Culex quinquefasciatus by the GPCR/Gαs/AC/cAMP/PKA signaling cascade.

    PubMed

    Li, Ting; Liu, Nannan

    2017-12-01

    This study explores the role of G-protein-coupled receptor-intracellular signaling in the development of P450-mediated insecticide resistance in mosquitoes, Culex quinquefasciatus , focusing on the essential function of the GPCRs and their downstream effectors of Gs alpha subunit protein (Gαs) and adenylyl cyclase (ACs) in P450-mediated insecticide resistance of Culex mosquitoes. Our RNAi-mediated functional study showed that knockdown of Gαs caused the decreased expression of the downstream effectors of ACs and PKAs in the GPCR signaling pathway and resistance P450 genes, whereas knockdown of ACs decreased the expression of PKAs and resistance P450 genes. Knockdown of either Gαs or ACs resulted in an increased susceptibility of mosquitoes to permethrin. These results add significantly to our understanding of the molecular basis of resistance P450 gene regulation through GPCR/Gαs/AC/cAMP-PKA signaling pathways in the insecticide resistance of mosquitoes. The temporal and spatial dynamic analyses of GPCRs, Gαs, ACs, PKAs, and P450s in two insecticide resistant mosquito strains revealed that all the GPCR signaling pathway components tested, namely GPCRs, Gαs, ACs and PKAs, were most highly expressed in the brain for both resistant strains, suggesting the role played by these genes in signaling transduction and regulation. The resistance P450 genes were mainly expressed in the brain, midgut and malpighian tubules (MTs), suggesting their critical function in the central nervous system and importance for detoxification. The temporal dynamics analysis for the gene expression showed a diverse expression profile during mosquito development, indicating their initially functional importance in response to exposure to insecticides during their life stages.

  13. In vitro characterization of sarizotan metabolism: hepatic clearance, identification and characterization of metabolites, drug-metabolizing enzyme identification, and evaluation of cytochrome p450 inhibition.

    PubMed

    Gallemann, Dieter; Wimmer, Elmar; Höfer, Constance C; Freisleben, Achim; Fluck, Markus; Ladstetter, Bernhard; Dolgos, Hugues

    2010-06-01

    In vitro biotransformation studies of sarizotan using human liver microsomes (HLM) showed aromatic and aliphatic monohydroxylation and dealkylation. Recombinant cytochromes P450 (P450) together with P450-selective inhibitors in HLM/hepatocyte cultures were used to evaluate the relative contribution of different P450s and revealed major involvement of CYP3A4, CYP2C9, CYP2C8, and CYP1A2 in sarizotan metabolism. The apparent K(m, u) and V(max) of sarizotan clearance, as investigated in HLM, were 9 microM and 3280 pmol/mg/min, predicting in vivo hepatic clearance of 0.94 l/h, which indicates that sarizotan is a low-clearance compound in humans and suggests nonsaturable metabolism at the targeted plasma concentration (< or =1 microM). This finding is confirmed by the reported human clearance (CL/F of 3.6-4.4 l/h) and by the dose-linear area under the curve increase observed with doses up to 25 mg. The inhibitory effect of sarizotan toward six major P450s was evaluated using P450-specific marker reactions in pooled HLM. K(i, u) values of sarizotan against CYP2C8, CYP2C19, and CYP3A4 were >10 microM, whereas those against CYP2D6 and CYP1A2 were 0.43 and 8.7 microM, respectively. Based on the estimates of sarizotan concentrations at the enzyme active sites, no clinically significant drug-drug interactions (DDIs) due to P450 inhibition are expected. This result has been confirmed in human DDI studies in which no inhibition of five major P450s was observed in terms of marker metabolite formation.

  14. Estimation of the binding modes with important human cytochrome P450 enzymes, drug interaction potential, pharmacokinetics, and hepatotoxicity of ginger components using molecular docking, computational, and pharmacokinetic modeling studies.

    PubMed

    Qiu, Jia-Xuan; Zhou, Zhi-Wei; He, Zhi-Xu; Zhang, Xueji; Zhou, Shu-Feng; Zhu, Shengrong

    2015-01-01

    Ginger is one of the most commonly used herbal medicines for the treatment of numerous ailments and improvement of body functions. It may be used in combination with prescribed drugs. The coadministration of ginger with therapeutic drugs raises a concern of potential deleterious drug interactions via the modulation of the expression and/or activity of drug-metabolizing enzymes and drug transporters, resulting in unfavorable therapeutic outcomes. This study aimed to determine the molecular interactions between 12 main active ginger components (6-gingerol, 8-gingerol, 10-gingerol, 6-shogaol, 8-shogaol, 10-shogaol, ar-curcumene, β-bisabolene, β-sesquiphelandrene, 6-gingerdione, (-)-zingiberene, and methyl-6-isogingerol) and human cytochrome P450 (CYP) 1A2, 2C9, 2C19, 2D6, and 3A4 and to predict the absorption, distribution, metabolism, excretion, and toxicity (ADMET) of the 12 ginger components using computational approaches and comprehensive literature search. Docking studies showed that ginger components interacted with a panel of amino acids in the active sites of CYP1A2, 2C9, 2C19, 2D6, and 3A4 mainly through hydrogen bond formation, to a lesser extent, via π-π stacking. The pharmacokinetic simulation studies showed that the [I]/[Ki ] value for CYP2C9, 2C19, and 3A4 ranged from 0.0002 to 19.6 and the R value ranged from 1.0002 to 20.6 and that ginger might exhibit a high risk of drug interaction via inhibition of the activity of human CYP2C9 and CYP3A4, but a low risk of drug interaction toward CYP2C19-mediated drug metabolism. Furthermore, it has been evaluated that the 12 ginger components possessed a favorable ADMET profiles with regard to the solubility, absorption, permeability across the blood-brain barrier, interactions with CYP2D6, hepatotoxicity, and plasma protein binding. The validation results showed that there was no remarkable effect of ginger on the metabolism of warfarin in humans, whereas concurrent use of ginger and nifedipine exhibited a

  15. Estimation of the binding modes with important human cytochrome P450 enzymes, drug interaction potential, pharmacokinetics, and hepatotoxicity of ginger components using molecular docking, computational, and pharmacokinetic modeling studies

    PubMed Central

    Qiu, Jia-Xuan; Zhou, Zhi-Wei; He, Zhi-Xu; Zhang, Xueji; Zhou, Shu-Feng; Zhu, Shengrong

    2015-01-01

    Ginger is one of the most commonly used herbal medicines for the treatment of numerous ailments and improvement of body functions. It may be used in combination with prescribed drugs. The coadministration of ginger with therapeutic drugs raises a concern of potential deleterious drug interactions via the modulation of the expression and/or activity of drug-metabolizing enzymes and drug transporters, resulting in unfavorable therapeutic outcomes. This study aimed to determine the molecular interactions between 12 main active ginger components (6-gingerol, 8-gingerol, 10-gingerol, 6-shogaol, 8-shogaol, 10-shogaol, ar-curcumene, β-bisabolene, β-sesquiphelandrene, 6-gingerdione, (−)-zingiberene, and methyl-6-isogingerol) and human cytochrome P450 (CYP) 1A2, 2C9, 2C19, 2D6, and 3A4 and to predict the absorption, distribution, metabolism, excretion, and toxicity (ADMET) of the 12 ginger components using computational approaches and comprehensive literature search. Docking studies showed that ginger components interacted with a panel of amino acids in the active sites of CYP1A2, 2C9, 2C19, 2D6, and 3A4 mainly through hydrogen bond formation, to a lesser extent, via π–π stacking. The pharmacokinetic simulation studies showed that the [I]/[Ki] value for CYP2C9, 2C19, and 3A4 ranged from 0.0002 to 19.6 and the R value ranged from 1.0002 to 20.6 and that ginger might exhibit a high risk of drug interaction via inhibition of the activity of human CYP2C9 and CYP3A4, but a low risk of drug interaction toward CYP2C19-mediated drug metabolism. Furthermore, it has been evaluated that the 12 ginger components possessed a favorable ADMET profiles with regard to the solubility, absorption, permeability across the blood–brain barrier, interactions with CYP2D6, hepatotoxicity, and plasma protein binding. The validation results showed that there was no remarkable effect of ginger on the metabolism of warfarin in humans, whereas concurrent use of ginger and nifedipine exhibited

  16. Human cytochromes P450 in health and disease

    PubMed Central

    Nebert, Daniel W.; Wikvall, Kjell; Miller, Walter L.

    2013-01-01

    There are 18 mammalian cytochrome P450 (CYP) families, which encode 57 genes in the human genome. CYP2, CYP3 and CYP4 families contain far more genes than the other 15 families; these three families are also the ones that are dramatically larger in rodent genomes. Most (if not all) genes in the CYP1, CYP2, CYP3 and CYP4 families encode enzymes involved in eicosanoid metabolism and are inducible by various environmental stimuli (i.e. diet, chemical inducers, drugs, pheromones, etc.), whereas the other 14 gene families often have only a single member, and are rarely if ever inducible or redundant. Although the CYP2 and CYP3 families can be regarded as largely redundant and promiscuous, mutations or other defects in one or more genes of the remaining 16 gene families are primarily the ones responsible for P450-specific diseases—confirming these genes are not superfluous or promiscuous but rather are more directly involved in critical life functions. P450-mediated diseases comprise those caused by: aberrant steroidogenesis; defects in fatty acid, cholesterol and bile acid pathways; vitamin D dysregulation and retinoid (as well as putative eicosanoid) dysregulation during fertilization, implantation, embryogenesis, foetogenesis and neonatal development. PMID:23297354

  17. Mobility of cytochrome P450 in the endoplasmic reticulum membrane.

    PubMed

    Szczesna-Skorupa, E; Chen, C D; Rogers, S; Kemper, B

    1998-12-08

    Cytochrome P450 2C2 is a resident endoplasmic reticulum (ER) membrane protein that is excluded from the recycling pathway and contains redundant retention functions in its N-terminal transmembrane signal/anchor sequence and its large, cytoplasmic domain. Unlike some ER resident proteins, cytochrome P450 2C2 does not contain any known retention/retrieval signals. One hypothesis to explain exclusion of resident ER proteins from the transport pathway is the formation of networks by interaction with other proteins that immobilize the proteins and are incompatible with packaging into the transport vesicles. To determine the mobility of cytochrome P450 in the ER membrane, chimeric proteins of either cytochrome P450 2C2, its catalytic domain, or the cytochrome P450 2C1 N-terminal signal/anchor sequence fused to green fluorescent protein (GFP) were expressed in transiently transfected COS1 cells. The laurate hydroxylase activities of cytochrome P450 2C2 or the catalytic domain with GFP fused to the C terminus were similar to the native enzyme. The mobilities of the proteins in the membrane were determined by recovery of fluorescence after photobleaching. Diffusion coefficients for all P450 chimeras were similar, ranging from 2.6 to 6.2 x 10(-10) cm2/s. A coefficient only slightly larger (7.1 x 10(-10) cm2/s) was determined for a GFP chimera that contained a C-terminal dilysine ER retention signal and entered the recycling pathway. These data indicate that exclusion of cytochrome P450 from the recycling pathway is not mediated by immobilization in large protein complexes.

  18. Relative contributions of the major human CYP450 to the metabolism of icotinib and its implication in prediction of drug-drug interaction between icotinib and CYP3A4 inhibitors/inducers using physiologically based pharmacokinetic modeling.

    PubMed

    Chen, Jia; Liu, Dongyang; Zheng, Xin; Zhao, Qian; Jiang, Ji; Hu, Pei

    2015-06-01

    Icotinib is an anticancer drug, but relative contributions of CYP450 have not been identified. This study was carried out to identify the contribution percentage of CYP450 to icotinib and use the results to develop a physiologically based pharmacokinetic (PBPK) model, which can help to predict drug-drug interaction (DDI). Human liver microsome (HLM) and supersome using relative activity factor (RAF) were employed to determine the relative contributions of the major human P450 to the net hepatic metabolism of icotinib. These values were introduced to develop a PBPK model using SimCYP. The model was validated by the observed data in a Phase I clinical trial in Chinese healthy subjects. Finally, the model was used to simulate the DDI with ketoconazole or rifampin. Final contribution of CYP450 isoforms determined by HLM showed that CYP3A4 provided major contributions to the metabolism of icotinib. The percentage contributions of the P450 to the net hepatic metabolism of icotinib were determined by HLM inhibition assay and RAF. The AUC ratio under concomitant use of ketoconazole and rifampin was 3.22 and 0.55, respectively. Percentage of contribution of CYP450 to icotinib metabolism was calculated by RAF. The model has been proven to fit the observed data and is used in predicting icotinib-ketoconazole/rifampin interaction.

  19. Human Liver Cytochrome P450 3A4 Ubiquitination

    PubMed Central

    Wang, YongQiang; Kim, Sung-Mi; Trnka, Michael J.; Liu, Yi; Burlingame, A. L.; Correia, Maria Almira

    2015-01-01

    CYP3A4 is an abundant and catalytically dominant human liver endoplasmic reticulum-anchored cytochrome P450 enzyme engaged in the biotransformation of endo- and xenobiotics, including >50% of clinically relevant drugs. Alterations of CYP3A4 protein turnover can influence clinically relevant drug metabolism and bioavailability and drug-drug interactions. This CYP3A4 turnover involves endoplasmic reticulum-associated degradation via the ubiquitin (Ub)-dependent 26 S proteasomal system that relies on two highly complementary E2 Ub-conjugating-E3 Ub-ligase (UBC7-gp78 and UbcH5a-C terminus of Hsc70-interacting protein (CHIP)-Hsc70-Hsp40) complexes, as well as protein kinases (PK) A and C. We have documented that CYP3A4 Ser/Thr phosphorylation (Ser(P)/Thr(P)) by PKA and/or PKC accelerates/enhances its Lys ubiquitination by either of these E2-E3 systems. Intriguingly, CYP3A4 Ser(P)/Thr(P) and ubiquitinated Lys residues reside within the cytosol-accessible surface loop and/or conformationally assembled acidic Asp/Glu clusters, leading us to propose that such post-translational Ser/Thr protein phosphorylation primes CYP3A4 for ubiquitination. Herein, this possibility was examined through various complementary approaches, including site-directed mutagenesis, chemical cross-linking, peptide mapping, and LC-MS/MS analyses. Our findings reveal that such CYP3A4 Asp/Glu/Ser(P)/Thr(P) surface clusters are indeed important for its intermolecular electrostatic interactions with each of these E2-E3 subcomponents. By imparting additional negative charge to these Asp/Glu clusters, such Ser/Thr phosphorylation would generate P450 phosphodegrons for molecular recognition by the E2-E3 complexes, thereby controlling the timing of CYP3A4 ubiquitination and endoplasmic reticulum-associated degradation. Although the importance of phosphodegrons in the CHIP targeting of its substrates is known, to our knowledge this is the first example of phosphodegron involvement in gp78-substrate

  20. Pyrethroid Activity-Based Probes for Profiling Cytochrome P450 Activities Associated with Insecticide Interactions

    SciTech Connect

    Ismail, Hanafy M.; O'Neill, Paul M.; Hong, David

    2014-01-18

    Pyrethroid insecticides are used to control a diverse spectrum of diseases spread by arthropods. We have developed a suite of pyrethroid mimetic activity based probes (PyABPs) to selectively label and identify P450s associated with pyrethroid metabolism. The probes were screened against pyrethroid metabolizing and non-metabolizing mosquito P450s, as well as rodent microsomes to measure labeling specificity, plus CPR and b5 knockout mouse livers to validate P450 activation and establish the role for b5 in probe activation. Using a deltamethrin mimetic PyABP we were able to profile active enzymes in rat liver microsomes and identify pyrethroid metabolizing enzymes in the targetmore » tissue. The most reactive enzyme was a P450, CYP2C11, which is known to metabolize deltamethrin. Furthermore, several other pyrethroid metabolizers were identified (CYPs 2C6, 3A4, 2C13 and 2D1) along with related detoxification enzymes, notably UDP-g’s 2B1 - 5, suggesting a network of associated pyrethroid metabolizing enzymes, or ‘pyrethrome’. Considering the central role that P450s play in metabolizing insecticides, we anticipate that PyABPs will aid the identification and profiling of P450s associated with insecticide pharmacology in a wide range of species, improving understanding of P450-insecticide interactions and aiding the development of new tools for disease control.« less

  1. Reduction in hepatic drug metabolizing CYP3A4 activities caused by P450 oxidoreductase mutations identified in patients with disordered steroid metabolism

    SciTech Connect

    Flueck, Christa E.; Mullis, Primus E.; Pandey, Amit V., E-mail: amit@pandeylab.org

    2010-10-08

    Research highlights: {yields} Cytochrome P450 3A4 (CYP3A4), metabolizes 50% of drugs in clinical use and requires NADPH-P450 reductase (POR). {yields} Mutations in human POR cause congenital adrenal hyperplasia from diminished activities of steroid metabolizing P450s. {yields} We are reporting that mutations in POR may reduce CYP3A4 activity. {yields} POR mutants Y181D, A457H, Y459H, V492E and R616X lost 99%, while A287P, C569Y and V608F lost 60-85% CYP3A4 activity. {yields} Reduction of CYP3A4 activity may cause increased risk of drug toxicities/adverse drug reactions in patients with POR mutations. -- Abstract: Cytochrome P450 3A4 (CYP3A4), the major P450 present in human liver metabolizesmore » approximately half the drugs in clinical use and requires electrons supplied from NADPH through NADPH-P450 reductase (POR, CPR). Mutations in human POR cause a rare form of congenital adrenal hyperplasia from diminished activities of steroid metabolizing P450s. In this study we examined the effect of mutations in POR on CYP3A4 activity. We used purified preparations of wild type and mutant human POR and in vitro reconstitution with purified CYP3A4 to perform kinetic studies. We are reporting that mutations in POR identified in patients with disordered steroidogenesis/Antley-Bixler syndrome (ABS) may reduce CYP3A4 activity, potentially affecting drug metabolism in individuals carrying mutant POR alleles. POR mutants Y181D, A457H, Y459H, V492E and R616X had more than 99% loss of CYP3A4 activity, while POR mutations A287P, C569Y and V608F lost 60-85% activity. Loss of CYP3A4 activity may result in increased risk of drug toxicities and adverse drug reactions in patients with POR mutations.« less

  2. Identification of human cytochrome P450 2D6 as major enzyme involved in the O-demethylation of the designer drug p-methoxymethamphetamine.

    PubMed

    Staack, Roland F; Theobald, Denis S; Paul, Liane D; Springer, Dietmar; Kraemer, Thomas; Maurer, Hans H

    2004-04-01

    p-Methoxymethamphetamine (PMMA) is a new designer drug, listed in many countries as a controlled substance. Several fatalities have been attributed to the abuse of this designer drug. Previous in vivo studies using Wistar rats had shown that PMMA was metabolized mainly by O-demethylation. The aim of the study presented here was to identify the human hepatic cytochrome P450 (P450) enzymes involved in the biotransformation of PMMA to p-hydroxymethamphetamine. Baculovirus-infected insect cell microsomes, pooled human liver microsomes (pHLMs), and CYP2D6 poor-metabolizer genotype human liver microsomes (PM HLMs) were used for this purpose. Only CYP2D6 catalyzed O-demethylation. The apparent K(m) and V(max) values in baculovirus-infected insect cell microsomes were 4.6 +/- 1.0 microM and 92.0 +/- 3.7 pmol/min/pmol P450, respectively, and 42.0 +/- 4.0 microM and 412.5 +/- 10.8 pmol/min/mg protein in pHLMs. Inhibition studies with 1 microM quinidine showed significant inhibition of the metabolite formation (67.2 +/- 0.6%; p < 0.0001), and comparison of the metabolite formation between pHLMs and PM HLMs revealed significantly lower metabolite formation in the incubations with PM HLMs (87.3 +/- 1.1%; p < 0.0001). According to these studies, CYP2D6 is the major P450 involved in O-demethylation of PMMA.

  3. [Pharmacokinetic interactions of telaprevir with other drugs].

    PubMed

    Berenguer Berenguer, Juan; González-García, Juan

    2013-07-01

    Telaprevir is a new direct-acting antiviral drug for the treatment of hepatitis C virus (HCV) infection and is both a substrate and an inhibitor of cytochrome P450 (CYP450) isoenzymes. With the introduction of this new drug, assessment of drug-drug interactions has become a key factor in the evaluation of patients under treatment for HCV infection. During the treatment of this infection, many patients require other drugs to mitigate the adverse effects of anti-HCV drugs and to control other comorbidities. Moreover, most patients coinfected with HIV and HCV require antiretroviral therapy during treatment for HCV. Physicians should therefore be familiar with the pharmacokinetic properties of direct-acting antivirals for HCV treatment and their potential drug-drug interactions. The present article reviews the available information to date on the interactions of telaprevir with other drugs and provides recommendations for daily clinical practice. Copyright © 2013 Elsevier España, S.L. All rights reserved.

  4. Drug Interactions and Antiretroviral Drug Monitoring

    PubMed Central

    Foy, Matthew; Sperati, C. John; Lucas, Gregory M.

    2014-01-01

    Due to the improved longevity afforded by combination antiretroviral therapy (cART), HIV-infected individuals are developing several non-AIDS related comorbid conditions. Consequently, medical management of the HIV-infected population is increasingly complex, with a growing list of potential drug-drug interactions (DDIs). This article reviews some of the most relevant and emerging potential interactions between antiretroviral medications and other agents. The most common DDIs are those involving protease inhibitors or non-nucleoside reverse transcriptase inhibitors which alter the cytochrome P450 enzyme system and/or drug transporters such as p-glycoprotein. Of note are the new agents for the treatment of chronic hepatitis C virus infection. These new classes of drugs and others drugs which are increasingly used in this patient population represent a significant challenge with regard to achieving the goals of effective HIV suppression and minimization of drug-related toxicities. Awareness of DDIs and a multidisciplinary approach are imperative in reaching these goals. PMID:24950731

  5. Engineering human cytochrome P450 enzymes into catalytically self-sufficient chimeras using molecular Lego.

    PubMed

    Dodhia, Vikash Rajnikant; Fantuzzi, Andrea; Gilardi, Gianfranco

    2006-10-01

    The membrane-bound human cytochrome P450s have essential roles in the metabolism of endogenous compounds and drugs. Presented here are the results on the construction and characterization of three fusion proteins containing the N-terminally modified human cytochrome P450s CYP2C9, CY2C19 and CYP3A4 fused to the soluble NADPH-dependent oxidoreductase domain of CYP102A1 from Bacillus megaterium. The constructs, CYP2C9/BMR, CYP2C19/BMR and CYP3A4/BMR are well expressed in Escherichia coli as holo proteins. The chimeras can be purified in the absence of detergent and the purified enzymes are both active and correctly folded in the absence of detergent, as demonstrated by circular dichroism and functional studies. Additionally, in comparison with the parent P450 enzyme, these chimeras have greatly improved solubility properties. The chimeras are catalytically self-sufficient and present turnover rates similar to those reported for the native enzymes in reconstituted systems, unlike previously reported mammalian cytochrome P450 fusion proteins. Furthermore the specific activities of these chimeras are not dependent on the enzyme concentration present in the reaction buffer and they do not require the addition of accessory proteins, detergents or phospholipids to be fully active. The solubility, catalytic self-sufficiency and wild-type like activities of these chimeras would greatly simplify the studies of cytochrome P450 mediated drug metabolism in solution.

  6. Cytochrome P450 monooxygenases: perspectives for synthetic application.

    PubMed

    Urlacher, Vlada B; Eiben, Sabine

    2006-07-01

    Cytochrome P450 monooxygenases are versatile biocatalysts that introduce oxygen into a vast range of molecules. These enzymes catalyze diverse reactions in a regio- and stereoselective manner, and their properties have been used for drug development, bioremediation and the synthesis of fine chemicals and other useful compounds. However, the potential of P450 monooxygenases has not been fully exploited; there are some drawbacks limiting the broader implementation of these catalysts for commercial needs. Protein engineering has produced P450 enzymes with widely altered substrate specificities, substantially increased activity and higher stability. Furthermore, electrochemical and enzymatic approaches for the replacement or regeneration of NAD(P)H have been developed, enabling the more cost-effective use of P450 enzymes. In this review, we focus on the aspects relevant to the synthetic applications of P450 enzymes and their optimization for commercial needs.

  7. Electrochemistry in the mimicry of oxidative drug metabolism by cytochrome P450s.

    PubMed

    Nouri-Nigjeh, Eslam; Bischoff, Rainer; Bruins, Andries P; Permentier, Hjalmar P

    2011-05-01

    Prediction of oxidative drug metabolism at the early stages of drug discovery and development requires fast and accurate analytical techniques to mimic the in vivo oxidation reactions by cytochrome P450s (CYP). Direct electrochemical oxidation combined with mass spectrometry, although limited to the oxidation reactions initiated by charge transfer, has shown promise in the mimicry of certain CYP-mediated metabolic reactions. The electrochemical approach may further be utilized in an automated manner in microfluidics devices facilitating fast screening of oxidative drug metabolism. A wide range of in vivo oxidation reactions, particularly those initiated by hydrogen atom transfer, can be imitated through the electrochemically-assisted Fenton reaction. This reaction is based on O-O bond activation in hydrogen peroxide and oxidation by hydroxyl radicals, wherein electrochemistry is used for the reduction of molecular oxygen to hydrogen peroxide, as well as the reduction of Fe(3+) to Fe(2+). Metalloporphyrins, as surrogates for the prosthetic group in CYP, utilizing metallo-oxo reactive species, can also be used in combination with electrochemistry. Electrochemical reduction of metalloporphyrins in solution or immobilized on the electrode surface activates molecular oxygen in a manner analogous to the catalytical cycle of CYP and different metalloporphyrins can mimic selective oxidation reactions. Chemoselective, stereoselective, and regioselective oxidation reactions may be mimicked using electrodes that have been modified with immobilized enzymes, especially CYP itself. This review summarizes the recent attempts in utilizing electrochemistry as a versatile analytical and preparative technique in the mimicry of oxidative drug metabolism by CYP. © 2011 Bentham Science Publishers Ltd.

  8. Examination of Zolpidem effects on AhR- and PXR-dependent expression of drug-metabolizing cytochromes P450 in primary cultures of human hepatocytes.

    PubMed

    Bachleda, Petr; Vrzal, Radim; Pivnicka, Jakub; Cvek, Boris; Dvorak, Zdenek

    2009-12-01

    A hypnotic drug Zolpidem is used in clinical practice for more than 25 years. Surprisingly, the effects of Zolpidem on the expression of drug-metabolizing cytochromes P450 (CYPs) were not examined yet. Recently, the unexpected capacity of several "old drugs", such as valproic acid or azoles, to induce CYPs was reported. Therefore, we tested whether Zolpidem induces the expression of important CYPs in primary cultures of human hepatocytes. Cells were treated for 24h with Zolpidem in therapeutic (0.1mg/L) and toxic (1mg/L) concentrations. The levels of CYP1A1, CYP1A2, CY2C9 and CYP3A4 mRNAs were not altered by Zolpidem, whereas model inducers dioxin and rifampicin significantly induced CYP1A and CYP2/3 gene expression, respectively. Consistently, Zolpidem did not activate aryl hydrocarbon receptor (AhR) and pregnane X receptor (PXR), the key regulators of cytochromes P450s, as revealed by transient transfection gene reporter assays using HepG2 cells. We conclude Zolpidem be considered a safe drug with respect to the possible interactions through AhR- and PXR-dependent induction of drug-metabolizing CYPs.

  9. The effect of Cree traditional medicinal teas on the activity of human cytochrome P450-mediated metabolism.

    PubMed

    Tam, Teresa W; Liu, Rui; Saleem, Ammar; Arnason, John T; Krantis, Anthony; Haddad, Pierre S; Foster, Brian C

    2014-08-08

    Rhododendron groenlandicum (Bog Labrador tea), Rhododendron tomentosum (Marsh Labrador tea) and Juniperus communis (Juniper) are used in medicinal teas by Canadian aboriginal cultures alone and in combination with conventional drug products. The safety of this combination had not been previously examined and this study was initiated to examine the potential of medicinal teas to inhibit the major human drug metabolizing enzyme, cytochrome P450 3A4 (CYP3A4). The decoctions of Rhododendron groenlandicum and Rhododendron tomentosum leaves and Juniperus communis berries were examined in a microtiter fluorometric assay to examine their potential to inhibit CYP-mediated metabolism. The decoctions showed progressive inhibition towards CYP3A4 the longer the leaves or berries were brewed. R. Rhododendron groenlandicum and Juniperus communis may have the potential to inhibit CYP3A4-mediated metabolism. The findings of this study with these traditional medicines are significant in that they provide mechanistic support that these products have the potential to affect the safety and efficacy of other health and medicinal products. As this study only examined CYP3A4, it is possible that these medicinals contain substances that could also affect other metabolic enzymes. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  10. High-Fat Diets Alter the Modulatory Effects of Xenobiotics on Cytochrome P450 Activities

    SciTech Connect

    Sadler, Natalie C.; Webb-Robertson, Bobbie-Jo M.; Clauss, Therese R.

    Cytochrome P450 monooxygenases (P450) are key to the metabolism of myriad endogenous chemicals and xenobiotics, including the majority of therapeutic drugs. Dysregulated P450 activities can lead to altered drug metabolism and toxicity, oxidative stress, and inflammation; all physiological states frequently charged as the impetus for various chronic pathologies. We characterized the impact of common xenobiotic exposures, specifically high-fat diet and active or passive cigarette smoke, on the functional capacity of hepatic and pulmonary P450s. We employed an activity-based protein profiling approach to characterize the identity and activity level of measured individual P450 isoforms. Our results confirm expectations of significant alterationsmore » in pulmonary P450s due to cigarette smoke, but now reveal the repressive impact of high-fat diet-induced obesity on many hepatic P450s activities, and the dynamic alterations due to concomitant diet and smoke exposures on liver and lung P450 activities impacting drug metabolism and pathways of inflammation.« less

  11. Structural basis for human NADPH-cytochrome P450 oxidoreductase deficiency

    SciTech Connect

    Xia, Chuanwu; Panda, Satya P.; Marohnic, Christopher C.

    2012-03-15

    NADPH-cytochrome P450 oxidoreductase (CYPOR) is essential for electron donation to microsomal cytochrome P450-mediated monooxygenation in such diverse physiological processes as drug metabolism (approximately 85-90% of therapeutic drugs), steroid biosynthesis, and bioactive metabolite production (vitamin D and retinoic acid metabolites). Expressed by a single gene, CYPOR's role with these multiple redox partners renders it a model for understanding protein-protein interactions at the structural level. Polymorphisms in human CYPOR have been shown to lead to defects in bone development and steroidogenesis, resulting in sexual dimorphisms, the severity of which differs significantly depending on the degree of CYPOR impairment. The atomic structure ofmore » human CYPOR is presented, with structures of two naturally occurring missense mutations, V492E and R457H. The overall structures of these CYPOR variants are similar to wild type. However, in both variants, local disruption of H bonding and salt bridging, involving the FAD pyrophosphate moiety, leads to weaker FAD binding, unstable protein, and loss of catalytic activity, which can be rescued by cofactor addition. The modes of polypeptide unfolding in these two variants differ significantly, as revealed by limited trypsin digestion: V492E is less stable but unfolds locally and gradually, whereas R457H is more stable but unfolds globally. FAD addition to either variant prevents trypsin digestion, supporting the role of the cofactor in conferring stability to CYPOR structure. Thus, CYPOR dysfunction in patients harboring these particular mutations may possibly be prevented by riboflavin therapy in utero, if predicted prenatally, or rescued postnatally in less severe cases.« less

  12. Insect P450 inhibitors and insecticides: challenges and opportunities.

    PubMed

    Feyereisen, René

    2015-06-01

    P450 enzymes are encoded by a large number of genes in insects, often over a hundred. They play important roles in insecticide metabolism and resistance, and growing numbers of P450 enzymes are now known to catalyse important physiological reactions, such as hormone metabolism or cuticular hydrocarbon synthesis. Ways to inhibit P450 enzymes specifically or less specifically are well understood, as P450 inhibitors are found as drugs, as fungicides, as plant growth regulators and as insecticide synergists. Yet there are no P450 inhibitors as insecticides on the market. As new modes of action are constantly needed to support insecticide resistance management, P450 inhibitors should be considered because of their high potential for insect selectivity, their well-known mechanisms of action and the increasing ease of rational design and testing. © 2014 Society of Chemical Industry.

  13. Pathophysiological implications of neurovascular P450 in brain disorders

    PubMed Central

    Ghosh, Chaitali; Hossain, Mohammed; Solanki, Jesal; Dadas, Aaron; Marchi, Nicola; Janigro, Damir

    2016-01-01

    Over the past decades, the significance of cytochrome P450 (CYP) enzymes has expanded beyond their role as peripheral drug metabolizers in the liver and gut. CYP enzymes are also functionally active at the neurovascular interface. CYP expression is modulated by disease states, impacting cellular functions, detoxification, and reactivity to toxic stimuli and brain drug biotransformation. Unveiling the physiological and molecular complexity of brain P450 enzymes will improve our understanding of the mechanisms underlying brain drug availability, pharmacological efficacy, and neurotoxic adverse effects from pharmacotherapy targeting brain disorders. PMID:27312874

  14. Oxidation of the endogenous cannabinoid arachidonoyl ethanolamide by the cytochrome P450 monooxygenases: physiological and pharmacological implications.

    PubMed

    Snider, Natasha T; Walker, Vyvyca J; Hollenberg, Paul F

    2010-03-01

    Arachidonoyl ethanolamide (anandamide) is an endogenous amide of arachidonic acid and an important signaling mediator of the endocannabinoid system. Given its numerous roles in maintaining normal physiological function and modulating pathophysiological responses throughout the body, the endocannabinoid system is an important pharmacological target amenable to manipulation directly by cannabinoid receptor ligands or indirectly by drugs that alter endocannabinoid synthesis and inactivation. The latter approach has the possible advantage of more selectivity, thus there is the potential for fewer untoward effects like those that are traditionally associated with cannabinoid receptor ligands. In that regard, inhibitors of the principal inactivating enzyme for anandamide, fatty acid amide hydrolase (FAAH), are currently in development for the treatment of pain and inflammation. However, several pathways involved in anandamide synthesis, metabolism, and inactivation all need to be taken into account when evaluating the effects of FAAH inhibitors and similar agents in preclinical models and assessing their clinical potential. Anandamide undergoes oxidation by several human cytochrome P450 (P450) enzymes, including CYP3A4, CYP4F2, CYP4X1, and the highly polymorphic CYP2D6, forming numerous structurally diverse lipids, which are likely to have important physiological roles, as evidenced by the demonstration that a P450-derived epoxide of anandamide is a potent agonist for the cannabinoid receptor 2. The focus of this review is to emphasize the need for a better understanding of the P450-mediated pathways of the metabolism of anandamide, because these are likely to be important in mediating endocannabinoid signaling as well as the pharmacological responses to endocannabinoid-targeting drugs.

  15. Diversity and evolution of cytochrome P450 monooxygenases in Oomycetes.

    PubMed

    Sello, Mopeli Marshal; Jafta, Norventia; Nelson, David R; Chen, Wanping; Yu, Jae-Hyuk; Parvez, Mohammad; Kgosiemang, Ipeleng Kopano Rosinah; Monyaki, Richie; Raselemane, Seiso Caiphus; Qhanya, Lehlohonolo Benedict; Mthakathi, Ntsane Trevor; Sitheni Mashele, Samson; Syed, Khajamohiddin

    2015-07-01

    Cytochrome P450 monooxygenases (P450s) are heme-thiolate proteins whose role as drug targets against pathogens, as well as in valuable chemical production and bioremediation, has been explored. In this study we performed comprehensive comparative analysis of P450s in 13 newly explored oomycete pathogens. Three hundred and fifty-six P450s were found in oomycetes. These P450s were grouped into 15 P450 families and 84 P450 subfamilies. Among these, nine P450 families and 31 P450 subfamilies were newly found in oomycetes. Research revealed that oomycetes belonging to different orders contain distinct P450 families and subfamilies in their genomes. Evolutionary analysis and sequence homology data revealed P450 family blooms in oomycetes. Tandem arrangement of a large number of P450s belonging to the same family indicated that P450 family blooming is possibly due to its members' duplications. A unique combination of amino acid patterns was observed at EXXR and CXG motifs for the P450 families CYP5014, CYP5015 and CYP5017. A novel P450 fusion protein (CYP5619 family) with an N-terminal P450 domain fused to a heme peroxidase/dioxygenase domain was discovered in Saprolegnia declina. Oomycete P450 patterns suggested host influence in shaping their P450 content. This manuscript serves as reference for future P450 annotations in newly explored oomycetes.

  16. PXR as a mediator of herb-drug interaction.

    PubMed

    Hogle, Brett C; Guan, Xiudong; Folan, M Maggie; Xie, Wen

    2018-04-01

    Medicinal herbs have been a part of human medicine for thousands of years. The herb-drug interaction is an extension of drug-drug interaction, in which the consumptions of herbs cause alterations in the metabolism of drugs the patients happen to take at the same time. The pregnane X receptor (PXR) has been established as one of the most important transcriptional factors that regulate the expression of phase I enzymes, phase II enzymes, and drug transporters in the xenobiotic responses. Since its initial discovery, PXR has been implicated in multiple herb-drug interactions that can lead to alterations of the drug's pharmacokinetic properties and cause fluctuating therapeutic efficacies, possibly leading to complications. Regions of the world that heavily incorporate herbalism into their primary health care and people turning to alternative medicines as a personal choice could be at risk for adverse reactions or unintended results from these interactions. This article is intended to highlight our understanding of the PXR-mediated herb-drug interactions. Copyright © 2017. Published by Elsevier B.V.

  17. A Transcriptional Regulatory Network Containing Nuclear Receptors and Long Noncoding RNAs Controls Basal and Drug-Induced Expression of Cytochrome P450s in HepaRG Cells.

    PubMed

    Chen, Liming; Bao, Yifan; Piekos, Stephanie C; Zhu, Kexin; Zhang, Lirong; Zhong, Xiao-Bo

    2018-07-01

    Cytochrome P450 (P450) enzymes are responsible for metabolizing drugs. Expression of P450s can directly affect drug metabolism, resulting in various outcomes in therapeutic efficacy and adverse effects. Several nuclear receptors are transcription factors that can regulate expression of P450s at both basal and drug-induced levels. Some long noncoding RNAs (lncRNAs) near a transcription factor are found to participate in the regulatory functions of the transcription factors. The aim of this study is to determine whether there is a transcriptional regulatory network containing nuclear receptors and lncRNAs controlling both basal and drug-induced expression of P450s in HepaRG cells. Small interfering RNAs or small hairpin RNAs were applied to knock down four nuclear receptors [hepatocyte nuclear factor 1 α (HNF1 α ), hepatocyte nuclear factor 4 α (HNF4 α ), pregnane X receptor (PXR), and constitutive androstane receptor (CAR)] as well as two lncRNAs [HNF1 α antisense RNA 1 (HNF1 α -AS1) and HNF4 α antisense RNA 1 (HNF4 α -AS1)] in HepaRG cells with or without treatment of phenobarbital or rifampicin. Expression of eight P450 enzymes was examined in both basal and drug-induced levels. CAR and PXR mainly regulated expression of specific P450s. HNF1 α and HNF4 α affected expression of a wide range of P450s as well as other transcription factors. HNF1 α and HNF4 α controlled the expression of their neighborhood lncRNAs, HNF1 α -AS1 and HNF4 α -AS1, respectively. HNF1 α -AS1 and HNF4 α -AS1 was also involved in the regulation of P450s and transcription factors in diverse manners. Altogether, our study concludes that a transcription regulatory network containing the nuclear receptors and lncRNAs controls both basal and drug-induced expression of P450s in HepaRG cells. Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.

  18. Electrochemistry of Canis familiaris cytochrome P450 2D15 with gold nanoparticles: An alternative to animal testing in drug discovery.

    PubMed

    Rua, Francesco; Sadeghi, Sheila J; Castrignanò, Silvia; Valetti, Francesca; Gilardi, Gianfranco

    2015-10-01

    This work reports for the first time the direct electron transfer of the Canis familiaris cytochrome P450 2D15 on glassy carbon electrodes to provide an analytical tool as an alternative to P450 animal testing in the drug discovery process. Cytochrome P450 2D15, that corresponds to the human homologue P450 2D6, was recombinantly expressed in Escherichia coli and entrapped on glassy carbon electrodes (GC) either with the cationic polymer polydiallyldimethylammonium chloride (PDDA) or in the presence of gold nanoparticles (AuNPs). Reversible electrochemical signals of P450 2D15 were observed with calculated midpoint potentials (E1/2) of −191 ± 5 and −233 ± 4 mV vs. Ag/AgCl for GC/PDDA/2D15 and GC/AuNPs/2D15, respectively. These experiments were then followed by the electro-catalytic activity of the immobilized enzyme in the presence of metoprolol. The latter drug is a beta-blocker used for the treatment of hypertension and is a specific marker of the human P450 2D6 activity. Electrocatalysis data showed that only in the presence of AuNps the expected α-hydroxy-metoprolol product was present as shown by HPLC. The successful immobilization of the electroactive C. familiaris cytochrome P450 2D15 on electrode surfaces addresses the ever increasing demand of developing alternative in vitromethods for amore detailed study of animal P450 enzymes' metabolism, reducing the number of animals sacrificed in preclinical tests.

  19. Insights into drug metabolism by cytochromes P450 from modelling studies of CYP2D6-drug interactions

    PubMed Central

    Maréchal, J-D; Kemp, C A; Roberts, G C K; Paine, M J I; Wolf, C R; Sutcliffe, M J

    2008-01-01

    The cytochromes P450 (CYPs) comprise a vast superfamily of enzymes found in virtually all life forms. In mammals, xenobiotic metabolizing CYPs provide crucial protection from the effects of exposure to a wide variety of chemicals, including environmental toxins and therapeutic drugs. Ideally, the information on the possible metabolism by CYPs required during drug development would be obtained from crystal structures of all the CYPs of interest. For some years only crystal structures of distantly related bacterial CYPs were available and homology modelling techniques were used to bridge the gap and produce structural models of human CYPs, and thereby obtain useful functional information. A significant step forward in the reliability of these models came seven years ago with the first crystal structure of a mammalian CYP, rabbit CYP2C5, followed by the structures of six human enzymes, CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2D6 and CYP3A4, and a second rabbit enzyme, CYP2B4. In this review we describe as a case study the evolution of a CYP2D6 model, leading to the validation of the model as an in silico tool for predicting binding and metabolism. This work has led directly to the successful design of CYP2D6 mutants with novel activity—including creating a testosterone hydroxylase, converting quinidine from inhibitor to substrate, creating a diclofenac hydroxylase and creating a dextromethorphan O-demethylase. Our modelling-derived hypothesis-driven integrated interdisciplinary studies have given key insight into the molecular determinants of CYP2D6 and other important drug metabolizing enzymes. PMID:18026129

  20. Mechanistic modeling to predict the transporter- and enzyme-mediated drug-drug interactions of repaglinide.

    PubMed

    Varma, Manthena V S; Lai, Yurong; Kimoto, Emi; Goosen, Theunis C; El-Kattan, Ayman F; Kumar, Vikas

    2013-04-01

    Quantitative prediction of complex drug-drug interactions (DDIs) is challenging. Repaglinide is mainly metabolized by cytochrome-P-450 (CYP)2C8 and CYP3A4, and is also a substrate of organic anion transporting polypeptide (OATP)1B1. The purpose is to develop a physiologically based pharmacokinetic (PBPK) model to predict the pharmacokinetics and DDIs of repaglinide. In vitro hepatic transport of repaglinide, gemfibrozil and gemfibrozil 1-O-β-glucuronide was characterized using sandwich-culture human hepatocytes. A PBPK model, implemented in Simcyp (Sheffield, UK), was developed utilizing in vitro transport and metabolic clearance data. In vitro studies suggested significant active hepatic uptake of repaglinide. Mechanistic model adequately described repaglinide pharmacokinetics, and successfully predicted DDIs with several OATP1B1 and CYP3A4 inhibitors (<10% error). Furthermore, repaglinide-gemfibrozil interaction at therapeutic dose was closely predicted using in vitro fraction metabolism for CYP2C8 (0.71), when primarily considering reversible inhibition of OATP1B1 and mechanism-based inactivation of CYP2C8 by gemfibrozil and gemfibrozil 1-O-β-glucuronide. This study demonstrated that hepatic uptake is rate-determining in the systemic clearance of repaglinide. The model quantitatively predicted several repaglinide DDIs, including the complex interactions with gemfibrozil. Both OATP1B1 and CYP2C8 inhibition contribute significantly to repaglinide-gemfibrozil interaction, and need to be considered for quantitative rationalization of DDIs with either drug.

  1. Novel approaches to mitigating parathion toxicity: targeting cytochrome P450mediated metabolism with menadione

    PubMed Central

    Jan, Yi-Hua; Richardson, Jason R.; Baker, Angela A.; Mishin, Vladimir; Heck, Diane E.; Laskin, Debra L.; Laskin, Jeffrey D.

    2016-01-01

    Accidental or intentional exposures to parathion, an organophosphorus (OP) pesticide, can cause severe poisoning in humans. Parathion toxicity is dependent on its metabolism by the cytochrome P450 (CYP) system to paraoxon (diethyl 4-nitrophenyl phosphate), a highly poisonous nerve agent and potent inhibitor of acetylcholinesterase (AChE). We have been investigating inhibitors of CYP-mediated bioactivation of OPs as a method of preventing or reversing progressive parathion toxicity. It is well recognized that NADPH–cytochrome P450 reductase, an enzyme required for the transfer of electrons to CYPs, mediates chemical redox cycling. In this process, the enzyme diverts electrons from CYPs to support chemical redox cycling, which results in inhibition of CYP-mediated biotransformation. Using menadione as the redox-cycling chemical, we discovered that this enzymatic reaction blocks metabolic activation of parathion in rat and human liver microsomes and in recombinant CYPs important to parathion metabolism, including CYP1A2, CYP2B6, and CYP3A4. Administration of menadione to rats reduces metabolism of parathion, as well as parathion-induced inhibition of brain cholinesterase activity. This resulted in inhibition of parathion neurotoxicity. Menadione has relatively low toxicity and is approved by the FDA for other indications. Its ability to block parathion metabolism makes it an attractive therapeutic candidate to mitigate parathion-induced neurotoxicity. PMID:27441453

  2. Single-Walled Carbon Nanotubes Inhibit the Cytochrome P450 Enzyme, CYP3A4

    NASA Astrophysics Data System (ADS)

    El-Sayed, Ramy; Bhattacharya, Kunal; Gu, Zonglin; Yang, Zaixing; Weber, Jeffrey K.; Li, Hu; Leifer, Klaus; Zhao, Yichen; Toprak, Muhammet S.; Zhou, Ruhong; Fadeel, Bengt

    2016-02-01

    We report a detailed computational and experimental study of the interaction of single-walled carbon nanotubes (SWCNTs) with the drug-metabolizing cytochrome P450 enzyme, CYP3A4. Dose-dependent inhibition of CYP3A4-mediated conversion of the model compound, testosterone, to its major metabolite, 6β-hydroxy testosterone was noted. Evidence for a direct interaction between SWCNTs and CYP3A4 was also provided. The inhibition of enzyme activity was alleviated when SWCNTs were pre-coated with bovine serum albumin. Furthermore, covalent functionalization of SWCNTs with polyethylene glycol (PEG) chains mitigated the inhibition of CYP3A4 enzymatic activity. Molecular dynamics simulations suggested that inhibition of the catalytic activity of CYP3A4 is mainly due to blocking of the exit channel for substrates/products through a complex binding mechanism. This work suggests that SWCNTs could interfere with metabolism of drugs and other xenobiotics and provides a molecular mechanism for this toxicity. Our study also suggests means to reduce this toxicity, eg., by surface modification.

  3. Single-Walled Carbon Nanotubes Inhibit the Cytochrome P450 Enzyme, CYP3A4

    PubMed Central

    El-Sayed, Ramy; Bhattacharya, Kunal; Gu, Zonglin; Yang, Zaixing; Weber, Jeffrey K.; Li, Hu; Leifer, Klaus; Zhao, Yichen; Toprak, Muhammet S.; Zhou, Ruhong; Fadeel, Bengt

    2016-01-01

    We report a detailed computational and experimental study of the interaction of single-walled carbon nanotubes (SWCNTs) with the drug-metabolizing cytochrome P450 enzyme, CYP3A4. Dose-dependent inhibition of CYP3A4-mediated conversion of the model compound, testosterone, to its major metabolite, 6β-hydroxy testosterone was noted. Evidence for a direct interaction between SWCNTs and CYP3A4 was also provided. The inhibition of enzyme activity was alleviated when SWCNTs were pre-coated with bovine serum albumin. Furthermore, covalent functionalization of SWCNTs with polyethylene glycol (PEG) chains mitigated the inhibition of CYP3A4 enzymatic activity. Molecular dynamics simulations suggested that inhibition of the catalytic activity of CYP3A4 is mainly due to blocking of the exit channel for substrates/products through a complex binding mechanism. This work suggests that SWCNTs could interfere with metabolism of drugs and other xenobiotics and provides a molecular mechanism for this toxicity. Our study also suggests means to reduce this toxicity, eg., by surface modification. PMID:26899743

  4. Understanding the determinants of selectivity in drug metabolism through modeling of dextromethorphan oxidation by cytochrome P450

    PubMed Central

    Oláh, Julianna; Mulholland, Adrian J.; Harvey, Jeremy N.

    2011-01-01

    Cytochrome P450 enzymes play key roles in the metabolism of the majority of drugs. Improved models for prediction of likely metabolites will contribute to drug development. In this work, two possible metabolic routes (aromatic carbon oxidation and O-demethylation) of dextromethorphan are compared using molecular dynamics (MD) simulations and density functional theory (DFT). The DFT results on a small active site model suggest that both reactions might occur competitively. Docking and MD studies of dextromethorphan in the active site of P450 2D6 show that the dextromethorphan is located close to heme oxygen in a geometry apparently consistent with competitive metabolism. In contrast, calculations of the reaction path in a large protein model [using a hybrid quantum mechanical–molecular mechanics (QM/MM) method] show a very strong preference for O-demethylation, in accordance with experimental results. The aromatic carbon oxidation reaction is predicted to have a high activation energy, due to the active site preventing formation of a favorable transition-state structure. Hence, the QM/MM calculations demonstrate a crucial role of many active site residues in determining reactivity of dextromethorphan in P450 2D6. Beyond substrate binding orientation and reactivity of Compound I, successful metabolite predictions must take into account the detailed mechanism of oxidation in the protein. These results demonstrate the potential of QM/MM methods to investigate specificity in drug metabolism. PMID:21444768

  5. Genomic organization of human fetal specific P-450IIIA7 (cytochrome P-450HFLa)-related gene(s) and interaction of transcriptional regulatory factor with its DNA element in the 5' flanking region.

    PubMed

    Itoh, S; Yanagimoto, T; Tagawa, S; Hashimoto, H; Kitamura, R; Nakajima, Y; Okochi, T; Fujimoto, S; Uchino, J; Kamataki, T

    1992-03-24

    P-450IIIA7 is a form of cytochrome P-450 which was isolated from human fetal livers and termed P-450HFLa. This form has been clarified to be expressed during fetal life specifically (Komori, M., Nishio, K., Kitada, M., Shiramatsu, K., Muroya, K., Soma, M., Nagashima, K. and Kamataki, T. (1990) Biochemistry 29, 4430-4433). In the present study, we isolated five independent clones which probably corresponded to the human P-450IIIA7 gene. These clones were completely sequenced, all exons, exon-intron junctions and the 5' flanking region from the cap site to-869. Although the sequences in the coding region were completely identical to P-450IIIA7, it is possible that genomic fragments sequenced in this study encode portions of other P-450IIIA7-related genes since we could not obtain a complete overlapping set of genomic clones. Within its 5' flanking sequence, the putative binding sites of several transcriptional regulatory factors existed. Among them, it was shown that a basic transcription element binding factor (BTEB) actually interacted with the 5' flanking region of this gene.

  6. P450 AND METABOLISM IN TOXICOLOGY

    EPA Science Inventory

    The cytochromes P450 catalyze the initial phase of detoxification of many environmental chemicals, xenobiotic, drugs and the secondary metabolic product of plants. Plant secondary chemicals can be highly toxic, and they evolved in a coevolving plant - animal warfare - the plants ...

  7. In vitro activity of commercial valerian root extracts against human cytochrome P450 3A4.

    PubMed

    Lefebvre, Tania; Foster, Brian C; Drouin, Cathy E; Krantis, Anthony; Livesey, John F; Jordan, Scott A

    2004-08-12

    Valerian root ( Valeriana officinalis L.) has been used since antiquity as a medicinal herb. Recent studies have found that certain herbal products used concomitantly with conventional therapeutic products can markedly affect drug disposition. The in vitro effect of aliquots from 14 commercially available single-entity and blended products containing valerian root on cytochrome P450 CYP3A4-mediated metabolism and P-glycoprotein transport has been determined with aqueous, ethanol and acetonitrile extracts. Hydroxyvalerenic acid, acetoxyvalerenic acid and valerenic acid content was analyzed and wide variation was found between samples and compared to the concentrations noted on the product labels. Valerian extracts from the products tested also exhibited a marked capacity to inhibit cytochrome P450 3A4-mediated metabolism and P-glycoprotein transport based upon the ATPase assay. There is wide variation between commercially available samples of valerian root. The findings from this study suggest that valerian root may have an initial inhibitory effect when taken with therapeutic products. Further work is warranted to determine whether valerian root can affect other CYP450 isozymes and how the results of this in vitro investigation can be extrapolated to in vivo situations.

  8. Studies on Pharmacokinetic Drug Interaction Potential of Vinpocetine

    PubMed Central

    Manda, Vamshi K.; Avula, Bharathi; Dale, Olivia R.; Chittiboyina, Amar G.; Khan, Ikhlas A.; Walker, Larry A.; Khan, Shabana I.

    2015-01-01

    Abstract Background Vinpocetine, a semi-synthetic derivative of vincamine, is a popular dietary supplement used for the treatment of several central nervous system related disorders. Despite its wide use, no pharmacokinetic drug interaction studies are reported in the literature. Due to increasing use of dietary supplements in combination with conventional drugs, the risk of adverse effects is on the rise. As a preliminary step to predict a possibility of drug interaction during concomitant use of vinpocetine and conventional drugs, this study was carried out to evaluate the effects of vinpocetine on three main regulators of pharmacokinetic drug interactions namely, cytochromes P450 (CYPs), P-glycoprotein (P-gp), and Pregnane X receptor (PXR). Methods Inhibition of CYPs was evaluated by employing recombinant enzymes. The inhibition of P-gp was determined by calcein-AM uptake method in transfected and wild type MDCKII cells. Modulation of PXR activity was monitored through a reporter gene assay in HepG2 cells. Results Vinpocetine showed a strong inhibition of P-gp (EC50 8 μM) and a moderate inhibition of recombinant CYP3A4 and CYP2D6 (IC50 2.8 and 6.5 μM) with no activity towards CYP2C9, CYP2C19 and CYP1A2 enzymes. In HLM, competitive inhibition of CYP3A4 (IC50 54 and Ki 19 μM) and non-competitive inhibition of CYP2D6 (IC50 19 and Ki 26 μM) was observed. Activation of PXR was observed only at the highest tested concentration of vinpocetine (30 μM) while lower doses were ineffective. Conclusion Strong inhibition of P-gp by vinpocetine is indicative of a possibility of drug interactions by altering the pharmacokinetics of drugs, which are the substrates of P-gp. However, the effects on CYPs and PXR indicate that vinpocetine may not affect CYP-mediated metabolism of drugs, as the inhibitory concentrations are much greater than the expected plasma concentrations in humans. PMID:28930203

  9. Studies on Pharmacokinetic Drug Interaction Potential of Vinpocetine.

    PubMed

    Manda, Vamshi K; Avula, Bharathi; Dale, Olivia R; Chittiboyina, Amar G; Khan, Ikhlas A; Walker, Larry A; Khan, Shabana I

    2015-06-05

    Background: Vinpocetine, a semi-synthetic derivative of vincamine, is a popular dietary supplement used for the treatment of several central nervous system related disorders. Despite its wide use, no pharmacokinetic drug interaction studies are reported in the literature. Due to increasing use of dietary supplements in combination with conventional drugs, the risk of adverse effects is on the rise. As a preliminary step to predict a possibility of drug interaction during concomitant use of vinpocetine and conventional drugs, this study was carried out to evaluate the effects of vinpocetine on three main regulators of pharmacokinetic drug interactions namely, cytochromes P450 (CYPs), P-glycoprotein (P-gp), and Pregnane X receptor (PXR). Methods: Inhibition of CYPs was evaluated by employing recombinant enzymes. The inhibition of P-gp was determined by calcein-AM uptake method in transfected and wild type MDCKII cells. Modulation of PXR activity was monitored through a reporter gene assay in HepG2 cells. Results: Vinpocetine showed a strong inhibition of P-gp (EC 50 8 µM) and a moderate inhibition of recombinant CYP3A4 and CYP2D6 (IC 50 2.8 and 6.5 µM) with no activity towards CYP2C9, CYP2C19 and CYP1A2 enzymes. In HLM, competitive inhibition of CYP3A4 (IC 50 54 and K i 19 µM) and non-competitive inhibition of CYP2D6 (IC 50 19 and K i 26 µM) was observed. Activation of PXR was observed only at the highest tested concentration of vinpocetine (30 µM) while lower doses were ineffective. Conclusion: Strong inhibition of P-gp by vinpocetine is indicative of a possibility of drug interactions by altering the pharmacokinetics of drugs, which are the substrates of P-gp. However, the effects on CYPs and PXR indicate that vinpocetine may not affect CYP-mediated metabolism of drugs, as the inhibitory concentrations are much greater than the expected plasma concentrations in humans.

  10. Differential effects of traumatic brain injury on the cytochrome p450 system: a perspective into hepatic and renal drug metabolism.

    PubMed

    Kalsotra, Auinash; Turman, Cheri M; Dash, Pramod K; Strobel, Henry W

    2003-12-01

    Traumatic brain injury is known to cause several secondary effects, one of which is altered drug clearance. Given the fact that patients who sustain TBI are subsequently treated with a variety of pharmacological agents for the purpose of either neuroprotection or physiological support, it is imperative to clarify changes in expression and/or activities of enzymes involved in clearing drugs. The mixed function oxidase system, which consists of cytochrome P450 and cytochrome P450 reductase, plays a vital role in phase I drug metabolism. This paper addresses the issue as to what extent TBI affects the levels and activity of various rat CYP450 subfamilies. Our results show that TBI induces tissue-specific and time-dependent alterations. Total hepatic CYP450 content showed a biphasic response with a decrease seen at 24 h followed by an increase at 2 weeks. CYP450 reductase, in contrast, showed an opposite temporal profile. Immunoblot analyses and marker substrate metabolism demonstrated a clear decrease in hepatic CYP1A levels while a significant increase in kidney was seen at both 24 h and 2 weeks. A dramatic induction of CYP3A was evident at 2 weeks in liver, while no changes were noticed in CYP2B or CYP2D subfamilies. CYP4F subfamily showed induction in kidney only. Collectively, the data reveal the differential effects of TBI on hepatic and renal drug metabolism.

  11. Part I---Evaluating Effects of Oligomer Formation on Cytochrome P450 2C9 Electron Transfer and Drug Metabolism, Part II---Utilizing Molecular Modeling Techniques to Study the Src-Interacting Proteins Actin Filament Associated Protein of 110 kDa (AFAP-110) and Cortactin

    NASA Astrophysics Data System (ADS)

    Jett, John Edward, Jr.

    The dissertation has been divided into two parts to accurately reflect the two distinct areas of interest pursued during my matriculation in the School of Pharmacy at West Virginia University. In Part I, I discuss research probing the nature of electron transfer in the Cytochrome P450 family of proteins, a group of proteins well-known for their role in drug metabolism. In Part II, I focus on in silico and in vitro work developed in concert to probe protein structure and protein-protein interactions involved in actin filament reorganization and cellular motility. Part I. Cytochrome P450s (P450s) are an important class of enzymes known to metabolize a variety of endogenous and xenobiotic compounds. P450s are most commonly found in liver and intestinal endothelial cells and are responsible for the metabolism of approximately 75% of pharmaceutical drugs on the market. CYP2C9---one of the six major P450 isoforms---is responsible for ˜20% of drug metabolism. Elucidation of the factors that affect in vitro drug metabolism is crucial to the accurate prediction of in vivo drug metabolism kinetics. Currently, the two major techniques for studying in vitro drug metabolism are solution-based. However, it is known that the results of solution-based studies can vary from in vivo drug metabolism. One reason suggested to account for this variation is the state of P450 oligomer formation in solution compared to the in vivo environment, where P450s are membrane-bound. To understand the details of how oligomer formation affects in vitro drug metabolism, it is imperative that techniques be developed which will allow for the unequivocal control of oligomer formation without altering other experimental parameters. Our long term goal of this research is to develop methods to more accurately predict in vivo drug metabolism from in vitro data. This section of the dissertation will discuss the development of a platform consisting of a doped silicon surface containing a large array of gold

  12. Whole genome co-expression analysis of soybean cytochrome P450 genes identifies nodulation-specific P450 monooxygenases

    PubMed Central

    2010-01-01

    co-expressed with several genes encoding isoflavonoid-related metabolic enzymes. We then focused on nodulation-induced P450s and found that CYP728H1 was co-expressed with the genes involved in phenylpropanoid metabolism. Similarly, CYP736A34 was highly co-expressed with lipoxygenase, lectin and CYP83D1, all of which are involved in root and nodule development. Conclusions The genome scale analysis of P450s in soybean reveals many unique features of these important enzymes in this crop although the functions of most of them are largely unknown. Gene co-expression analysis proves to be a useful tool to infer the function of uncharacterized genes. Our work presented here could provide important leads toward functional genomics studies of soybean P450s and their regulatory network through the integration of reverse genetics, biochemistry, and metabolic profiling tools. The identification of nodule-specific P450s and their further exploitation may help us to better understand the intriguing process of soybean and rhizobium interaction. PMID:21062474

  13. Simultaneous Assessment of Transporter-Mediated Drug-Drug Interactions Using a Probe Drug Cocktail in Cynomolgus Monkey.

    PubMed

    Kosa, Rachel E; Lazzaro, Sarah; Bi, Yi-An; Tierney, Brendan; Gates, Dana; Modi, Sweta; Costales, Chester; Rodrigues, A David; Tremaine, Larry M; Varma, Manthena V

    2018-06-07

    We aim to establish an in vivo preclinical model to enable simultaneous assessment of inhibition potential of an investigational drug on clinically relevant drug transporters, organic anion transporting polypeptide (OATP)1B, breast cancer resistance protein (BCRP), P-glycoprotein (P-gp) and organic anion transporter (OAT)3. Pharmacokinetics of substrate cocktail consisting of pitavastatin (OATP1B substrate), rosuvastatin (OATP1B/BCRP/OAT3), sulfasalazine (BCRP) and talinolol (P-gp) were obtained in cynomolgus monkey - alone or in combination with transporter inhibitors. Single dose rifampicin (30 mg/kg) significantly (p<0.01) increased the plasma exposure of all four drugs, with a marked effect on pitavastatin and rosuvastatin (AUC ratio ~21-39). Elacridar, BCRP/P-gp inhibitor, increased the AUC of sulfasalazine, talinolol, as well as rosuvastatin and pitavastatin. An OAT1/3 inhibitor (probenecid) significantly (p<0.05) impacted the renal clearance of rosuvastatin (~8-fold). In vitro, rifampicin (10μM) inhibited uptake of pitavastatin, rosuvastatin and sulfasalazine by monkey and human primary hepatocytes. Transport studies using membrane vesicles suggested that all probe substrates, except talinolol, are transported by cynoBCRP; while talinolol is a cynoP-gp substrate. Elacridar and rifampicin inhibited both cynoBCRP and cynoP-gp in vitro, indicating potential for in vivo intestinal efflux inhibition. In conclusion, a probe substrate cocktail was validated to simultaneously evaluate perpetrator impact on multiple clinically relevant transporters using the cynomolgus monkey. The results support the use of the cynomolgus monkey as a model that could enable drug-drug interaction risk assessment, before advancing a new molecular entity into clinical development, as well as providing mechanistic insights on transporter-mediated interactions. The American Society for Pharmacology and Experimental Therapeutics.

  14. Pharmacokinetic Drug Interactions with Panax ginseng.

    PubMed

    Ramanathan, Meenakshi R; Penzak, Scott R

    2017-08-01

    Panax ginseng is widely used as an adaptogen throughout the world. The major active constituents of P. ginseng are ginsenosides. Most naturally occurring ginsenosides are deglycosylated by colonic bacteria to intestinal metabolites. Ginsenosides along with these metabolites are widely accepted as being responsible for the pharmacologic activity and drug interaction potential of ginseng. Numerous preclinical studies have assessed the influence of various ginseng components on cytochrome P450 (CYP), glucuronidation, and drug transport activity. Results from these investigations have been largely inconclusive due to the use of different ginseng products and variations in methodology between studies. Drug interaction studies in humans have been conflicting and have largely yielded negative results or results that suggest only a weak interaction. One study using a midazolam probe found weak CYP3A induction and another using a fexofenadine probe found weak P-gp inhibition. Despite several case reports indicating a drug interaction between warfarin and P. ginseng, pharmacokinetic studies involving these agents in combination have failed to find significant pharmacokinetic or pharmacodynamic interactions. To this end, drug interactions involving P. ginseng appear to be rare; however, close clinical monitoring is still suggested for patients taking warfarin or CYP3A or P-gp substrates with narrow therapeutic indices.

  15. Transporter-mediated natural product-drug interactions for the treatment of cardiovascular diseases.

    PubMed

    Zha, Weibin

    2018-04-01

    The growing use of natural products in cardiovascular (CV) patients has been greatly raising the concerns about potential natural product-CV drug interactions. Some of these may lead to unexpected cardiovascular adverse effects and it is, therefore, essential to identify or predict potential natural product-CV drug interactions, and to understand the underlying mechanisms. Drug transporters are important determinants for the pharmacokinetics of drugs and alterations of drug transport has been recognized as one of the major causes of natural product-drug interactions. In last two decades, many CV drugs (e.g., angiotensin II receptor blockers, beta-blockers and statins) have been identified to be substrates and inhibitors of the solute carrier (SLC) transporters and the ATP-binding cassette (ABC) transporters, which are two major transporter superfamilies. Meanwhile, in vitro and in vivo studies indicate that a growing number of natural products showed cardioprotective effects (e.g., gingko biloba, danshen and their active ingredients) are also substrates and inhibitors of drug transporters. Thus, to understand transporter-mediated natural product-CV drug interactions is important and some transporter-mediated interactions have already shown to have clinical relevance. In this review, we review the current knowledge on the role of ABC and SLC transporters in CV therapy, as well as transporter modulation by natural products used in CV diseases and their induced natural product-CV drug interactions through alterations of drug transport. We hope our review will aid in a comprehensive summary of transporter-mediated natural product-CV drug interactions and help public and physicians understand these type of interactions. Copyright © 2017. Published by Elsevier B.V.

  16. Overcoming heterologous protein interdependency to optimize P450-mediated Taxol precursor synthesis in Escherichia coli

    PubMed Central

    Biggs, Bradley Walters; Lim, Chin Giaw; Sagliani, Kristen; Shankar, Smriti; Stephanopoulos, Gregory; Ajikumar, Parayil Kumaran

    2016-01-01

    Recent advances in metabolic engineering have demonstrated the potential to exploit biological chemistry for the synthesis of complex molecules. Much of the progress to date has leveraged increasingly precise genetic tools to control the transcription and translation of enzymes for superior biosynthetic pathway performance. However, applying these approaches and principles to the synthesis of more complex natural products will require a new set of tools for enabling various classes of metabolic chemistries (i.e., cyclization, oxygenation, glycosylation, and halogenation) in vivo. Of these diverse chemistries, oxygenation is one of the most challenging and pivotal for the synthesis of complex natural products. Here, using Taxol as a model system, we use nature’s favored oxygenase, the cytochrome P450, to perform high-level oxygenation chemistry in Escherichia coli. An unexpected coupling of P450 expression and the expression of upstream pathway enzymes was discovered and identified as a key obstacle for functional oxidative chemistry. By optimizing P450 expression, reductase partner interactions, and N-terminal modifications, we achieved the highest reported titer of oxygenated taxanes (∼570 ± 45 mg/L) in E. coli. Altogether, this study establishes E. coli as a tractable host for P450 chemistry, highlights the potential magnitude of protein interdependency in the context of synthetic biology and metabolic engineering, and points to a promising future for the microbial synthesis of complex chemical entities. PMID:26951651

  17. Overcoming heterologous protein interdependency to optimize P450-mediated Taxol precursor synthesis in Escherichia coli.

    PubMed

    Biggs, Bradley Walters; Lim, Chin Giaw; Sagliani, Kristen; Shankar, Smriti; Stephanopoulos, Gregory; De Mey, Marjan; Ajikumar, Parayil Kumaran

    2016-03-22

    Recent advances in metabolic engineering have demonstrated the potential to exploit biological chemistry for the synthesis of complex molecules. Much of the progress to date has leveraged increasingly precise genetic tools to control the transcription and translation of enzymes for superior biosynthetic pathway performance. However, applying these approaches and principles to the synthesis of more complex natural products will require a new set of tools for enabling various classes of metabolic chemistries (i.e., cyclization, oxygenation, glycosylation, and halogenation) in vivo. Of these diverse chemistries, oxygenation is one of the most challenging and pivotal for the synthesis of complex natural products. Here, using Taxol as a model system, we use nature's favored oxygenase, the cytochrome P450, to perform high-level oxygenation chemistry in Escherichia coli. An unexpected coupling of P450 expression and the expression of upstream pathway enzymes was discovered and identified as a key obstacle for functional oxidative chemistry. By optimizing P450 expression, reductase partner interactions, and N-terminal modifications, we achieved the highest reported titer of oxygenated taxanes (∼570 ± 45 mg/L) in E. coli. Altogether, this study establishes E. coli as a tractable host for P450 chemistry, highlights the potential magnitude of protein interdependency in the context of synthetic biology and metabolic engineering, and points to a promising future for the microbial synthesis of complex chemical entities.

  18. Cytochrome P450 systems--biological variations of electron transport chains.

    PubMed

    Hannemann, Frank; Bichet, Andreas; Ewen, Kerstin M; Bernhardt, Rita

    2007-03-01

    Cytochromes P450 (P450) are hemoproteins encoded by a superfamily of genes nearly ubiquitously distributed in different organisms from all biological kingdoms. The reactions carried out by P450s are extremely diverse and contribute to the biotransformation of drugs, the bioconversion of xenobiotics, the bioactivation of chemical carcinogens, the biosynthesis of physiologically important compounds such as steroids, fatty acids, eicosanoids, fat-soluble vitamins and bile acids, the conversion of alkanes, terpenes and aromatic compounds as well as the degradation of herbicides and insecticides. Cytochromes P450 belong to the group of external monooxygenases and thus receive the necessary electrons for oxygen cleavage and substrate hydroxylation from different redox partners. The classical as well as the recently discovered P450 redox systems are compiled in this paper and classified according to their composition.

  19. Inhibition of cytochrome P450 by furanocoumarins in grapefruit juice and herbal medicines.

    PubMed

    Guo, Lian-qing; Yamazoe, Yasushi

    2004-02-01

    Furanocoumarins (psoralens) exist in various plants and some of them are used to cure skin diseases. These chemicals draw attentions recently because of their abilities to arouse drug interaction through inhibition of cytochrome P450. Grapefruit juice is a well-known example for food-drug interaction. But in vitro and in vivo studies have shown that the causative components are mainly furanocoumarin derivatives with geranyloxy side chains. In vitro experiments confirmed that furanocoumarins from grapefruit juice are both competitive and mechanism-based inhibitors of CYP3A4. Although the inhibition appeared to be stronger in the dimers than that in the monomers, all contribute comprehensively to the grapefruit juice-drug interaction. Further experiments with other furanocoumarins and related citrus fruits or umbelliferous herbal medicines indicate that drug interaction might also occur with stuffs other than grapefruit juice, especially with traditional medicine.

  20. Pharmacokinetic drug interactions of morphine, codeine, and their derivatives: theory and clinical reality, Part II.

    PubMed

    Armstrong, Scott C; Cozza, Kelly L

    2003-01-01

    Pharmacokinetic drug-drug interactions with codeine, dihydrocodeine, hydrocodone, oxycodone, and buprenorphine are reviewed in this column. These compounds have a very similar chemical structure to morphine. Unlike morphine, which is metabolized chiefly through conjugation reactions with uridine diphosphate glucuronosyl transferase (UGT) enzymes, these five drugs are metabolized both through oxidative reactions by the cytochrome P450 (CYP450) enzyme and conjugation by UGT enzymes. There is controversy as to whether codeine, dihydrocodeine, and hydrocodone are actually prodrugs requiring activation by the CYP450 2D6 enzyme or UGT enzymes. Oxycodone and buprenorphine, however, are clearly not prodrugs and are metabolized by the CYP450 2D6 and 3A4 enzymes, respectively. Knowledge of this metabolism assists in the understanding for the potential of drug-drug interactions with these drugs. This understanding is important so that clinicians can choose the proper dosages for analgesia and anticipate potential drug-drug interactions.

  1. Involvement of Cytochrome P450 in Pentachlorophenol Transformation in a White Rot Fungus Phanerochaete chrysosporium

    PubMed Central

    Ning, Daliang; Wang, Hui

    2012-01-01

    The occurrence of cytochrome P450 and P450-mediated pentachlorophenol oxidation in a white rot fungus Phanerochaete chrysosporium was demonstrated in this study. The carbon monoxide difference spectra indicated induction of P450 (103±13 pmol P450 per mg protein in the microsomal fraction) by pentachlorophenol. The pentachlorophenol oxidation by the microsomal P450 was NADPH-dependent at a rate of 19.0±1.2 pmol min−1 (mg protein)−1, which led to formation of tetrachlorohydroquinone and was significantly inhibited by piperonyl butoxide (a P450 inhibitor). Tetrachlorohydroquinone was also found in the cultures, while the extracellular ligninases which were reported to be involved in tetrachlorohydroquinone formation were undetectable. The formation of tetrachlorohydroquinone was not detectable in the cultures added with either piperonyl butoxide or cycloheximide (an inhibitor of de novo protein synthesis). These results revealed the pentachlorophenol oxidation by induced P450 in the fungus, and it should be the first time that P450-mediated pentachlorophenol oxidation was demonstrated in a microorganism. Furthermore, the addition of the P450 inhibitor to the cultures led to obvious increase of pentachlorophenol, suggesting that the relationship between P450 and pentachlorophenol methylation is worthy of further research. PMID:23029295

  2. Functional characterisation of an engineered multidomain human P450 2E1 by molecular Lego.

    PubMed

    Fairhead, Michael; Giannini, Silva; Gillam, Elizabeth M J; Gilardi, Gianfranco

    2005-12-01

    The human cytochrome P450s constitute an important family of monooxygenase enzymes that carry out essential roles in the metabolism of endogenous compounds and foreign chemicals. We present here results of a fusion between a human P450 enzyme and a bacterial reductase that for the first time is shown does not require the addition of lipids or detergents to achieve wild-type-like activities. The fusion enzyme, P450 2E1-BMR, contains the N-terminally modified residues 22-493 of the human P450 2E1 fused at the C-terminus to residues 473-1049 of the P450 BM3 reductase (BMR). The P450 2E1-BMR enzyme is active, self-sufficient and presents the typical marker activities of the native human P450 2E1: the hydroxylation of p-nitrophenol (KM=1.84+/-0.09 mM and kcat of 2.98+/-0.04 nmol of p-nitrocatechol formed per minute per nanomole of P450) and chlorzoxazone (KM=0.65+/-0.08 mM and kcat of 0.95+/-0.10 nmol of 6-hydroxychlorzoxazone formed per minute per nanomole of P450). A 3D model of human P450 2E1 was generated to rationalise the functional data and to allow an analysis of the surface potentials. The distribution of charges on the model of P450 2E1 compared with that of the FMN domain of BMR provides the ground for the understanding of the interaction between the fused domains. The results point the way to successfully engineer a variety of catalytically self-sufficient human P450 enzymes for drug metabolism studies in solution.

  3. Survey of Human Oxidoreductases and Cytochrome P450 Enzymes Involved in the Metabolism of Xenobiotic and Natural Chemicals

    PubMed Central

    2015-01-01

    Analyzing the literature resources used in our previous reports, we calculated the fractions of the oxidoreductase enzymes FMO (microsomal flavin-containing monooxygenase), AKR (aldo-keto reductase), MAO (monoamine oxidase), and cytochrome P450 participating in metabolic reactions. The calculations show that the fractions of P450s involved in the metabolism of all chemicals (general chemicals, natural, and physiological compounds, and drugs) are rather consistent in the findings that >90% of enzymatic reactions are catalyzed by P450s. Regarding drug metabolism, three-fourths of the human P450 reactions can be accounted for by a set of five P450s: 1A2, 2C9, 2C19, 2D6, and 3A4, and the largest fraction of the P450 reactions is catalyzed by P450 3A enzymes. P450 3A4 participation in metabolic reactions of drugs varied from 13% for general chemicals to 27% for drugs. PMID:25485457

  4. The use of electrochemistry for the synthesis of 17 alpha-hydroxyprogesterone by a fusion protein containing P450c17.

    PubMed

    Estabrook, R W; Shet, M S; Faulkner, K; Fisher, C W

    1996-11-01

    A method has been developed for the commercial application of the unique oxygen chemistry catalyzed by various cytochrome P450s. This is illustrated here for the synthesis of hydroxylated steroids. This method requires the preparation of large amounts of enzymatically functional P450 proteins that can serve as catalysts and a technique for providing electrons at an economically acceptable cost. To generate large amounts of enzymatically active recombinant P450s we have engineered the cDNAs for various P450s, including bovine adrenal P450c17, by linking them to a modified cDNA for rat NADPH-P450 reductase and placing them in the plasmid pCWori+. Transformation of E. coli results in the high level expression of an enzymatically active protein that can be easily purified by affinity chromatography. Incubation of the purified enzyme with steroid in a reaction vessel containing a platinum electrode and a Ag/AgCl electrode couple poised at -650 mV, together with the electromotively active redox mediator, cobalt sepulchrate, results in the 17 alpha-hydroxylation of progesterone at rates as high as 25 nmoles of progesterone hydroxylated/min/nmole of P450. Thus, high concentrations of hydroxylated steroids can be produced with incubation conditions of hours duration without the use of costly NADPH. Similar experiments have been carried out for the generation of the 6 beta-hydroxylation product of testosterone (using a fusion protein containing human P450 3A4). It is apparent that this method is applicable to many other P450 catalyzed reactions for the synthesis of large amounts of hydroxylated steroid metabolites. The electrochemical system is also applicable to drug discovery studies for the characterization of drug metabolites.

  5. P450 oxidoreductase deficiency: a disorder of steroidogenesis with multiple clinical manifestations.

    PubMed

    Miller, Walter L

    2012-10-23

    Cytochrome P450 enzymes catalyze the biosynthesis of steroid hormones and metabolize drugs. There are seven human type I P450 enzymes in mitochondria and 50 type II enzymes in endoplasmic reticulum. Type II enzymes, including both drug-metabolizing and some steroidogenic enzymes, require electron donation from a two-flavin protein, P450 oxidoreductase (POR). Although knockout of the POR gene causes embryonic lethality in mice, we discovered human POR deficiency as a disorder of steroidogenesis associated with the Antley-Bixler skeletal malformation syndrome and found mild POR mutations in phenotypically normal adults with infertility. Assay results of mutant forms of POR using the traditional but nonphysiologic assay (reduction of cytochrome c) did not correlate with patient phenotypes; assays based on the 17,20 lyase activity of P450c17 (CYP17) correlated with clinical phenotypes. The POR sequence in 842 normal individuals revealed many polymorphisms; amino acid sequence variant A503V is encoded by ~28% of human alleles. POR A503V has about 60% of wild-type activity in assays with CYP17, CYP2D6, and CYP3A4, but nearly wild-type activity with P450c21, CYP1A2, and CYP2C19. Activity of a particular POR variant with one P450 enzyme will not predict its activity with another P450 enzyme: Each POR-P450 combination must be studied individually. Human POR transcription, initiated from an untranslated exon, is regulated by Smad3/4, thyroid receptors, and the transcription factor AP-2. A promoter polymorphism reduces transcription to 60% in liver cells and to 35% in adrenal cells. POR deficiency is a newly described disorder of steroidogenesis, and POR variants may account for some genetic variation in drug metabolism.

  6. Cancer Activation and Polymorphisms of Human Cytochrome P450 1B1

    PubMed Central

    Chun, Young-Jin; Kim, Donghak

    2016-01-01

    Human cytochrome P450 enzymes (P450s, CYPs) are major oxidative catalysts that metabolize various xenobiotic and endogenous compounds. Many carcinogens induce cancer only after metabolic activation and P450 enzymes play an important role in this phenomenon. P450 1B1 mediates bioactivation of many procarcinogenic chemicals and carcinogenic estrogen. It catalyzes the oxidation reaction of polycyclic aromatic carbons, heterocyclic and aromatic amines, and the 4-hydroxylation reaction of 17β-estradiol. Enhanced expression of P450 1B1 promotes cancer cell proliferation and metastasis. There are at least 25 polymorphic variants of P450 1B1 and some of these have been reported to be associated with eye diseases. In addition, P450 1B1 polymorphisms can greatly affect the metabolic activation of many procarcinogenic compounds. It is necessary to understand the relationship between metabolic activation of such substances and P450 1B1 polymorphisms in order to develop rational strategies for the prevention of its toxic effect on human health. PMID:27123158

  7. Cytochrome p450 turnover: regulation of synthesis and degradation, methods for determining rates, and implications for the prediction of drug interactions.

    PubMed

    Yang, Jiansong; Liao, Mingxiang; Shou, Magang; Jamei, Masoud; Yeo, Karen Rowland; Tucker, Geoffrey T; Rostami-Hodjegan, Amin

    2008-06-01

    In vivo enzyme levels are governed by the rates of de novo enzyme synthesis and degradation. A current lack of consensus on values of the in vivo turnover half-lives of human cytochrome P450 (CYP) enzymes places a significant limitation on the accurate prediction of changes in drug concentration-time profiles associated with interactions involving enzyme induction and mechanism (time)-based inhibition (MBI). In the case of MBI, the full extent of inhibition is also sensitive to values of enzyme turnover half-life. We review current understanding of CYP regulation, discuss the pros and cons of various in vitro and in vivo approaches used to estimate the turnover of specific CYPs and, by simulation, consider the impact of variability in estimates of CYP turnover on the prediction of enzyme induction and MBI in vivo. In the absence of consensus on values for the in vivo turnover half-lives of key CYPs, a sensitivity analysis of predictions of the pharmacokinetic effects of enzyme induction and MBI to these values should be an integral part of the modelling exercise, and the selective use of values should be avoided.

  8. Herb-drug, food-drug, nutrient-drug, and drug-drug interactions: mechanisms involved and their medical implications.

    PubMed

    Sørensen, Janina Maria

    2002-06-01

    Adverse drug reactions (ADRs) and iatrogenic diseases have been identified as significant factors responsible for patient morbidity and mortality. Significant studies on drug metabolism in humans have been published during the last few years, offering a deeper comprehension of the mechanisms underlying adverse drug reactions and interactions. More understanding of these mechanisms, and of recent advances in laboratory technology, can help to evaluate potential drug interactions when drugs are prescribed concurrently. Increasing knowledge of interindividual variation in drug breakdown capacity and recent findings concerning the influence of environment, diet, nutrients, and herbal products can be used to reduce ADRs and iatrogenic diseases. Reviewed data suggest that drug treatment should be increasingly custom tailored to suit the individual patient and that appropriately co-prescribed diet and herbal remedies, could increase drug efficacy and lessen drug toxicity. This review focuses mainly on recently published research material. The cytochrome p450 enzymes, their role in metabolism, and their mechanisms of action are reviewed, and their role in drug-drug interactions are discussed. Drug-food and drug-herb interactions have garnered attention. Interdisciplinary communication among medical herbalists, medical doctors, and dietetic experts needs to be improved and encouraged. Internet resources for obtaining current information regarding drug-drug, drug-herb, and drug-nutrient interactions are provided.

  9. CW EPR parameters reveal cytochrome P450 ligand binding modes.

    PubMed

    Lockart, Molly M; Rodriguez, Carlo A; Atkins, William M; Bowman, Michael K

    2018-06-01

    Cytochrome P450 (CYP) monoxygenses utilize heme cofactors to catalyze oxidation reactions. They play a critical role in metabolism of many classes of drugs, are an attractive target for drug development, and mediate several prominent drug interactions. Many substrates and inhibitors alter the spin state of the ferric heme by displacing the heme's axial water ligand in the resting enzyme to yield a five-coordinate iron complex, or they replace the axial water to yield a nitrogen-ligated six-coordinate iron complex, which are traditionally assigned by UV-vis spectroscopy. However, crystal structures and recent pulsed electron paramagnetic resonance (EPR) studies find a few cases where molecules hydrogen bond to the axial water. The water-bridged drug-H 2 O-heme has UV-vis spectra similar to nitrogen-ligated, six-coordinate complexes, but are closer to "reverse type I" complexes described in older liteature. Here, pulsed and continuous wave (CW) EPR demonstrate that water-bridged complexes are remarkably common among a range of nitrogenous drugs or drug fragments that bind to CYP3A4 or CYP2C9. Principal component analysis reveals a distinct clustering of CW EPR spectral parameters for water-bridged complexes. CW EPR reveals heterogeneous mixtures of ligated states, including multiple directly-coordinated complexes and water-bridged complexes. These results suggest that water-bridged complexes are under-represented in CYP structural databases and can have energies similar to other ligation modes. The data indicates that water-bridged binding modes can be identified and distinguished from directly-coordinated binding by CW EPR. Copyright © 2018 Elsevier Inc. All rights reserved.

  10. The Effects of Milk Thistle (Silybum marianum) on Human Cytochrome P450 Activity

    PubMed Central

    Kawaguchi-Suzuki, Marina; Frye, Reginald F.; Zhu, Hao-Jie; Brinda, Bryan J.; Chavin, Kenneth D.; Bernstein, Hilary J.

    2014-01-01

    Milk thistle (Silybum marianum) extracts are widely used as a complementary and alternative treatment of various hepatic conditions and a host of other diseases/disorders. The active constituents of milk thistle supplements are believed to be the flavonolignans contained within the extracts. In vitro studies have suggested that some milk thistle components may significantly inhibit specific cytochrome P450 (P450) enzymes. However, determining the potential for clinically significant drug interactions with milk thistle products has been complicated by inconsistencies between in vitro and in vivo study results. The aim of the present study was to determine the effect of a standardized milk thistle supplement on major P450 drug-metabolizing enzymes after a 14-day exposure period. CYP1A2, CYP2C9, CYP2D6, and CYP3A4/5 activities were measured by simultaneously administering the four probe drugs, caffeine, tolbutamide, dextromethorphan, and midazolam, to nine healthy volunteers before and after exposure to a standardized milk thistle extract given thrice daily for 14 days. The three most abundant falvonolignans found in plasma, following exposure to milk thistle extracts, were silybin A, silybin B, and isosilybin B. The concentrations of these three major constituents were individually measured in study subjects as potential perpetrators. The peak concentrations and areas under the time-concentration curves of the four probe drugs were determined with the milk thistle administration. Exposure to milk thistle extract produced no significant influence on CYP1A2, CYP2C9, CYP2D6, or CYP3A4/5 activities. PMID:25028567

  11. Cytochrome P450 peroxidase/peroxygenase mediated xenobiotic metabolic activation and cytotoxicity in isolated hepatocytes.

    PubMed

    Anari, M R; Khan, S; Liu, Z C; O'Brien, P J

    1995-12-01

    Cytochrome P450 (P450) can utilize organic hydroperoxides and peracids to support hydroxylation and dealkylation of various P450 substrates. However, the biological significance of this P450 peroxygenase/peroxidase activity in the bioactivation of xenobiotics in intact cells has not been demonstrated. We have shown that tert-butyl hydroperoxide (tBHP) markedly enhances 3-20-fold the cytotoxicity of various aromatic hydrocarbons and their phenolic metabolites. The tBHP-enhanced hepatocyte cytotoxicity of 4-nitroanisole (4-NA) and 4-hydroxyanisole (4-HA) was also accompanied by an increase in the hepatocyte O-demethylation of 4-NA and 4-HA up to 7.5- and 21-fold, respectively. Hepatocyte GSH conjugation by 4-HA was also markedly increased by tBHP. An LC/MS analysis of the GSH conjugates identified hydroquinone-GSH and 4-methoxy-catechol:GSH conjugates as the predominant adducts. Pretreatment of hepatocytes with P450 inhibitors, e.g., phenylimidazole, prevented tBHP-enhanced 4-HA metabolism, GSH depletion, and cytotoxicity. In conclusion, hydroperoxides can therefore be used by intact cells to support the bioactivation of xenobiotics through the P450 peroxidase/peroxygenase system.

  12. Interactions between recreational drugs and antiretroviral agents.

    PubMed

    Antoniou, Tony; Tseng, Alice Lin-In

    2002-10-01

    To summarize existing data regarding potential interactions between recreational drugs and drugs commonly used in the management of HIV-positive patients. Information was obtained via a MEDLINE search (1966-August 2002) using the MeSH headings human immunodeficiency virus, drug interactions, cytochrome P450, medication names commonly prescribed for the management of HIV and related opportunistic infections, and names of commonly used recreational drugs. Abstracts of national and international conferences, review articles, textbooks, and references of all articles were also reviewed. Literature on pharmacokinetic interactions was considered for inclusion. Pertinent information was selected and summarized for discussion. In the absence of specific data, prediction of potential clinically significant interactions was based on pharmacokinetic and pharmacodynamic properties. All protease inhibitors (PIs) and nonnucleoside reverse transcriptase inhibitors are substrates and potent inhibitors or inducers of the cytochrome P450 system. Many classes of recreational drugs, including benzodiazepines, amphetamines, and opioids, are also metabolized by the liver and can potentially interact with antiretrovirals. Controlled interaction studies are often not available, but clinically significant interactions have been observed in a number of case reports. Overdoses secondary to interactions between the "rave" drugs methylenedioxymethamphetamine (MDMA) or gamma-hydroxybutyrate (GHB) and PIs have been reported. PIs, particularly ritonavir, may also inhibit metabolism of amphetamines, ketamine, lysergic acid diethylmide (LSD), and phencyclidine (PCP). Case series and pharmacokinetic studies suggest that nevirapine and efavirenz induce methadone metabolism, which may lead to symptoms of opiate withdrawal. A similar interaction may exist between methadone and the PIs ritonavir and nelfinavir, although the data are less consistent. Opiate metabolism can be inhibited or induced by

  13. Phorate can reverse P450 metabolism-based herbicide resistance in Lolium rigidum.

    PubMed

    Busi, Roberto; Gaines, Todd Adam; Powles, Stephen

    2017-02-01

    Organophosphate insecticides can inhibit specific cytochrome P450 enzymes involved in metabolic herbicide resistance mechanisms, leading to synergistic interactions between the insecticide and the herbicide. In this study we report synergistic versus antagonistic interactions between the organophosphate insecticide phorate and five different herbicides observed in a population of multiple herbicide-resistant Lolium rigidum. Phorate synergised with three different herbicide modes of action, enhancing the activity of the ALS inhibitor chlorsulfuron (60% LD 50 reduction), the VLCFAE inhibitor pyroxasulfone (45% LD 50 reduction) and the mitosis inhibitor trifluralin (70% LD 50 reduction). Conversely, phorate antagonised the two thiocarbamate herbicides prosulfocarb and triallate with a 12-fold LD 50 increase. We report the selective reversal of P450-mediated metabolic multiple resistance to chlorsulfuron and trifluralin in the grass weed L. rigidum by synergistic interaction with the insecticide phorate, and discuss the putative mechanistic basis. This research should encourage diversity in herbicide use patterns for weed control as part of a long-term integrated management effort to reduce the risk of selection of metabolism-based multiple herbicide resistance in L. rigidum. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

  14. [Interaction between CYP450 enzymes and metabolism of traditional Chinese medicine as well as enzyme activity assay].

    PubMed

    Lu, Tu-lin; Su, Lian-lin; Ji, De; Gu, Wei; Mao, Chun-qin

    2015-09-01

    Drugs are exogenous compounds for human bodies, and will be metabolized by many enzymes after administration. CYP450 enzyme, as a major metabolic enzyme, is an important phase I drug metabolizing enzyme. In human bodies, about 75% of drug metabolism is conducted by CYP450 enzymes, and CYP450 enzymes is the key factor for drug interactions between traditional Chinese medicine( TCM) -TCM, TCM-medicine and other drug combination. In order to make clear the interaction between metabolic enzymes and TCM metabolism, we generally chose the enzymatic activity as an evaluation index. That is to say, the enhancement or reduction of CYP450 enzyme activity was used to infer the inducing or inhibitory effect of active ingredients and extracts of traditional Chinese medicine on enzymes. At present, the common method for measuring metabolic enzyme activity is Cocktail probe drugs, and it is the key to select the suitable probe substrates. This is of great significance for study drug's absorption, distribution, metabolism and excretion (ADME) process in organisms. The study focuses on the interaction between TCMs, active ingredients, herbal extracts, cocktail probe substrates as well as CYP450 enzymes, in order to guide future studies.

  15. Hepatic cytochrome P450 activity, abundance, and expression throughout human development

    SciTech Connect

    Sadler, Natalie C.; Nandhikonda, Premchendar; Webb-Robertson, Bobbie-Jo M.

    Cytochrome P450s are Phase I metabolic enzymes that play critical roles in the biotransformation of endogenous compounds and xenobiotics. The expression and activity of P450 enzymes can vary considerably throughout human development, especially when comparing fetal development to neonates, children, and adults. In an effort to develop a more comprehensive understanding of the ontogeny of P450 expression and activity we employed a multi-omic characterization of P450 transcript expression, protein abundance, and functional activity. To quantify the functional activity of individual P450s we employ activity-based protein profiling, which uses modified mechanism-based inhibitors of P450s as chemical probes, in tandem with proteomicmore » analyses to quantify activity. Our results reveal life-stage-dependent variability in P450 expression, abundance, and activity throughout human development and frequent discordant relationships between expression and activity. The results were used to distribute P450s into three general classes based upon developmental stage of expression and activity. We have significantly expanded the knowledge of P450 ontogeny, particularly at the level of individual P450 activity. We anticipate that our ontogeny results will be useful for enabling predictive therapeutic dosing, and for avoiding potentially adverse and harmful reactions during maturation from both therapeutic drugs and environmental xenobiotics.« less

  16. Inhibition of human P450 enzymes by natural extracts used in traditional medicine.

    PubMed

    Rodeiro, Idania; Donato, María T; Jimenez, Nuria; Garrido, Gabino; Molina-Torres, Jorge; Menendez, Roberto; Castell, José V; Gómez-Lechón, María J

    2009-02-01

    Different medicinal plants are widely used in Cuba and Mexico to treat several disorders. This paper reports in vitro inhibitory effects on the P450 system of herbal products commonly used by people in Cuba and Mexico in traditional medicine for decades. Experiments were conducted in human liver microsomes. The catalytic activities of CYP1A1/2, 2D6, and 3A4 were measured using specific probe substrates. The Heliopsis longipes extract exhibited a concentration-dependent inhibition of the three enzymes, and similar effects were produced by affinin (an alkamide isolated from the H. longipes extract) and two catalytically reduced alkamides. Mangifera indica L. and Thalassia testudinum extracts, two natural polyphenol-rich extracts, diminished CYP1A1/2 and 3A4 activities, but not the CYP2D6 activity. These results suggest that these herbs inhibit the major human P450 enzymes involved in drug metabolism and could induce potential herbal-drug interactions. Copyright (c) 2008 John Wiley & Sons, Ltd.

  17. Cytochrome P450 3A4 activity after surgical stress.

    PubMed

    Haas, Curtis E; Kaufman, David C; Jones, Carolyn E; Burstein, Aaron H; Reiss, William

    2003-05-01

    To evaluate the relationship between the acute inflammatory response after surgical trauma and changes in hepatic cytochrome P450 3A4 activity, compare changes in cytochrome P450 3A4 activity after procedures with varying degrees of surgical stress, and to explore the time course of any potential drug-cytokine interaction after surgery. Prospective, open-label study with each patient serving as his or her own control. University-affiliated, acute care, general hospital. A total of 16 patients scheduled for elective repair of an abdominal aortic aneurysm (n = 5), complete or partial colectomy (n = 6), or peripheral vascular surgery with graft (n = 5). Cytochrome P450 3A4 activity was estimated using the carbon-14 [14C]erythromycin breath test (ERMBT) before surgery and 24, 48, and 72 hrs after surgery. Abdominal aortic aneurysm and colectomy patients also had an ERMBT performed at discharge. Blood samples were obtained before surgery, immediately after surgery, and 6, 24, 32, 48, and 72 hrs after surgery for determination of plasma concentrations of interleukin-6, interleukin-1beta, and tumor necrosis factor-alpha. Clinical markers of surgical stress that were collected included duration of surgery, estimated blood loss, and volume of fluids administered in the operating room. ERMBT results significantly declined in all three surgical groups, with the lowest value at the time of the 72-hr study in all three groups. There was a trend toward differences in ERMBT results among groups that did not reach statistical significance (p =.06). The nadir ERMBT result was significantly and negatively correlated with both peak interleukin-6 concentration (r(s) = -.541, p =.03) and log interleukin-6 area under the curve from 0 to 72 hrs (r(s) = -.597, p =.014). Subjects with a peak interleukin-6 of >100 pg/mL had a significantly lower nadir ERMBT compared with subjects with a peak interleukin-6 of <100 pg/mL (35.5% +/- 5.2% vs. 74.7% +/- 5.1%, p <.001). Acute inflammation after

  18. Predicting the effect of cytochrome P450 inhibitors on substrate drugs: analysis of physiologically based pharmacokinetic modeling submissions to the US Food and Drug Administration.

    PubMed

    Wagner, Christian; Pan, Yuzhuo; Hsu, Vicky; Grillo, Joseph A; Zhang, Lei; Reynolds, Kellie S; Sinha, Vikram; Zhao, Ping

    2015-01-01

    The US Food and Drug Administration (FDA) has seen a recent increase in the application of physiologically based pharmacokinetic (PBPK) modeling towards assessing the potential of drug-drug interactions (DDI) in clinically relevant scenarios. To continue our assessment of such approaches, we evaluated the predictive performance of PBPK modeling in predicting cytochrome P450 (CYP)-mediated DDI. This evaluation was based on 15 substrate PBPK models submitted by nine sponsors between 2009 and 2013. For these 15 models, a total of 26 DDI studies (cases) with various CYP inhibitors were available. Sponsors developed the PBPK models, reportedly without considering clinical DDI data. Inhibitor models were either developed by sponsors or provided by PBPK software developers and applied with minimal or no modification. The metric for assessing predictive performance of the sponsors' PBPK approach was the R predicted/observed value (R predicted/observed = [predicted mean exposure ratio]/[observed mean exposure ratio], with the exposure ratio defined as [C max (maximum plasma concentration) or AUC (area under the plasma concentration-time curve) in the presence of CYP inhibition]/[C max or AUC in the absence of CYP inhibition]). In 81 % (21/26) and 77 % (20/26) of cases, respectively, the R predicted/observed values for AUC and C max ratios were within a pre-defined threshold of 1.25-fold of the observed data. For all cases, the R predicted/observed values for AUC and C max were within a 2-fold range. These results suggest that, based on the submissions to the FDA to date, there is a high degree of concordance between PBPK-predicted and observed effects of CYP inhibition, especially CYP3A-based, on the exposure of drug substrates.

  19. Summary of Information on the Effects of Ionizing and Non-ionizing Radiation on Cytochrome P450 and Other Drug Metabolizing Enzymes and Transporters

    PubMed Central

    Rendic, Slobodan; Guengerich, F. Peter

    2014-01-01

    The present paper is an update of data on the effects of ionizing radiation (γ-rays, X-rays, high energy UV, fast neutron) caused by environmental pollution or clinical treatments and the effects of non-ionizing radiation (low energy UV) on the expression and/or activity of drug metabolism (e.g., cytochrome P450,, glutathione transferase), enzymes involved in oxidative stress (e.g., peroxidases, catalase,, aconitase, superoxide dismutase), and transporters. The data are presented in tabular form (Tables 1–3) and are a continuation of previously published summaries on the effects of drugs and other chemicals on cytochrome P450 enzymes (Rendic, S.; Di Carlo, F. Drug Metab. Rev., 1997, 29 (1–2), 413–580, Rendic, S. Drug Metab. Rev., 2002, 34 (1–2), 83–448) and of the data on the effects of diseases and environmental factors on the expression and/or activity of human cytochrome P450 enzymes and transporters (Guengerich, F.P.; Rendic, S. Curr. Drug Metab., 2010, 11(1), 1–3, Rendic, S.; Guengerich, F.P. Curr. Drug Metab., 2010, 11 (1), 4–84). The collective information is as presented by the cited author(s) in cases where several references are cited the latest published information is included. Remarks and conclusions suggesting clinically important impacts are highlighted, followed by discussion of the major findings. The searchable database is available as an Excel file (for information about file availability contact the corresponding author). PMID:22571481

  20. Investigation of sarizotan's impact on the pharmacokinetics of probe drugs for major cytochrome P450 isoenzymes: a combined cocktail trial.

    PubMed

    Krösser, Sonja; Neugebauer, Roland; Dolgos, Hugues; Fluck, Markus; Rost, Karl-Ludwig; Kovar, Andreas

    2006-04-01

    The 5HT(1A) receptor agonist sarizotan is in clinical development for the treatment of dyskinesia, a potentially disabling complication in Parkinson's disease. We investigated the effect of sarizotan on the clinical pharmacokinetics of probe drugs for cytochrome P450 (CYP) to evaluate the risk of CYP-related drug-drug interactions. This was a double-blind, randomised, two-period cross-over interaction study with repeated administration of 5 mg sarizotan HCl or placebo b.i.d. for 8 days in 18 healthy volunteers. On day 4, a single dose of 100 mg metoprolol (CYP2D6 probe) was administered. On day 8, single doses of 100 mg caffeine (CYP1A2 probe), 50 mg diclofenac (CYP2C9 probe), 100 mg mephenytoin (CYP2C19 probe) and 7.5 mg midazolam (CYP3A4 probe) were simultaneously applied. Pharmacokinetic parameters for probe drugs and their metabolites in plasma and urinary recovery were determined. Concentration-time profiles and pharmacokinetic parameters of all probes and their metabolites remained unchanged after co-administration of sarizotan, compared with placebo. Analysis of variance of the area under the plasma concentration-time curve for probe drugs/metabolites, metabolic ratios and urinary excretion resulted in 90% confidence intervals within the acceptance range (0.8-1.25), indicating the absence of drug-drug interactions. At a dose higher than that intended for clinical use (1 mg b.i.d.), sarizotan had no effect on the metabolism and pharmacokinetics of specific probe drugs for CYP isoenzymes 1A2, 2C19, 2C9, 2D6 and 3A4. Pharmacokinetic interactions with co-administered drugs metabolised by these CYP isoforms are not expected, and dose adjustment of co-administered CYP substrates is not necessary.

  1. Drug Interactions of Direct-Acting Oral Anticoagulants.

    PubMed

    Fitzgerald, John Leonard; Howes, Laurence Guy

    2016-09-01

    In recent years, new direct-acting oral anticoagulants (DOACs) have been introduced into clinical practice that specifically inhibit either factor Ia or Xa. These drugs have, to a large extent, replaced warfarin for the treatment of venous thrombosis, pulmonary embolism, and non-valvular atrial fibrillation. They have potential advantages over warfarin in providing more stable anticoagulation and the lack of a need for regular venesection to monitor activity. They also have the promise of less drug and food interactions. All of these drugs are substrates for the permeability glycoprotein (P-gp) excretion system, and several are metabolised, in part, by cytochrome P450 (CYP) 3A4. This current article assesses the interactions that do or may occur with the DOACs, particularly with respect to the P-gp and CYP3A4 systems.

  2. [Interactions of food and drug metabolism].

    PubMed

    Delzenne, N M; Verbeeck, R K

    2001-01-01

    The nutritional state, and/or the ingestion of specific nutrients, is/are able to modify drug disposition, by interfering with drug absorption, distribution, storage, and metabolism. Recent data report that nutrients interfere with drug metabolism either by modifying key enzymes of phase I (cytochromeP450 dependent mixed function oxidase) and II (glucuronosyl, sulfonyl- ... transferases), or by modulating coenzymes availability (NADPH, UDPglucuronic acid...). Food components involved in drug metabolism modifications are either macro-nutrients (carbohydrates, lipids, proteins, ethanol), micronutriments (vitamins, minerals), or phytochemicals. Drug-nutrients interactions may be beneficials, and thus could constitute, i.e. a way to improve drug therapeutic index, or generate adverse effects.

  3. Bioavailable flavonoids: cytochrome P450-mediated metabolism of methoxyflavones.

    PubMed

    Walle, U Kristina; Walle, Thomas

    2007-11-01

    Methoxylated flavones were recently shown to be promising cancer chemopreventive agents. Their high metabolic stability compared with the hydroxylated analogs was shown in our laboratory using the human hepatic S9 fraction with cofactors for glucuronidation, sulfation, and oxidation. In the present study, the resistance of methoxylated flavones toward oxidative metabolism was investigated with human liver microsomes and recombinant cytochrome P450 (P450) isoforms. Among 15 methoxylated flavones investigated, the two partially methylated compounds, tectochrysin and kaempferide, were among the most susceptible to microsomal oxidation (Cl(int) 283 and 82 ml/min/kg). Of the fully methylated compounds, 5,7-dimethoxyflavone and 5-methoxyflavone were the most stable (Cl(int) 13 and 18 ml/min/kg, respectively), whereas 4'-methoxyflavone, 3'-methoxyflavone, 5,4'-dimethoxyflavone, and 7,3'-dimethoxyflavone were the least stable (Cl(int) 161, 140, 119, and 92 ml/min/kg, respectively), emphasizing the importance of the positions of the methoxy substituents in the flavone ring system. Among the five P450 isoforms tested, CYP1A1 showed the highest rate of metabolism of fully methylated compounds, followed by CYP1A2 and CYP3A4. CYP2C9 and CYP2D6 gave minimal disappearance of the parent compound. Finally, in incubations with hepatic S9 fraction with cofactors for oxidation and both conjugation reactions, partially methylated flavones, as expected, were much less metabolically stable than fully methylated flavones, confirming that oxidative demethylation is the rate-limiting metabolic reaction for fully methylated flavones only. In summary, the rate of oxidative metabolism of methoxylated flavones, mainly involving CYP1A1 and CYP1A2, varied widely, even between compounds with very similar structures.

  4. Identification of putative substrates for cynomolgus monkey cytochrome P450 2C8 by substrate depletion assays with 22 human P450 substrates and inhibitors.

    PubMed

    Hosaka, Shinya; Murayama, Norie; Satsukawa, Masahiro; Uehara, Shotaro; Shimizu, Makiko; Iwasaki, Kazuhide; Iwano, Shunsuke; Uno, Yasuhiro; Yamazaki, Hiroshi

    2016-07-01

    Cynomolgus monkeys are widely used in drug developmental stages as non-human primate models. Previous studies used 89 compounds to investigate species differences associated with cytochrome P450 (P450 or CYP) function that reported monkey specific CYP2C76 cleared 19 chemicals, and homologous CYP2C9 and CYP2C19 metabolized 17 and 30 human CYP2C9 and/or CYP2C19 substrates/inhibitors, respectively. In the present study, 22 compounds selected from viewpoints of global drug interaction guidances and guidelines were further evaluated to seek potential substrates for monkey CYP2C8, which is highly homologous to human CYP2C8 (92%). Amodiaquine, montelukast, quercetin and rosiglitazone, known as substrates or competitive inhibitors of human CYP2C8, were metabolically depleted by recombinant monkey CYP2C8 at relatively high rates. Taken together with our reported findings of the slow eliminations of amodiaquine and montelukast by monkey CYP2C9, CYP2C19 and CYP2C76, the present results suggest that these at least four chemicals may be good marker substrates for monkey CYP2C8. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

  5. The effects of milk thistle (Silybum marianum) on human cytochrome P450 activity.

    PubMed

    Kawaguchi-Suzuki, Marina; Frye, Reginald F; Zhu, Hao-Jie; Brinda, Bryan J; Chavin, Kenneth D; Bernstein, Hilary J; Markowitz, John S

    2014-10-01

    Milk thistle (Silybum marianum) extracts are widely used as a complementary and alternative treatment of various hepatic conditions and a host of other diseases/disorders. The active constituents of milk thistle supplements are believed to be the flavonolignans contained within the extracts. In vitro studies have suggested that some milk thistle components may significantly inhibit specific cytochrome P450 (P450) enzymes. However, determining the potential for clinically significant drug interactions with milk thistle products has been complicated by inconsistencies between in vitro and in vivo study results. The aim of the present study was to determine the effect of a standardized milk thistle supplement on major P450 drug-metabolizing enzymes after a 14-day exposure period. CYP1A2, CYP2C9, CYP2D6, and CYP3A4/5 activities were measured by simultaneously administering the four probe drugs, caffeine, tolbutamide, dextromethorphan, and midazolam, to nine healthy volunteers before and after exposure to a standardized milk thistle extract given thrice daily for 14 days. The three most abundant falvonolignans found in plasma, following exposure to milk thistle extracts, were silybin A, silybin B, and isosilybin B. The concentrations of these three major constituents were individually measured in study subjects as potential perpetrators. The peak concentrations and areas under the time-concentration curves of the four probe drugs were determined with the milk thistle administration. Exposure to milk thistle extract produced no significant influence on CYP1A2, CYP2C9, CYP2D6, or CYP3A4/5 activities. Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.

  6. Electrochemistry of cytochrome P450 17α-hydroxylase/17,20-lyase (P450c17).

    PubMed

    Martin, Lisandra L; Kubeil, Clemens; Simonov, Alexandr N; Kuznetsov, Vladimir L; Corbin, C Jo; Auchus, Richard J; Conley, Alan J; Bond, Alan M; Rodgers, Raymond J

    2017-02-05

    Within the superfamily of cytochrome P450 enzymes (P450s), there is a small class which is functionally employed for steroid biosynthesis. The enzymes in this class appear to have a small active site to accommodate the steroid substrates specifically and snuggly, prior to the redox transformation or hydroxylation to form a product. Cytochrome P450c17 is one of these and is also a multi-functional P450, with two activities, the first 17α-hydroxylation of pregnenolone is followed by a subsequent 17,20-lyase transformation to dehydroepiandrosterone (DHEA) as the dominant pathways to cortisol precursors or androgens in humans, respectively. How P450c17 regulates these two redox reactions is of special interest. There is a paucity of direct electrochemical studies on steroidogenic P450s, and in this mini-review we provide an overview of these studies with P450c17. Historical consideration as to the difficulties in obtaining reliable electrochemistry due to issues of handling proteins on an electrode, together with advances in the electrochemical techniques are addressed. Recent work using Fourier transformed alternating current voltammetry is highlighted as this technique can provide both catalytic information simultaneously with the underlying redox transfer with the P450 haem. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  7. [Convulsions due to an interaction between anti-epileptic drugs and rifampicin].

    PubMed

    Hanrath, Maarten A; Swart, Eleonora L

    2014-01-01

    Anti-epileptic drugs (AEDs) have a small therapeutic window, so it is important to monitor plasma levels. Inadequate plasma levels may lead to convulsions. Many AEDs are cleared hepatically, and there are many drug interactions that are known to lead to changes in plasma levels. A 54-year-old woman with known epilepsy developed convulsions after using rifampicin and flucloxacillin, despite the use of maintenance treatment in the form of carbamazepine, valproic acid and clonazepam. Since rifampicin is known to induce several cytochrome P450 enzymes and clearance of the anti-epileptic drug used may be affected by this, it can be assumed that the convulsions were caused by rifampicin. This interaction is however not mentioned in the Dutch 'G-standard' database. Rifampicin is known to be a strong inducer of various cytochrome P450 enzymes. This case description shows that the use of rifampicin may lead to convulsions. For this reason, these interactions should be included in the Dutch G-standard database.

  8. Inhibitors of steroidal cytochrome p450 enzymes as targets for drug development.

    PubMed

    Baston, Eckhard; Leroux, Frédéric R

    2007-01-01

    Cytochrome P450's are enzymes which catalyze a large number of biological reactions, for example hydroxylation, N-, O-, S- dealkylation, epoxidation or desamination. Their substrates include fatty acids, steroids or prostaglandins. In addition, a high number of various xenobiotics are metabolized by these enzymes. The enzyme 17alpha-hydroxylase-C17,20-lyase (P450(17), CYP 17, androgen synthase), a cytochrome P450 monooxygenase, is the key enzyme for androgen biosynthesis. It catalyzes the last step of the androgen biosynthesis in the testes and adrenal glands and produces androstenedione and dehydroepiandrosterone from progesterone and pregnenolone. The microsomal enzyme aromatase (CYP19) transforms these androgens to estrone and estradiol. Estrogens stimulate tumor growth in hormone dependent breast cancer. In addition, about 80 percent of prostate cancers are androgen dependent. Selective inhibitors of these enzymes are thus important alternatives to treatment options like antiandrogens or antiestrogens. The present article deals with recent patents (focus on publications from 2000 - 2006) concerning P450 inhibitor design where steroidal substrates are involved. In this context a special focus is provided for CYP17 and CYP19. Mechanisms of action will also be discussed. Inhibitors of CYP11B2 (aldosterone synthase) will also be dealt with.

  9. The effects of Andrographis paniculata (Burm.f.) Nees extract and diterpenoids on the CYP450 isoforms' activities, a review of possible herb-drug interaction risks.

    PubMed

    Tan, Mei Lan; Lim, Lin Ee

    2015-01-01

    Andrographis paniculata (Burm.f.) Nees is a popular medicinal plant and its components are used in various traditional product preparations. However, its herb-drug interactions risks remain unclear. This review specifically discusses the various published studies carried out to evaluate the effects of Andrographis paniculata (Burm.f.) Nees plant extracts and diterpenoids on the CYP450 metabolic enzyme and if the plant components pose a possible herb-drug interaction risk. Unfortunately, the current data are insufficient to indicate if the extracts or diterpenoids can be labeled as in vitro CYP1A2, CYP2C9 or CYP3A4 inhibitors. A complete CYP inhibition assay utilizing human liver microsomes and the derivation of relevant parameters to predict herb-drug interaction risks may be necessary for these isoforms. However, based on the current studies, none of the extracts and diterpenoids exhibited CYP450 induction activity in human hepatocytes or human-derived cell lines. It is crucial that a well-defined experimental design is needed to make a meaningful herb-drug interaction prediction.

  10. Cytochromes P450

    PubMed Central

    Bak, Søren; Beisson, Fred; Bishop, Gerard; Hamberger, Björn; Höfer, René; Paquette, Suzanne; Werck-Reichhart, Danièle

    2011-01-01

    There are 244 cytochrome P450 genes (and 28 pseudogenes) in the Arabidopsis genome. P450s thus form one of the largest gene families in plants. Contrary to what was initially thought, this family diversification results in very limited functional redundancy and seems to mirror the complexity of plant metabolism. P450s sometimes share less than 20% identity and catalyze extremely diverse reactions leading to the precursors of structural macromolecules such as lignin, cutin, suberin and sporopollenin, or are involved in biosynthesis or catabolism of all hormone and signaling molecules, of pigments, odorants, flavors, antioxidants, allelochemicals and defense compounds, and in the metabolism of xenobiotics. The mechanisms of gene duplication and diversification are getting better understood and together with co-expression data provide leads to functional characterization. PMID:22303269

  11. Constitutive Androgen Receptor-Null Mice Are Sensitive to the Toxic Effects of Parathion: Association with Reduced Cytochrome P450-Mediated Parathion MetabolismS⃞

    PubMed Central

    Mota, Linda C.; Hernandez, Juan P.

    2010-01-01

    Constitutive androgen receptor (CAR) is activated by several chemicals and in turn regulates multiple detoxification genes. Our research demonstrates that parathion is one of the most potent, environmentally relevant CAR activators with an EC50 of 1.43 μM. Therefore, animal studies were conducted to determine whether CAR was activated by parathion in vivo. Surprisingly, CAR-null mice, but not wild-type (WT) mice, showed significant parathion-induced toxicity. However, parathion did not induce Cyp2b expression, suggesting that parathion is not a CAR activator in vivo, presumably because of its short half-life. CAR expression is also associated with the expression of several drug-metabolizing cytochromes P450 (P450). CAR-null mice demonstrate lower expression of Cyp2b9, Cyp2b10, Cyp2c29, and Cyp3a11 primarily, but not exclusively in males. Therefore, we incubated microsomes from untreated WT and CAR-null mice with parathion in the presence of esterase inhibitors to determine whether CAR-null mice show perturbed P450-mediated parathion metabolism compared with that in WT mice. The metabolism of parathion to paraoxon and p-nitrophenol (PNP) was reduced in CAR-null mice with male CAR-null mice showing reduced production of both paraoxon and PNP, and female CAR-null mice showing reduced production of only PNP. Overall, the data indicate that CAR-null mice metabolize parathion slower than WT mice. These results provide a potential mechanism for increased sensitivity of individuals with lower CAR activity such as newborns to parathion and potentially other chemicals due to decreased metabolic capacity. PMID:20573718

  12. Inhibition of cytochrome P450 2B4 by environmentally persistent free radical-containing particulate matter

    PubMed Central

    Reed, James R.; dela Cruz, Albert Leo N.; Lomnicki, Slawo M.; Backes, Wayne L.

    2015-01-01

    Combustion processes generate particulate matter (PM) that can affect human health. The presence of redox-active metals and aromatic hydrocarbons in the post-combustion regions results in the formation of air-stable, environmentally persistent free radicals (EPFRs) on entrained particles. Exposure to EPFRs has been shown to negatively influence pulmonary and cardiovascular functions. Cytochromes P450 (P450/CYP) are endoplasmic reticulum resident proteins that are responsible for the metabolism of foreign compounds. Previously, it was shown that model EPFRs, generated by exposure of silica containing 5% copper oxide (CuO-Si) to either dicholorobenzene (DCB230) or 2-monochlorophenol (MCP230) at ≥ 230°C, inhibited six forms of P450 in rat liver microsomes (Toxicol. Appl. Pharmacol. (2014) 277:200-209). In this study, the inhibition of P450 by MCP230 was examined in more detail by measuring its effect on the rate of metabolism of 7-ethoxy-4-trifluoromethylcoumarin (7EFC) and 7-benzyloxyresorufin (7BRF) by the purified, reconstituted CYP2B4 system. MCP230 inhibited the CYP2B4-mediated metabolism of 7EFC at least 10-fold more potently than non-EPFR controls (CuO-Si, silica, and silica generated from heating silica and MCP at 50°C, so that EPFRs were not formed (MCP50)). The inhibition by EPFRs was specific for the P450 and did not affect the ability of the redox partner, P450 reductase (CPR) from reducing cytochrome c. All of the PM inhibited CYP2B4-mediated metabolism noncompetitively with respect to substrate. When CYP2B4-mediated metabolism of 7EFC was measured as a function of the CPR concentration, the mechanism of inhibition was competitive. EPFRs likely inhibit CYP2B4-mediated substrate metabolism by physically disrupting the CPR•P450 complex. PMID:25817938

  13. Luminogenic cytochrome P450 assays.

    PubMed

    Cali, James J; Ma, Dongping; Sobol, Mary; Simpson, Daniel J; Frackman, Susan; Good, Troy D; Daily, William J; Liu, David

    2006-08-01

    Luminogenic cytochrome P450 (CYP) assays couple CYP enzyme activity to firefly luciferase luminescence in a technology called P450-Glo(TM) (Promega). Luminogenic substrates are used in assays of human CYP1A1, -1A2, -1B1, -2C8, -2C9, -2C19, -2D6, -2J2, -3A4, -3A7, -4A11, -4F3B, -4F12 and -19. The assays detect dose-dependent CYP inhibition by test compounds against recombinant CYP enzymes or liver microsomes. Induction or inhibition of CYP activities in cultured hepatocytes is measured in a nonlytic approach that leaves cells intact for additional analysis. Luminogenic CYP assays offer advantages of speed and safety over HPLC and radiochemical-based methods. Compared with fluorogenic methods the approach offers advantages of improved sensitivity and decreased interference between optical properties of test compound and CYP substrate. These homogenous assays are sensitive and robust tools for high-throughput CYP screening in early drug discovery.

  14. P450 monooxygenases (P450ome) of the model white rot fungus Phanerochaete chrysosporium.

    PubMed

    Syed, Khajamohiddin; Yadav, Jagjit S

    2012-11-01

    Phanerochaete chrysosporium, the model white rot fungus, has been the focus of research for the past about four decades for understanding the mechanisms and processes of biodegradation of the natural aromatic polymer lignin and a broad range of environmental toxic chemicals. The ability to degrade this vast array of xenobiotic compounds was originally attributed to its lignin-degrading enzyme system, mainly the extracellular peroxidases. However, subsequent physiological, biochemical, and/or genetic studies by us and others identified the involvement of a peroxidase-independent oxidoreductase system, the cytochrome P450 monooxygenase system. The whole genome sequence revealed an extraordinarily large P450 contingent (P450ome) with an estimated 149 P450s in this organism. This review focuses on the current status of understanding on the P450 monooxygenase system of P. chrysosproium in terms of pre-genomic and post-genomic identification, structural and evolutionary analysis, transcriptional regulation, redox partners, and functional characterization for its biodegradative potential. Future research on this catalytically diverse oxidoreductase enzyme system and its major role as a newly emerged player in xenobiotic metabolism/degradation is discussed.

  15. Prediction of Human Cytochrome P450 Inhibition Using a Multitask Deep Autoencoder Neural Network.

    PubMed

    Li, Xiang; Xu, Youjun; Lai, Luhua; Pei, Jianfeng

    2018-05-30

    Adverse side effects of drug-drug interactions induced by human cytochrome P450 (CYP450) inhibition is an important consideration in drug discovery. It is highly desirable to develop computational models that can predict the inhibitive effect of a compound against a specific CYP450 isoform. In this study, we developed a multitask model for concurrent inhibition prediction of five major CYP450 isoforms, namely, 1A2, 2C9, 2C19, 2D6, and 3A4. The model was built by training a multitask autoencoder deep neural network (DNN) on a large dataset containing more than 13 000 compounds, extracted from the PubChem BioAssay Database. We demonstrate that the multitask model gave better prediction results than that of single-task models, previous reported classifiers, and traditional machine learning methods on an average of five prediction tasks. Our multitask DNN model gave average prediction accuracies of 86.4% for the 10-fold cross-validation and 88.7% for the external test datasets. In addition, we built linear regression models to quantify how the other tasks contributed to the prediction difference of a given task between single-task and multitask models, and we explained under what conditions the multitask model will outperform the single-task model, which suggested how to use multitask DNN models more effectively. We applied sensitivity analysis to extract useful knowledge about CYP450 inhibition, which may shed light on the structural features of these isoforms and give hints about how to avoid side effects during drug development. Our models are freely available at http://repharma.pku.edu.cn/deepcyp/home.php or http://www.pkumdl.cn/deepcyp/home.php .

  16. Predicting transporter-mediated drug interactions: Commentary on: "Pharmacokinetic evaluation of a drug transporter cocktail consisting of digoxin, furosemide, metformin and rosuvastatin" and "Validation of a microdose probe drug cocktail for clinical drug interaction assessments for drug transporters and CYP3A".

    PubMed

    Zhang, L; Sparreboom, A

    2017-04-01

    Transporters, expressed in various tissues, govern the absorption, distribution, metabolism, and excretion of drugs, and consequently their inherent safety and efficacy profiles. Drugs may interact with a transporter as a substrate and/or an inhibitor. Understanding transporter-mediated drug-drug interactions (DDIs), in addition to enzyme-mediated DDIs, is an integral part of risk assessment in drug development and regulatory review because the concomitant use of more than one medication in patients is common. © 2016 ASCPT.

  17. Role of reactive oxygen intermediates in the interferon-mediated depression of hepatic drug metabolism and protective effect of N-acetylcysteine in mice.

    PubMed

    Ghezzi, P; Bianchi, M; Gianera, L; Landolfo, S; Salmona, M

    1985-08-01

    Interferon (IFN) and IFN inducers are known to depress hepatic microsomal cytochrome P-450 levels, and the liver toxicity of IFN was reported to be lethal in newborn mice. We have observed that administration to mice of IFN and IFN inducers caused a marked increase in liver xanthine oxidase activity. Because this enzyme is well known to produce reactive oxygen intermediates and cytochrome P-450 was reported to be sensitive to the oxidative damage, we have tested the hypothesis that a free radical mechanism could mediate the depression of cytochrome P-450 levels by IFN. Administration to mice of the IFN inducer polyinosinic-polycytidylic acid (2 mg/kg i.p.) caused a 29 to 52% decrease in liver cytochrome P-450. Concomitant p.o. administration of the free radical scavenger, N-acetylcysteine (as a 2.5% solution in drinking water), or the xanthine oxidase inhibitor, allopurinol (100 mg/kg), protected against the IFN-mediated depression of P-450 kg), protected against the IFN-mediated depression of P-450 levels. The results suggest that an increased endogenous generation of free radicals, possibly due to the induction of xanthine oxidase, is implicated in the IFN-mediated depression of liver drug metabolism. The relevance of these data also extends to cases in which this side effect is observed in pathological situations (e.g., viral diseases and administration of vaccines) associated with an induction of IFN.

  18. Piperine activates human pregnane X receptor to induce the expression of cytochrome P450 3A4 and multidrug resistance protein 1

    SciTech Connect

    Wang, Yue-Ming; Lin, Wenwei; Chai, Sergio C.

    2013-10-01

    Activation of the pregnane X receptor (PXR) and subsequently its target genes, including those encoding drug transporters and metabolizing enzymes, while playing substantial roles in xenobiotic detoxification, might cause undesired drug-drug interactions. Recently, an increased awareness has been given to dietary components for potential induction of diet–drug interactions through activation of PXR. Here, we studied, whether piperine (PIP), a major component extracted from the widely-used daily spice black pepper, could induce PXR-mediated expression of cytochrome P450 3A4 (CYP3A4) and multidrug resistance protein 1 (MDR1). Our results showed that PIP activated human PXR (hPXR)-mediated CYP3A4 and MDR1 expression in human hepatocytes,more » intestine cells, and a mouse model; PIP activated hPXR by recruiting its coactivator SRC-1 in both cellular and cell-free systems; PIP bound to the hPXR ligand binding domain in a competitive ligand binding assay in vitro. The dichotomous effects of PIP on induction of CYP3A4 and MDR1 expression observed here and inhibition of their activity reported elsewhere challenges the potential use of PIP as a bioavailability enhancer and suggests that caution should be taken in PIP consumption during drug treatment in patients, particularly those who favor daily pepper spice or rely on certain pepper remedies. - Highlights: • Piperine induces PXR-mediated CYP3A4 and MDR1 expression. • Piperine activates PXR by binding to PXR and recruiting coactivator SRC-1. • Piperine induces PXR activation in vivo. • Caution should be taken in piperine consumption during drug treatment.« less

  19. Exposure to benzo[a]pyrene of Hepatic Cytochrome P450 Reductase Null (HRN) and P450 Reductase Conditional Null (RCN) mice: Detection of benzo[a]pyrene diol epoxide-DNA adducts by immunohistochemistry and 32P-postlabelling.

    PubMed

    Arlt, Volker M; Poirier, Miriam C; Sykes, Sarah E; John, Kaarthik; Moserova, Michaela; Stiborova, Marie; Wolf, C Roland; Henderson, Colin J; Phillips, David H

    2012-09-03

    Benzo[a]pyrene (BaP) is a widespread environmental carcinogen activated by cytochrome P450 (P450) enzymes. In Hepatic P450 Reductase Null (HRN) and Reductase Conditional Null (RCN) mice, P450 oxidoreductase (Por) is deleted specifically in hepatocytes, resulting in the loss of essentially all hepatic P450 function. Treatment of HRN mice with a single i.p. or oral dose of BaP (12.5 or 125mg/kg body weight) resulted in higher DNA adduct levels in liver (up to 10-fold) than in wild-type (WT) mice, indicating that hepatic P450s appear to be more important for BaP detoxification in vivo. Similar results were obtained in RCN mice. We tested whether differences between hepatocytes and non-hepatocytes in P450 activity may underlie the increased liver BaP-DNA binding in HRN mice. Cellular localisation by immunohistochemistry of BaP-DNA adducts showed that HRN mice have ample capacity for formation of BaP-DNA adducts in liver, indicating that the metabolic process does not result in the generation of a reactive species different from that formed in WT mice. However, increased protein expression of cytochrome b(5) in hepatic microsomes of HRN relative to WT mice suggests that cytochrome b(5) may modulate the P450-mediated bioactivation of BaP in HRN mice, partially substituting the function of Por. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  20. In vitro metabolism and drug-drug interaction potential of UTL-5g, a novel chemo- and radioprotective agent.

    PubMed

    Wu, Jianmei; Shaw, Jiajiu; Dubaisi, Sarah; Valeriote, Frederick; Li, Jing

    2014-12-01

    N-(2,4-dichlorophenyl)-5-methyl-1,2-oxazole-3-carboxamide (UTL-5g), a potential chemo- and radioprotective agent, acts as a prodrug requiring bioactivation to the active metabolite 5-methylisoxazole-3-carboxylic acid (ISOX). UTL-5g hydrolysis to ISOX and 2,4-dichloroaniline (DCA) has been identified in porcine and rabbit liver esterases. The purpose of this study was to provide insights on the metabolism and drug interaction potential of UTL-5g in humans. The kinetics of UTL-5g hydrolysis was determined in human liver microsomes (HLM) and recombinant human carboxylesterases (hCE1b and hCE2). The potential of UTL-5g and its metabolites for competitive inhibition and time-dependent inhibition of microsomal cytochrome P450 (P450) was examined in HLM. UTL-5g hydrolysis to ISOX and DCA in HLM were NADPH-independent, with a maximum rate of reaction (Vmax) of 11.1 nmol/min per mg and substrate affinity (Km) of 41.6 µM. Both hCE1b and hCE2 effectively catalyzed UTL-5g hydrolysis, but hCE2 exhibited ∼30-fold higher catalytic efficiency (Vmax/Km) than hCE1b. UTL-5g and DCA competitively inhibited microsomal CYP1A2, CYP2B6, and CYP2C19 (IC50 values <50 µM), and exhibited time-dependent inhibition of microsomal CYP1A2 with the inactivation efficiency (kinact/KI) of 0.68 and 0.51 minute(-1)·mM(-1), respectively. ISOX did not inhibit or inactivate any tested microsomal P450. In conclusion, hCE1b and hCE2 play a key role in the bioactivation of UTL-5g. Factors influencing carboxylesterase activities may have a significant impact on the pharmacological and therapeutic effects of UTL-5g. UTL-5g has the potential to inhibit P450-mediated metabolism through competitive inhibition or time-dependent inhibition. Caution is particularly needed for potential drug interactions involving competitive inhibition or time-dependent inhibition of CYP1A2 in the future clinical development of UTL-5g. Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.

  1. In Vitro Metabolism and Drug-Drug Interaction Potential of UTL-5g, a Novel Chemo- and Radioprotective Agent

    PubMed Central

    Wu, Jianmei; Shaw, Jiajiu; Dubaisi, Sarah; Valeriote, Frederick

    2014-01-01

    N-(2,4-dichlorophenyl)-5-methyl-1,2-oxazole-3-carboxamide (UTL-5g), a potential chemo- and radioprotective agent, acts as a prodrug requiring bioactivation to the active metabolite 5-methylisoxazole-3-carboxylic acid (ISOX). UTL-5g hydrolysis to ISOX and 2,4-dichloroaniline (DCA) has been identified in porcine and rabbit liver esterases. The purpose of this study was to provide insights on the metabolism and drug interaction potential of UTL-5g in humans. The kinetics of UTL-5g hydrolysis was determined in human liver microsomes (HLM) and recombinant human carboxylesterases (hCE1b and hCE2). The potential of UTL-5g and its metabolites for competitive inhibition and time-dependent inhibition of microsomal cytochrome P450 (P450) was examined in HLM. UTL-5g hydrolysis to ISOX and DCA in HLM were NADPH-independent, with a maximum rate of reaction (Vmax) of 11.1 nmol/min per mg and substrate affinity (Km) of 41.6 µM. Both hCE1b and hCE2 effectively catalyzed UTL-5g hydrolysis, but hCE2 exhibited ∼30-fold higher catalytic efficiency (Vmax/Km) than hCE1b. UTL-5g and DCA competitively inhibited microsomal CYP1A2, CYP2B6, and CYP2C19 (IC50 values <50 µM), and exhibited time-dependent inhibition of microsomal CYP1A2 with the inactivation efficiency (kinact/KI) of 0.68 and 0.51 minute−1·mM−1, respectively. ISOX did not inhibit or inactivate any tested microsomal P450. In conclusion, hCE1b and hCE2 play a key role in the bioactivation of UTL-5g. Factors influencing carboxylesterase activities may have a significant impact on the pharmacological and therapeutic effects of UTL-5g. UTL-5g has the potential to inhibit P450-mediated metabolism through competitive inhibition or time-dependent inhibition. Caution is particularly needed for potential drug interactions involving competitive inhibition or time-dependent inhibition of CYP1A2 in the future clinical development of UTL-5g. PMID:25249693

  2. Exploring the Role of CYP3A4 Mediated Drug Metabolism in the Pharmacological Modulation of Nitric Oxide Production

    PubMed Central

    Pérez-del Palacio, José; Díaz, Caridad; Vergara, Noemí; Algieri, Francesca; Rodríguez-Nogales, Alba; de Pedro, Nuria; Rodríguez-Cabezas, M. Elena; Genilloud, Olga; Gálvez, Julio; Vicente, Francisca

    2017-01-01

    Nitric-oxide synthase, the enzyme responsible for mammalian nitric oxide generation, and cytochrome P450, the major enzymes involved in drug metabolism, share striking similarities. Therefore, it makes sense that cytochrome P450 drug mediated biotransformations might play an important role in the pharmacological modulation of nitric oxide synthase. In this work, we have undertaken an integrated in vitro assessment of the hepatic metabolism and nitric oxide modulation of previously described dual inhibitors (imidazoles and macrolides) of these enzymes in order assess the implication of CYP450 activities over production of nitric oxide. In vitro systems based in human liver microsomes and activated mouse macrophages were developed for these purposes. Additionally in vitro production the hepatic metabolites of dual inhibitor, roxithromycin, was investigated achieving the identification and isolation of main hepatic biotransformation products. Our results suggested that for some macrolide compounds, the cytochrome P450 3A4 derived drug metabolites have an important effect on nitric oxide production and might critically contribute to the pharmacological immunomodulatory activity observed. PMID:28446877

  3. Relationships among Ergot Alkaloids, Cytochrome P450 Activity, and Beef Steer Growth

    NASA Astrophysics Data System (ADS)

    Rosenkrans, Charles; Ezell, Nicholas

    2015-03-01

    Determining a grazing animal’s susceptibility to ergot alkaloids has been a research topic for decades. Our objective was to determine if the Promega™ P450-Glo assay could be used to indirectly detect ergot alkaloids or their metabolites in urine of steers. The first experiment validated the effects of ergot alkaloids [0, 20, and 40 μM of ergotamine (ET), dihydroergotamine (DHET), and ergonovine (EN)] on human CYP3A4 using the P450-Glo assay (Promega™ V9800). With this assay, luminescence is directly proportional to CYP450 activity. Relative inhibition of in vitro cytochrome P450 activity was affected (P < 0.001) by an interaction between alkaloids and concentration. That interaction resulted in no concentration effect of EN, but within ET and DHET 20 and 40 µM concentrations inhibited CYP450 activity when compared with controls. In experiment 2, urine was collected from Angus-sired crossbred steers (n = 39; 216 ± 2.6 d of age; 203 ± 1.7 kg) after grazing tall fescue pastures for 105 d. Non-diluted urine was added to the Promega™ P450-Glo assay, and observed inhibition (3.7 % ± 2.7 of control). Urine content of total ergot alkaloids (331.1 ng/mg of creatinine ± 325.7) was determined using enzyme linked immunosorbent assay. Urine inhibition of CYP450 activity and total alkaloids were correlated (r = -0.31; P < 0.05). Steers were genotyped at CYP450 single nucleotide polymorphism, C994G. Steer genotype affected (P < 0.03) inhibition of CYP450 activity by urine; heterozygous steers had the least amount of CYP450 inhibition suggesting that genotyping cattle may be a method of identifying animals that are susceptible to ergot alkaloids. Although, additional research is needed, we demonstrate that the Promega™ P450-Glo assay is sensitive to ergot alkaloids and urine from steers grazing tall fescue. With some refinement the P450-Glo assay has potential as a tool for screening cattle for their exposure to fescue toxins.

  4. Development, validation and utility of an in vitro technique for assessment of potential clinical drug-drug interactions involving P-glycoprotein.

    PubMed

    Keogh, John P; Kunta, Jeevan R

    2006-04-01

    Regulatory interest is increasing for drug transporters generally and P-glycoprotein (Pgp) in particular, primarily in the area of drug-drug interactions. To aid in both identifying and discharging the potential liabilities associated with drug-transporter interactions, the pharmaceutical industry has a growing requirement for routine and robust non-clinical assays. An assay was designed, optimised and validated to determine the in vitro inhibitory potency of new chemical entities (NCEs) towards human Pgp-mediated transport. [3H]-Digoxin was established as a suitable probe substrate by investigating its characteristics in the in vitro system (MDCKII-MDR1 cells grown in 24-multiwell inserts). The inhibitory potencies (apparent IC50) of known Pgp inhibitors astemizole, GF120918, ketoconazole, itraconazole, quinidine, verapamil and quinine were determined over at least a 1000-fold concentration range. Validation was carried out using manual and automatic techniques. [3H]-Digoxin was found to be stable and have good mass balance in the system. In contrast to [A-->B] transport, [3H]-digoxin [B-->A] transport rates were readily measured with good reproducibility. There was no evidence of saturation of transport up to 10 microM digoxin and 30 nM digoxin was selected for routine assay use, reflecting clinical therapeutic concentrations. IC50 values ranged over approximately 100-fold with excellent reproducibility. Results from manual and automated versions were in close agreement. This method is suitable for routine use to assess the in vitro inhibitory potency of NCEs on Pgp-mediated digoxin transport. Comparison of IC50 values against clinical interaction profiles for the probe inhibitors indicated the in vitro assay is predictive of clinical digoxin-drug interactions mediated via Pgp.

  5. Venetoclax (ABT-199) Might Act as a Perpetrator in Pharmacokinetic Drug-Drug Interactions.

    PubMed

    Weiss, Johanna; Gajek, Thomas; Köhler, Bruno Christian; Haefeli, Walter Emil

    2016-02-24

    Venetoclax (ABT-199) represents a specific B-cell lymphoma 2 (Bcl-2) inhibitor that is currently under development for the treatment of lymphoid malignancies. So far, there is no published information on its interaction potential with important drug metabolizing enzymes and drug transporters, or its efficacy in multidrug resistant (MDR) cells. We therefore scrutinized its drug-drug interaction potential in vitro. Inhibition of cytochrome P450 enzymes (CYPs) was quantified by commercial kits. Inhibition of drug transporters (P-glycoprotein (P-gp, ABCB1), breast cancer resistance protein (BCRP), and organic anion transporting polypeptides (OATPs)) was evaluated by the use of fluorescent probe substrates. Induction of drug transporters and drug metabolizing enzymes was quantified by real-time RT-PCR. The efficacy of venetoclax in MDR cells lines was evaluated with proliferation assays. Venetoclax moderately inhibited P-gp, BCRP, OATP1B1, OATP1B3, CYP3A4, and CYP2C19, whereas CYP2B6 activity was increased. Venetoclax induced the mRNA expression of CYP1A1, CYP1A2, UGT1A3, and UGT1A9. In contrast, expression of ABCB1 was suppressed, which might revert tumor resistance towards antineoplastic P-gp substrates. P-gp over-expression led to reduced antiproliferative effects of venetoclax. Effective concentrations for inhibition and induction lay in the range of maximum plasma concentrations of venetoclax, indicating that it might act as a perpetrator drug in pharmacokinetic drug-drug interactions.

  6. Monkey liver cytochrome P450 2C9 is involved in caffeine 7-N-demethylation to form theophylline.

    PubMed

    Utoh, Masahiro; Murayama, Norie; Uno, Yasuhiro; Onose, Yui; Hosaka, Shinya; Fujino, Hideki; Shimizu, Makiko; Iwasaki, Kazuhide; Yamazaki, Hiroshi

    2013-12-01

    Caffeine (1,3,7-trimethylxanthine) is a phenotyping substrate for human cytochrome P450 1A2. 3-N-Demethylation of caffeine is the main human metabolic pathway, whereas monkeys extensively mediate the 7-N-demethylation of caffeine to form pharmacological active theophylline. Roles of monkey P450 enzymes in theophylline formation from caffeine were investigated using individual monkey liver microsomes and 14 recombinantly expressed monkey P450 enzymes, and the results were compared with those for human P450 enzymes. Caffeine 7-N-demethylation activity in microsomes from 20 monkey livers was not strongly inhibited by α-naphthoflavone, quinidine or ketoconazole, and was roughly correlated with diclofenac 4'-hydroxylation activities. Monkey P450 2C9 had the highest activity for caffeine 7-N-demethylation. Kinetic analysis revealed that monkey P450 2C9 had a high Vmax/Km value for caffeine 7-N-demethylation, comparable to low Km value for monkey liver microsomes. Caffeine could dock favorably with monkey P450 2C9 modeled for 7-N-demethylation and with human P450 1A2 for 3-N-demethylation. The primary metabolite theophylline was oxidized to 8-hydroxytheophylline in similar ways by liver microsomes and by recombinant P450s in both humans and monkeys. These results collectively suggest a high activity for monkey liver P450 2C9 toward caffeine 7-N-demethylation, whereas, in humans, P450 1A2-mediated caffeine 3-N-demethylation is dominant.

  7. Cycle affects imidacloprid efficiency by mediating cytochrome P450 expression in the brown planthopper Nilaparvata lugens.

    PubMed

    Kang, K; Yang, P; Pang, R; Yue, L; Zhang, W

    2017-10-01

    Circadian clocks influence most behaviours and physiological activities in animals, including daily fluctuations in metabolism. However, how the clock gene cycle influences insects' responses to pesticides has rarely been reported. Here, we provide evidence that cycle affects imidacloprid efficacy by mediating the expression of cytochrome P450 genes in the brown planthopper (BPH) Nilaparvata lugens, a serious insect pest of rice. Survival bioassays showed that the susceptibility of BPH adults to imidacloprid differed significantly between the two time points tested [Zeitgeber Time 8 (ZT8) and ZT4]. After cloning the cycle gene in the BPH (Nlcycle), we found that Nlcycle was expressed at higher levels in the fat body and midgut, and its expression was rhythmic with two peaks. Knockdown of Nlcycle affected the expression levels and rhythms of cytochrome P450 genes as well as susceptibility to imidacloprid. The survival rates of BPH adults after treatment with imidacloprid did not significantly differ between ZT4 and ZT8 after double-stranded Nlcycle treatment. These findings can be used to improve pesticide use and increase pesticide efficiency in the field. © 2017 The Royal Entomological Society.

  8. Cytochrome P450 pharmacogenetics in drug development: in vitro studies and clinical consequences.

    PubMed

    Rodrigues, A David; Rushmore, Thomas H

    2002-06-01

    Members of the human cytochrome P450 (CYP) superfamily play a role in the metabolism of many drugs and several of them, CYP2D6, CYP2C9 and CYP2C19, have been shown to be polymorphic as a result of single nucleotide polymorphisms (SNPs), gene deletions, and gene duplications. These polymorphisms can impact the pharmacokinetics (PK), metabolism, safety and efficacy of drugs, and because of the availability of automation, genotyped human tissue, recombinant CYP preparations (rCYPs) and reagents, most pharmaceutical companies have increasingly screened out compounds that are metabolized solely by polymorphic CYPs. In the absence of suitable animal models, it has been widely accepted that such in vitro data are useful because one can obtain information prior to dosing in man and select the most appropriate clinical studies with prospectively genotyped and phenotyped subjects. Overall, current trends in the industry have been fueled by increased managed healthcare, the desire to minimize the need for therapeutic drug monitoring and CYP genotyping in medical practice, and a very competitive market place. In the past, such paradigms have not been as influential and there are numerous examples of marketed drugs that are metabolized by polymorphic CYPs.

  9. Human Hepatic Cytochrome P450-Specific Metabolism of the Organophosphorus Pesticides Methyl Parathion and Diazinon

    PubMed Central

    Tian, Yuan; Knaak, James B.; Kostyniak, Paul J.; Olson, James R.

    2012-01-01

    Organophosphorus pesticides (OPs) are a public health concern due to their worldwide use and documented human exposures. Phosphorothioate OPs are metabolized by cytochrome P450s (P450s) through either a dearylation reaction to form an inactive metabolite, or through a desulfuration reaction to form an active oxon metabolite, which is a potent cholinesterase inhibitor. This study investigated the rate of desulfuration (activation) and dearylation (detoxification) of methyl parathion and diazinon in human liver microsomes. In addition, recombinant human P450s were used to determine the P450-specific kinetic parameters (Km and Vmax) for each compound for future use in refining human physiologically based pharmacokinetic/pharmacodynamic (PBPK/PD) models of OP exposure. The primary enzymes involved in bioactivation of methyl parathion were CYP2B6 (Km = 1.25 μM; Vmax = 9.78 nmol · min−1 · nmol P450−1), CYP2C19 (Km = 1.03 μM; Vmax = 4.67 nmol · min−1 · nmol P450−1), and CYP1A2 (Km = 1.96 μM; Vmax = 5.14 nmol · min−1 · nmol P450−1), and the bioactivation of diazinon was mediated primarily by CYP1A1 (Km = 3.05 μM; Vmax = 2.35 nmol · min−1 · nmol P450−1), CYP2C19 (Km = 7.74 μM; Vmax = 4.14 nmol · min−1 · nmol P450−1), and CYP2B6 (Km = 14.83 μM; Vmax = 5.44 nmol · min−1 · nmol P450−1). P450-mediated detoxification of methyl parathion only occurred to a limited extent with CYP1A2 (Km = 16.8 μM; Vmax = 1.38 nmol · min−1 · nmol P450−1) and 3A4 (Km = 104 μM; Vmax = 5.15 nmol · min−1 · nmol P450−1), whereas the major enzyme involved in diazinon detoxification was CYP2C19 (Km = 5.04 μM; Vmax = 5.58 nmol · min−1 · nmol P450−1). The OP- and P450-specific kinetic values will be helpful for future use in refining human PBPK/PD models of OP exposure. PMID:21969518

  10. Nonclinical Pharmacokinetics, Disposition, and Drug-Drug Interaction Potential of a Novel d-Amino Acid Peptide Agonist of the Calcium-Sensing Receptor AMG 416 (Etelcalcetide).

    PubMed

    Subramanian, Raju; Zhu, Xiaochun; Kerr, Savannah J; Esmay, Joel D; Louie, Steven W; Edson, Katheryne Z; Walter, Sarah; Fitzsimmons, Michael; Wagner, Mylo; Soto, Marcus; Pham, Roger; Wilson, Sarah F; Skiles, Gary L

    2016-08-01

    AMG 416 (etelcalcetide) is a novel synthetic peptide agonist of the calcium-sensing receptor composed of a linear chain of seven d-amino acids (referred to as the d-amino acid backbone) with a d-cysteine linked to an l-cysteine via a disulfide bond. AMG 416 contains four basic d-arginine residues and is a +4 charged peptide at physiologic pH with a mol. wt. of 1048.3 Da. The pharmacokinetics (PK), disposition, and potential of AMG 416 to cause drug-drug interaction were investigated in nonclinical studies with two single (14)C-labels placed either at a potentially metabolically labile acetyl position or on the d-alanine next to d-cysteine in the interior of the d-amino acid backbone. After i.v. dosing, the PK and disposition of AMG 416 were similar in male and female rats. Radioactivity rapidly distributed to most tissues in rats with intact kidneys, and renal elimination was the predominant clearance pathway. No strain-dependent differences were observed. In bilaterally nephrectomized rats, minimal radioactivity (1.2%) was excreted via nonrenal pathways. Biotransformation occurred primarily via disulfide exchange with endogenous thiol-containing molecules in whole blood rather than metabolism by enzymes, such as proteases or cytochrome P450s; the d-amino acid backbone remained unaltered. A substantial proportion of the plasma radioactivity was covalently conjugated to albumin. AMG 416 presents a low risk for P450 or transporter-mediated drug-drug interactions because it showed no interactions in vitro. These studies demonstrated a (14)C label on either the acetyl or the d-alanine in the d-amino acid backbone would be appropriate for clinical studies. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

  11. Recollection of the early years of the research on cytochrome P450

    PubMed Central

    OMURA, Tsuneo

    2011-01-01

    Since the publication of the first paper on “cytochrome P450” in 1962, the biochemical research on this novel hemoprotein expanded rapidly in the 1960s and the 1970s as its principal roles in various important metabolic processes including steroid hormone biosynthesis in the steroidogenic organs and drug metabolism in the liver were elucidated. Establishment of the purification procedures of microsomal and mitochondrial P450s in the middle of the 1970s together with the introduction of molecular biological techniques accelerated the remarkable expansion of the research on P450 in the following years. This review paper summarizes the important developments in the research on P450 in the early years, for about two decades from the beginning, together with my personal recollections. PMID:22156409

  12. Camptothecin Attenuates Cytochrome P450 3A4 Induction by Blocking the Activation of Human Pregnane X ReceptorS⃞

    PubMed Central

    Chen, Yakun; Tang, Yong; Robbins, Gregory T.

    2010-01-01

    Differential regulation of drug-metabolizing enzymes (DMEs) is a common cause of adverse drug effects in cancer therapy. Due to the extremely important role of cytochrome P450 3A4 (CYP3A4) in drug metabolism and the dominant regulation of human pregnane X receptor (hPXR) on CYP3A4, finding inhibitors for hPXR could provide a unique tool to control drug efficacies in cancer therapy. Camptothecin (CPT) was demonstrated as a novel and potent inhibitor (IC50 = 0.58 μM) of an hPXR-mediated transcriptional regulation on CYP3A4 in this study. In contrast, one of its analogs, irinotecan (CPT-11), was found to be an hPXR agonist in the same tests. CPT disrupted the interaction of hPXR with steroid receptor coactivator-1 but had effects on neither the competition of ligand binding nor the formation of the hPXR and retinoid X receptor α heterodimer, nor the interaction between the regulatory complex and DNA-responsive elements. CPT treatment resulted in delayed metabolism of nifedipine in human hepatocytes treated with rifampicin, suggesting a potential prevention of drug-drug interactions between CYP3A4 inducers and CYP3A4-metabolized drugs. Because CPT is the leading compound of topoisomerase I inhibitors, which comprise a quickly developing class of anticancer agents, the findings indicate the potential of a new class of compounds to modify hPXR activity as agonists/inhibitors and are important in the development of CPT analogs. PMID:20504912

  13. Pungent ginger components modulates human cytochrome P450 enzymes in vitro

    PubMed Central

    Li, Mian; Chen, Pei-zhan; Yue, Qing-xi; Li, Jing-quan; Chu, Rui-ai; Zhang, Wei; Wang, Hui

    2013-01-01

    Aim: Ginger rhizome is used worldwide as a spicy flavor agent. This study was designed to explore the potential effects of pungent ginger components, 6-, 8-, and 10-gingerol, on human cytochrome P450 (CYP450) enzymes that are responsible for the metabolism of many prescription drugs. Methods: The activities of human CYP2C9, CYP2C19, CYP2D6, and CYP3A4 were analyzed using Vivid P450 assay kits. The mRNA expression of CYP3A4 in human hepatocellular carcinoma cell line HepG2 was measured using quantitative real-time PCR assay. Results: All three gingerols potently inhibited CYP2C9 activity, exerted moderate inhibition on CYP2C19 and CYP3A4, and weak inhibion on CYP2D6. 8-Gingerol was the most potent in inhibition of P450 enzymes with IC50 values of 6.8, 12.5, 8.7, and 42.7 μmol/L for CYP2C9, CYP2C19, CYP3A4, and CYP2D6, respectively. By comparing the effects of gingerols on CYP3A4 with three different fluorescent substrate probes, it was demonstrated that the inhibition of gingerols on CYP3A4 had no substrate-dependence. In HepG2 cells, 8-gingerol and 10-gingerol inhibited, but 6-gingerol induced mRNA expression of CYP3A4. Conclusion: 6-, 8-, and 10-gingerol suppress human cytochrome P450 activity, while 8- and 10-gingerol inhibit CYP3A4 expression. The results may have an implication for the use of ginger or ginger products when combined with therapeutic drugs that are metabolized by cytochrome P450 enzymes. PMID:23770984

  14. Nutritional Status Differentially Alters Cytochrome P450 3A4 (CYP3A4) and Uridine 5'-Diphospho-Glucuronosyltransferase (UGT) Mediated Drug Metabolism: Effect of Short-Term Fasting and High Fat Diet on Midazolam Metabolism.

    PubMed

    Lammers, Laureen A; Achterbergh, Roos; Romijn, Johannes A; Mathôt, Ron A A

    2018-06-06

    Previous studies have shown that nutritional status can alter drug metabolism which may result in treatment failure or untoward side effects. This study assesses the effect of two nutritional conditions, short-term fasting, and a short-term high fat diet (HFD) on cytochrome P450 3A4 (CYP3A4) and uridine 5'-diphospho-glucuronosyltransferase (UGT) mediated drug metabolism by studying the pharmacokinetics of midazolam and its main metabolites. In a randomized-controlled cross-over trial, nine healthy subjects received a single intravenous administration of 0.015 mg/kg midazolam after: (1) an overnight fast (control); (2) 36 h of fasting; and (3) an overnight fast after 3 days of a HFD consisting of 500 ml of cream supplemented to their regular diet. Pharmacokinetic parameters were analyzed simultaneously using non-linear mixed-effects modeling. Short-term fasting increased CYP3A4-mediated midazolam clearance by 12% (p < 0.01) and decreased UGT-mediated metabolism apparent 1-OH-midazolam clearance by 13% (p < 0.01) by decreasing the ratio of clearance and the fraction metabolite formed (ΔCL 1-OH-MDZ /f 1-OH-MDZ ). Furthermore, short-term fasting decreased apparent clearance of 1-OH-midazolam-O-glucuronide (CL 1-OH-MDZ-glucuronide /(f 1-OH-MDZ-glucuronide  × f 1-OH-MDZ )) by 20% (p < 0.01). The HFD did not affect systemic clearance of midazolam or metabolites. Short-term fasting differentially alters midazolam metabolism by increasing CYP3A4-mediated metabolism but by decreasing UGT-mediated metabolism. In contrast, a short-term HFD did not affect systemic clearance of midazolam.

  15. Clinical Confirmation that the Selective JAK1 Inhibitor Filgotinib (GLPG0634) has a Low Liability for Drug-drug Interactions.

    PubMed

    Namour, Florence; Desrivot, Julie; Van der Aa, Annegret; Harrison, Pille; Tasset, Chantal; van't Klooster, Gerben

    2016-01-01

    The selective Janus kinase 1 inhibitor filgotinib (GLPG0634), which is currently in clinical development for the treatment of rheumatoid arthritis (RA) and Crohn's disease, demonstrated encouraging safety and efficacy profiles in RA patients after 4 weeks of daily dosing. As RA patients might be treated with multiple medications simultaneously, possible drug-drug interactions of filgotinib with cytochrome P450 enzymes and with key drug transporters were evaluated in vitro and in clinical studies. The enzymes involved in filgotinib's metabolism and the potential interactions of the parent and its active major metabolite with drug-metabolizing enzymes and drug transporters, were identified using recombinant enzymes, human microsomes, and cell systems. Furthermore, filgotinib's interaction potential with CYP3A4 was examined in an open-label study in healthy volunteers, which evaluated the impact of filgotinib co-administration on the CYP3A4-sensitive substrate midazolam. The potential interaction with the common RA drug methotrexate was investigated in a clinical study in RA patients. In vitro, filgotinib and its active metabolite at clinically relevant concentrations did not interact with cytochrome P450 enzymes and uridine 5'-diphospho-glucuronosyltransferases, and did not inhibit key drug transporters. In the clinic, a lack of relevant pharmacokinetic drug interactions by filgotinib and its active metabolite with substrates of CYP3A4, as well as with organic anion transporters involved in methotrexate elimination were found. the collective in vivo and in vitro data on drug-metabolizing enzymes and on key drug transporters, support co-administration of filgotinib with commonly used RA drugs to patients without the need for dose adjustments.

  16. Structural and Kinetic Basis of Steroid 17α,20-Lyase Activity in Teleost Fish Cytochrome P450 17A1 and Its Absence in Cytochrome P450 17A2*

    PubMed Central

    Pallan, Pradeep S.; Nagy, Leslie D.; Lei, Li; Gonzalez, Eric; Kramlinger, Valerie M.; Azumaya, Caleigh M.; Wawrzak, Zdzislaw; Waterman, Michael R.; Guengerich, F. Peter; Egli, Martin

    2015-01-01

    Cytochrome P450 (P450) 17A enzymes play a critical role in the oxidation of the steroids progesterone (Prog) and pregnenolone (Preg) to glucocorticoids and androgens. In mammals, a single enzyme, P450 17A1, catalyzes both 17α-hydroxylation and a subsequent 17α,20-lyase reaction with both Prog and Preg. Teleost fish contain two 17A P450s; zebrafish P450 17A1 catalyzes both 17α-hydroxylation and lyase reactions with Prog and Preg, and P450 17A2 is more efficient in pregnenolone 17α-hydroxylation but does not catalyze the lyase reaction, even in the presence of cytochrome b5. P450 17A2 binds all substrates and products, although more loosely than P450 17A1. Pulse-chase and kinetic spectral experiments and modeling established that the two-step P450 17A1 Prog oxidation is more distributive than the Preg reaction, i.e. 17α-OH product dissociates more prior to the lyase step. The drug orteronel selectively blocked the lyase reaction of P450 17A1 but only in the case of Prog. X-ray crystal structures of zebrafish P450 17A1 and 17A2 were obtained with the ligand abiraterone and with Prog for P450 17A2. Comparison of the two fish P450 17A-abiraterone structures with human P450 17A1 (DeVore, N. M., and Scott, E. E. (2013) Nature 482, 116–119) showed only a few differences near the active site, despite only ∼50% identity among the three proteins. The P450 17A2 structure differed in four residues near the heme periphery. These residues may allow the proposed alternative ferric peroxide mechanism for the lyase reaction, or residues removed from the active site may allow conformations that lead to the lyase activity. PMID:25533464

  17. The burden and management of cytochrome P450 2D6 (CYP2D6)-mediated drug-drug interaction (DDI): co-medication of metoprolol and paroxetine or fluoxetine in the elderly.

    PubMed

    Bahar, Muh Akbar; Hak, Eelko; Bos, Jens H J; Borgsteede, Sander D; Wilffert, Bob

    2017-07-01

    Metoprolol and paroxetine/fluoxetine are inevitably co-prescribed because cardiovascular disorders and depression often coexist in the elderly. This leads to CYP2D6-mediated drug-drug interactions (DDI). Because systematic evaluations are lacking, we assessed the burden of metoprolol-paroxetine/fluoxetine interaction in the elderly and how these interactions are managed in Dutch community pharmacies. Dispensing data were collected from the University of Groningen pharmacy database (IADB.nl, 1999-2014) for elderly patients (≥60 years) starting beta-blockers and/or antidepressants. Based on the two main DDI alert systems (G-Standard and Pharmabase), incidences were divided between signalled (metoprolol-fluoxetine/paroxetine) and not-signalled (metoprolol-alternative antidepressants and alternative beta-blockers-paroxetine/fluoxetine) combinations. Incident users were defined as patients starting at least one signalled or a non-signalled combination. G-Standard signalled throughout the study period, whereas Pharmabase stopped after 2005. A total of 1763 patients had 2039 metoprolol-paroxetine/fluoxetine co-prescriptions, despite DDI alert systems, and about 57.3% were signalled. The number of metoprolol-alternative antidepressant combinations (incidences = 3150) was higher than alternative beta-blocker-paroxetine/fluoxetine combinations (incidences = 1872). Metoprolol users are more likely to be co-medicated with an alternative antidepressant (incidences = 2320) than paroxetine/fluoxetine users (incidences = 1232) are. The number of paroxetine/fluoxetine users co-prescribed with alternative beta-blockers was comparable to those co-medicated with metoprolol (about 50%). Less than 5% of patients received a substitute therapy after using metoprolol-paroxetine/fluoxetine. Most of the metoprolol users (90%) received a low dose (mean DDD = 0.47) regardless whether they were prescribed paroxetine/fluoxetine. Despite the signalling software, metoprolol

  18. Adrenodoxin supports reactions catalyzed by microsomal steroidogenic cytochrome P450s

    SciTech Connect

    Pechurskaya, Tatiana A.; Harnastai, Ivan N.; Grabovec, Irina P.

    2007-02-16

    The interaction of adrenodoxin (Adx) and NADPH cytochrome P450 reductase (CPR) with human microsomal steroidogenic cytochrome P450s was studied. It is found that Adx, mitochondrial electron transfer protein, is able to support reactions catalyzed by human microsomal P450s: full length CYP17, truncated CYP17, and truncated CYP21. CPR, but not Adx, supports activity of truncated CYP19. Truncated and the full length CYP17s show distinct preference for electron donor proteins. Truncated CYP17 has higher activity with Adx compared to CPR. The alteration in preference to electron donor does not change product profile for truncated enzymes. The electrostatic contacts play a major rolemore » in the interaction of truncated CYP17 with either CPR or Adx. Similarly electrostatic contacts are predominant in the interaction of full length CYP17 with Adx. We speculate that Adx might serve as an alternative electron donor for CYP17 at the conditions of CPR deficiency in human.« less

  19. Clinical relevance of cimetidine drug interactions.

    PubMed

    Shinn, A F

    1992-01-01

    The excellent efficacy and tolerability profiles of H2-antagonists have established these agents as the leading class of antiulcer drugs. Attention has been focused on drug interactions with H2-antagonists as a means of product differentiation and because many patients are receiving multiple drug therapy. The main mechanism of most drug interactions involving cimetidine appears to be inhibition of the hepatic microsomal enzyme cytochrome P450, an effect which may be related to the different structures of H2-antagonists. Ranitidine appears to have less affinity than cimetidine for this system. There have been many published case reports and studies of drug interactions with cimetidine, but many of these have provided pharmacokinetic data only, with little information concerning the clinical significance of these findings. Nevertheless, the coadministration of cimetidine with drugs that have a narrow therapeutic margin (such as theophylline) may potentially result in clinically significant adverse effects. The monitoring of serum concentrations of drugs coadministered with cimetidine may reduce the risk of adverse events but does not abolish the problem. However, for most patients, concomitant administration of cimetidine with drugs possessing a wide therapeutic margin is unlikely to pose a significant problem.

  20. Similar substrate specificity of cynomolgus monkey cytochrome P450 2C19 to reported human P450 2C counterpart enzymes by evaluation of 89 drug clearances.

    PubMed

    Hosaka, Shinya; Murayama, Norie; Satsukawa, Masahiro; Uehara, Shotaro; Shimizu, Makiko; Iwasaki, Kazuhide; Iwano, Shunsuke; Uno, Yasuhiro; Yamazaki, Hiroshi

    2015-12-01

    Cynomolgus monkeys are used widely in preclinical studies as non-human primate species. The amino acid sequence of cynomolgus monkey cytochrome P450 (P450 or CYP) 2C19 is reportedly highly correlated to that of human CYP2C19 (92%) and CYP2C9 (93%). In the present study, 89 commercially available compounds were screened to find potential substrates for cynomolgus monkey CYP2C19. Of 89 drugs, 34 were metabolically depleted by cynomolgus monkey CYP2C19 with relatively high rates. Among them, 30 compounds have been reported as substrates or inhibitors of, either or both, human CYP2C19 and CYP2C9. Several compounds, including loratadine, showed high selectivity to cynomolgus monkey CYP2C19, and all of these have been reported as human CYP2C19 and/or CYP2C9 substrates. In addition, cynomolgus monkey CYP2C19 formed the same loratadine metabolite as human CYP2C19, descarboethoxyloratadine. These results suggest that cynomolgus monkey CYP2C19 is generally similar to human CYP2C19 and CYP2C9 in its substrate recognition functionality. Copyright © 2015 John Wiley & Sons, Ltd.

  1. The cytochrome p450 homepage.

    PubMed

    Nelson, David R

    2009-10-01

    The Cytochrome P450 Homepage is a universal resource for nomenclature and sequence information on cytochrome P450 ( CYP ) genes. The site has been in continuous operation since February 1995. Currently, naming information for 11,512 CYPs are available on the web pages. The P450 sequences are manually curated by David Nelson, and the nomenclature system conforms to an evolutionary scheme such that members of CYP families and subfamilies share common ancestors. The organisation and content of the Homepage are described.

  2. The Cytochrome P450 Homepage

    PubMed Central

    2009-01-01

    The Cytochrome P450 Homepage is a universal resource for nomenclature and sequence information on cytochrome P450 (CYP) genes. The site has been in continuous operation since February 1995. Currently, naming information for 11,512 CYPs are available on the web pages. The P450 sequences are manually curated by David Nelson, and the nomenclature system conforms to an evolutionary scheme such that members of CYP families and subfamilies share common ancestors. The organisation and content of the Homepage are described. PMID:19951895

  3. Benzylic oxidation of gemfibrozil-1-O-beta-glucuronide by P450 2C8 leads to heme alkylation and irreversible inhibition.

    PubMed

    Baer, Brian R; DeLisle, Robert Kirk; Allen, Andrew

    2009-07-01

    Gemfibrozil-1-O-beta-glucuronide (GEM-1-O-gluc), a major metabolite of the antihyperlipidemic drug gemfibrozil, is a mechanism-based inhibitor of P450 2C8 in vitro, and this irreversible inactivation may lead to clinical drug-drug interactions between gemfibrozil and other P450 2C8 substrates. In light of this in vitro finding and the observation that the glucuronide conjugate does not contain any obvious structural alerts, the current study was conducted to determine the potential site of GEM-1-O-gluc bioactivation and the subsequent mechanism of P450 2C8 inhibition (i.e., modification of apoprotein or heme). LC/MS analysis of a reaction mixture containing recombinant P450 2C8 and GEM-1-O-gluc revealed that the substrate was covalently linked to the heme prosthetic heme group during catalysis. A combination of mass spectrometry and deuterium isotope effects revealed that a benzylic carbon on the 2',5'-dimethylphenoxy group of GEM-1-O-gluc was covalently bound to the heme of P450 2C8. The regiospecificity of substrate addition to the heme group was not confirmed experimentally, but computational modeling experiments indicated that the gamma-meso position was the most likely site of modification. The metabolite profile, which consisted of two benzyl alcohol metabolites and a 4'-hydroxy-GEM-1-O-gluc metabolite, indicated that oxidation of GEM-1-O-gluc was limited to the 2',5'-dimethylphenoxy group. These results are consistent with an inactivation mechanism wherein GEM-1-O-gluc is oxidized to a benzyl radical intermediate, which evades oxygen rebound, and adds to the gamma-meso position of heme. Mechanism-based inhibition of P450 2C8 can be rationalized by the formation of the GEM-1-O-gluc-heme adduct and the consequential restriction of additional substrate access to the catalytic iron center.

  4. Studies on the interactions between drugs and estrogen: analytical method for prediction system of gynecomastia induced by drugs on the inhibitory metabolism of estradiol using Escherichia coli coexpressing human CYP3A4 with human NADPH-cytochrome P450 reductase.

    PubMed

    Satoh, T; Fujita, K I; Munakata, H; Itoh, S; Nakamura, K; Kamataki, T; Itoh, S; Yoshizawa, I

    2000-11-15

    To establish a prediction system for drug-induced gynecomastia in clinical fields, a model reaction system was developed to explain numerically this side effect. The principle is based on the assumption that 50% inhibition concentration (IC(50)) of drugs on the in vitro metabolism of estradiol (E2) to its major product 2-hydroxyestradiol (2-OH-E2) can be regarded as the index for achieving this purpose. By using human cytochrome P450s coexpressed with human NADPH-cytochrome P450 reductase in Escherichia coli as the enzyme, the reaction was examined. Among the nine enzymes (CYP1A1, 1A2, 2A6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4) tested, CYP3A4 having a V(max)/K(m) (ml/min/nmol P450) value of 0.32 for production of 2-OH-E2 was shown to be the most suitable enzyme as the reagent. The inhibitory effects of ketoconazole, cyclosporin A, and cimetidine toward the 2-hydroxylation of E2 catalyzed by CYP3A4 were obtained, and their IC(50) values were 7 nM, 64 nM, and 290 microM, respectively. The present results suggest that IC(50) values thus obtained can be substituted as the prediction index for gynecomastia induced by drugs, considering the patients' individual information. Copyright 2000 Academic Press.

  5. Improved Predictions of Drug-Drug Interactions Mediated by Time-Dependent Inhibition of CYP3A.

    PubMed

    Yadav, Jaydeep; Korzekwa, Ken; Nagar, Swati

    2018-05-07

    Time-dependent inactivation (TDI) of cytochrome P450s (CYPs) is a leading cause of clinical drug-drug interactions (DDIs). Current methods tend to overpredict DDIs. In this study, a numerical approach was used to model complex CYP3A TDI in human-liver microsomes. The inhibitors evaluated included troleandomycin (TAO), erythromycin (ERY), verapamil (VER), and diltiazem (DTZ) along with the primary metabolites N-demethyl erythromycin (NDE), norverapamil (NV), and N-desmethyl diltiazem (NDD). The complexities incorporated into the models included multiple-binding kinetics, quasi-irreversible inactivation, sequential metabolism, inhibitor depletion, and membrane partitioning. The resulting inactivation parameters were incorporated into static in vitro-in vivo correlation (IVIVC) models to predict clinical DDIs. For 77 clinically observed DDIs, with a hepatic-CYP3A-synthesis-rate constant of 0.000 146 min -1 , the average fold difference between the observed and predicted DDIs was 3.17 for the standard replot method and 1.45 for the numerical method. Similar results were obtained using a synthesis-rate constant of 0.000 32 min -1 . These results suggest that numerical methods can successfully model complex in vitro TDI kinetics and that the resulting DDI predictions are more accurate than those obtained with the standard replot approach.

  6. CHARACTERIZATION OF THE ALKANE-INDUCIBLE CYTOCHROME P450 (P450ALK) GENE FROM THE YEAST CANDIDA TROPICALIS: IDENTIFICATION OF A NEW P450 FAMILY

    EPA Science Inventory

    The P450alk gene, which is inducible by the assimilation of alkane in Candida tropicalis, was sequenced and characterized. Structural features described in promoter and terminator regions of Saccharomyces yeast genes are present in the P450alk gene and some particular structures ...

  7. Cytochrome P450s and molecular epidemiology

    NASA Astrophysics Data System (ADS)

    Gonzalez, Frank J.; Gelboin, Harry V.

    1993-03-01

    Cytochrome P450 (P450) represent a superfamily of heme-containing monooxygenases that are found throughout the animal and plant kingdoms and in many microorganisms. A number of these enzymes are involved in biosynthetic pathways of steroid synthesis but in mammals the vast majority of P450s function to metabolize foreign chemicals or xenobiotics. In the classical phase I reactions on the latter, a membrane-bound P450 will hydroxylate a compound, usually hydrophobic in nature, and the hydroxyl group will serve as a substrate for the various transferases or phase II enzymes that attach hydrophilic substituents such as glutathione, sulfate or glucuronic acid. Some chemicals, however, are metabolically-activated by P450s to electrophiles capable of reacting with cellular macromolecules. The cellular concentrations of the chemical and P450, reactivity of the active metabolite with nucleic acid and the repairability of the resultant adducts, in addition to the nature of the cell type, likely determines whether a chemical will be toxic and kill the cell or will transform the cell. Immunocorrelative and cDNA-directed expression have been used to define the substrate specificities of numerous human P450s. Levels of expression of different human P450 forms have been measured by both in vivo and in vitro methodologies leading to the realization that a large degree of interindividual differences occur in P450 expression. Reliable procedures for measuring P450 expression in healthy and diseased subjects will lead to prospective and case- cohort studies to determine whether interindividual differences in levels of P450 are associated with susceptibility or resistance to environmentally-based disease.

  8. Evaluation of Memory Enhancing Clinically Available Standardized Extract of Bacopa monniera on P-Glycoprotein and Cytochrome P450 3A in Sprague-Dawley Rats

    PubMed Central

    Singh, Rajbir; Panduri, Jagadeesh; Kumar, Devendra; Kumar, Deepak; Chandsana, Hardik; Ramakrishna, Rachumallu; Bhatta, Rabi Sankar

    2013-01-01

    Bacopa monniera is a traditional Ayurvedic herbal medicine used to treat various mental ailments from ancient times. Recently, chemically standardized alcoholic extract of Bacopa monniera (BM) has been developed and currently available as over the counter herbal remedy for memory enhancement in children and adults. However, the consumption of herbal drugs has been reported to alter the expression of drug metabolizing enzymes and membrane transporters. Present study in male Sprague-Dawley rat was performed to evaluate the effect of memory enhancing standardized extract of BM on hepatic and intestinal cytochrome P450 3A and P-glycoprotein expression and activity. The BM (31 mg/kg/day) was orally administered for one week in BM pre-treated group while the control group received the same amount of vehicle for the same time period. The BM treatment decreased the cytochrome P450 3A (CYP3A) mediated testosterone 6β-hydroxylation activity of the liver and intestine by 2 and 1.5 fold, respectively compared to vehicle treated control. Similarly pretreatment with BM extract decreased the expression of intestinal P-glycoprotein (Pgp) as confirmed by Western blot analysis but did not alter the expression of hepatic Pgp. To investigate whether this BM pretreatment mediated decrease in activity of CYP3A and Pgp would account for the alteration of respective substrate or not, pharmacokinetic study with carbamazepine and digoxin was performed in BM pre-treated rats and vehicle treated rats. Carbamazepine and digoxin were used as CYP3A and Pgp probe drugs, respectively. Significant increase in AUC and Cmax of carbamazepine (4 and 1.8 fold) and digoxin (1.3 and 1.2 fold), respectively following the BM pre-treatment confirmed the down regulation of CYP3A and Pgp. PMID:24015255

  9. Evaluation of memory enhancing clinically available standardized extract of Bacopa monniera on P-glycoprotein and cytochrome P450 3A in Sprague-Dawley rats.

    PubMed

    Singh, Rajbir; Panduri, Jagadeesh; Kumar, Devendra; Kumar, Deepak; Chandsana, Hardik; Ramakrishna, Rachumallu; Bhatta, Rabi Sankar

    2013-01-01

    Bacopa monniera is a traditional Ayurvedic herbal medicine used to treat various mental ailments from ancient times. Recently, chemically standardized alcoholic extract of Bacopa monniera (BM) has been developed and currently available as over the counter herbal remedy for memory enhancement in children and adults. However, the consumption of herbal drugs has been reported to alter the expression of drug metabolizing enzymes and membrane transporters. Present study in male Sprague-Dawley rat was performed to evaluate the effect of memory enhancing standardized extract of BM on hepatic and intestinal cytochrome P450 3A and P-glycoprotein expression and activity. The BM (31 mg/kg/day) was orally administered for one week in BM pre-treated group while the control group received the same amount of vehicle for the same time period. The BM treatment decreased the cytochrome P450 3A (CYP3A) mediated testosterone 6β-hydroxylation activity of the liver and intestine by 2 and 1.5 fold, respectively compared to vehicle treated control. Similarly pretreatment with BM extract decreased the expression of intestinal P-glycoprotein (Pgp) as confirmed by Western blot analysis but did not alter the expression of hepatic Pgp. To investigate whether this BM pretreatment mediated decrease in activity of CYP3A and Pgp would account for the alteration of respective substrate or not, pharmacokinetic study with carbamazepine and digoxin was performed in BM pre-treated rats and vehicle treated rats. Carbamazepine and digoxin were used as CYP3A and Pgp probe drugs, respectively. Significant increase in AUC and Cmax of carbamazepine (4 and 1.8 fold) and digoxin (1.3 and 1.2 fold), respectively following the BM pre-treatment confirmed the down regulation of CYP3A and Pgp.

  10. Cytochrome P450BM-3 reduces aldehydes to alcohols through a direct hydride transfer

    SciTech Connect

    Kaspera, Ruediger; Sahele, Tariku; Lakatos, Kyle

    Highlights: Black-Right-Pointing-Pointer Cytochrome P450BM-3 reduced aldehydes to alcohols efficiently (k{sub cat} {approx} 25 min{sup -1}). Black-Right-Pointing-Pointer Reduction is a direct hydride transfer from R-NADP{sup 2}H to the carbonyl moiety. Black-Right-Pointing-Pointer P450 domain variants enhance reduction through potential allosteric/redox interactions. Black-Right-Pointing-Pointer Novel reaction will have implications for metabolism of xenobiotics. -- Abstract: Cytochrome P450BM-3 catalyzed the reduction of lipophilic aldehydes to alcohols efficiently. A k{sub cat} of {approx}25 min{sup -1} was obtained for the reduction of methoxy benzaldehyde with wild type P450BM-3 protein which was higher than in the isolated reductase domain (BMR) alone and increased in specific P450-domain variants. Themore » reduction was caused by a direct hydride transfer from preferentially R-NADP{sup 2}H to the carbonyl moiety of the substrate. Weak substrate-P450-binding of the aldehyde, turnover with the reductase domain alone, a deuterium incorporation in the product from NADP{sup 2}H but not D{sub 2}O, and no inhibition by imidazole suggests the reductase domain of P450BM-3 as the potential catalytic site. However, increased aldehyde reduction by P450 domain variants (P450BM-3 F87A T268A) may involve allosteric or redox mechanistic interactions between heme and reductase domains. This is a novel reduction of aldehydes by P450BM-3 involving a direct hydride transfer and could have implications for the metabolism of endogenous substrates or xenobiotics.« less

  11. Inhibitory effects of herbal constituents on P-glycoprotein in vitro and in vivo: herb-drug interactions mediated via P-gp.

    PubMed

    Li, Xue; Hu, Jinping; Wang, Baolian; Sheng, Li; Liu, Zhihao; Yang, Shuang; Li, Yan

    2014-03-01

    Modulation of drug transporters via herbal medicines which have been widely used in combination with conventional prescription drugs may result in herb-drug interactions in clinical practice. The present study was designed to investigate the inhibitory effects of 50 major herbal constituents on P-glycoprotein (P-gp) in vitro and in vivo as well as related inhibitory mechanisms. Among these herbal medicines, four constituents, including emodin, 18β-glycyrrhetic acid (18β-GA), dehydroandrographolide (DAG), and 20(S)-ginsenoside F₁ [20(S)-GF₁] exhibited significant inhibition (>50%) on P-gp in MDR1-MDCKII and Caco-2 cells. Emodin was the strongest inhibitor of P-gp (IC₅₀=9.42 μM), followed by 18β-GA (IC₅₀=21.78 μM), 20(S)-GF₁ (IC₅₀=76.08 μM) and DAG (IC₅₀=77.80 μM). P-gp ATPase activity, which was used to evaluate the affinity of substrates to P-gp, was stimulated by emodin and DAG with Km and Vmax values of 48.61, 29.09 μM and 71.29, 38.45 nmol/min/mg protein, respectively. However, 18β-GA and 20(S)-GF₁ exhibited significant inhibition on both basal and verapamil-stimulated P-gp ATPase activities at high concentration. Molecular docking analysis (CDOCKER) further elucidated the mechanism for structure-inhibition relationships of herbal constituents with P-gp. When digoxin was co-administered to male SD rats with emodin or 18β-GA, the AUC(₀₋t) and Cmax of digoxin were increased by approximately 51% and 58%, respectively. Furthermore, 18β-GA, DAG, 20(S)-GF₁ and Rh₁ at 10 μM significantly inhibited CYP3A4/5 activity, while emodin activated the metabolism of midazolam in human liver microsomes. In conclusion, four herbal constituents demonstrated inhibition of P-gp to specific extents in vitro and in vivo. Taken together, our findings provided the basis for the reliable assessment of the potential risks of herb-drug interactions in humans. Copyright © 2014 Elsevier Inc. All rights reserved.

  12. Drug & Gene Interaction Risk Analysis With & Without Genetic Testing Among Patients Undergoing MTM

    ClinicalTrials.gov

    2017-02-22

    Cytochrome P450 CYP2D6 Enzyme Deficiency; Poor Metabolizer Due to Cytochrome P450 CYP2D6 Variant; Ultrarapid Metabolizer Due to Cytochrome P450 CYP2D6 Variant; Extensive Metabolizer Due to Cytochrome P450 CYP2D6 Variant; Cytochrome P450 CYP2C9 Enzyme Deficiency; Cytochrome P450 CYP2C19 Enzyme Deficiency; Drug Metabolism, Poor, CYP2D6-RELATED; Drug Metabolism, Poor, CYP2C19-RELATED; CYP2D6 Polymorphism

  13. Phytomonitoring and phytoremediation of agrochemicals and related compounds based on recombinant cytochrome P450s and aryl hydrocarbon receptors (AhRs).

    PubMed

    Shimazu, Sayuri; Inui, Hideyuki; Ohkawa, Hideo

    2011-04-13

    Molecular mechanisms of metabolism and modes of actions of agrochemicals and related compounds are important for understanding selective toxicity, biodegradability, and monitoring of biological effects on nontarget organisms. It is well-known that in mammals, cytochrome P450 (P450 or CYP) monooxygenases metabolize lipophilic foreign compounds. These P450 species are inducible, and both CYP1A1 and CYP1A2 are induced by aryl hydrocarbon receptor (AhR) combined with a ligand. Gene engineering of P450 and NADPH cytochrome P450 oxidoreductase (P450 reductase) was established for bioconversion. Also, gene modification of AhRs was developed for recombinant AhR-mediated β-glucronidase (GUS) reporter assay of AhR ligands. Recombinant P450 genes were transformed into plants for phytoremediation, and recombinant AhR-mediated GUS reporter gene expression systems were each transformed into plants for phytomonitoring. Transgenic rice plants carrying CYP2B6 metabolized the herbicide metolachlor and remarkably reduced the residues in the plants and soils under paddy field conditions. Transgenic Arabidopsis plants carrying recombinant guinea pig (g) AhR-mediated GUS reporter genes detected PCB126 at the level of 10 ng/g soils in the presence of biosurfactants MEL-B. Both phytomonitoring and phytoremediation plants were each evaluated from the standpoint of practical uses.

  14. NADPH–Cytochrome P450 Oxidoreductase: Roles in Physiology, Pharmacology, and Toxicology

    PubMed Central

    Ding, Xinxin; Wolf, C. Roland; Porter, Todd D.; Pandey, Amit V.; Zhang, Qing-Yu; Gu, Jun; Finn, Robert D.; Ronseaux, Sebastien; McLaughlin, Lesley A.; Henderson, Colin J.; Zou, Ling; Flück, Christa E.

    2013-01-01

    This is a report on a symposium sponsored by the American Society for Pharmacology and Experimental Therapeutics and held at the Experimental Biology 2012 meeting in San Diego, California, on April 25, 2012. The symposium speakers summarized and critically evaluated our current understanding of the physiologic, pharmacological, and toxicological roles of NADPH–cytochrome P450 oxidoreductase (POR), a flavoprotein involved in electron transfer to microsomal cytochromes P450 (P450), cytochrome b5, squalene mono-oxygenase, and heme oxygenase. Considerable insight has been derived from the development and characterization of mouse models with conditional Por deletion in particular tissues or partial suppression of POR expression in all tissues. Additional mouse models with global or conditional hepatic deletion of cytochrome b5 are helping to clarify the P450 isoform- and substrate-specific influences of cytochrome b5 on P450 electron transfer and catalytic function. This symposium also considered studies using siRNA to suppress POR expression in a hepatoma cell–culture model to explore the basis of the hepatic lipidosis phenotype observed in mice with conditional deletion of Por in liver. The symposium concluded with a strong translational perspective, relating the basic science of human POR structure and function to the impacts of POR genetic variation on human drug and steroid metabolism. PMID:23086197

  15. Application of Physiologically Based Pharmacokinetic Modeling in Understanding Bosutinib Drug-Drug Interactions: Importance of Intestinal P-Glycoprotein.

    PubMed

    Yamazaki, Shinji; Loi, Cho-Ming; Kimoto, Emi; Costales, Chester; Varma, Manthena V

    2018-05-08

    Bosutinib is an orally available Src/Abl tyrosine kinase inhibitor indicated for the treatment of patients with Ph+ chronic myelogenous leukemia at a clinically recommended dose of 500 mg once daily. Clinical results indicated that increases in bosutinib oral exposures were supra-proportional at the lower doses (50 to 200 mg) and approximately dose-proportional at the higher doses (200 to 600 mg). Bosutinib is a substrate of CYP3A4 and P-glycoprotein and exhibits pH-dependent solubility with moderate intestinal permeability. These findings led us to investigate the factors influencing the underlying pharmacokinetic mechanisms of bosutinib with physiologically-based pharmacokinetic (PBPK) models. Our primary objectives were to: 1) refine the previously developed bosutinib PBPK model based on the latest oral bioavailability data and 2) verify the refined PBPK model with P-glycoprotein kinetics based on the bosutinib drug-drug interaction (DDI) results with ketoconazole and rifampin. Additionally, the verified PBPK model was applied to predict bosutinib DDIs with dual CYP3A/P-glycoprotein inhibitors. The results indicated that 1) the refined PBPK model adequately described the observed plasma concentration-time profiles of bosutinib and 2) the verified PBPK model reasonably predicted the effects of ketoconazole and rifampin on bosutinib exposures by accounting for intestinal P-gp inhibition/induction. These results suggested that bosutinib DDI mechanism could involve not only CYP3A4-mediated metabolism but also P-glycoprotein-mediated efflux on absorption. In summary, P-glycoprotein kinetics could constitute a critical element in the PBPK models to understand the pharmacokinetic mechanism of dual CYP3A/P-glycoprotein substrates such as bosutinib exhibiting nonlinear pharmacokinetics due largely to a saturation of intestinal P-glycoprotein-mediated efflux. The American Society for Pharmacology and Experimental Therapeutics.

  16. Potential drug-drug interactions between anti-cancer agents and community pharmacy dispensed drugs.

    PubMed

    Voll, Marsha L; Yap, Kim D; Terpstra, Wim E; Crul, Mirjam

    2010-10-01

    To identify the prevalence of potential drug-drug interactions between hospital pharmacy dispensed anti-cancer agents and community pharmacy dispensed drugs. A retrospective cohort study was conducted on the haematology/oncology department of the internal medicine ward in a large teaching hospital in Amsterdam, the Netherlands. Prescription data from the last 100 patients treated with anti-cancer agents were obtained from Paracelsus, the chemotherapy prescribing system in the hospital. The community pharmacy dispensed drugs of these patients were obtained by using OZIS, a system that allows regionally linked pharmacies to call up active medication on any patient. Both medication lists were manually screened for potential drug-drug interactions by using several information sources on interactions, e.g. Pubmed, the Flockhart P450 table, Micromedex and Dutch reference books. Prevalence of potential drug-drug interactions between anti-cancer agents provided by the hospital pharmacy and drugs dispensed by the community pharmacy. Ninety-one patients were included in the study. A total of 31 potential drug-drug interactions were found in 16 patients, of which 15 interactions were clinically relevant and would have required an intervention. Of these interactions 1 had a level of severity ≥ D, meaning the potential drug-drug interaction could lead to long lasting or permanent damage, or even death. The majority of the interactions requiring an intervention (67%) had a considerable level of evidence (≥ 2) and were based on well-documented case reports or controlled interaction studies. Most of the potential drug-drug interactions involved the antiretroviral drugs (40%), proton pump inhibitors (20%) and antibiotics (20%). The anti-cancer drug most involved in the drug-drug interactions is methotrexate (33%). This study reveals a high prevalence of potential drug-drug interactions between anti-cancer agents provided by the hospital pharmacy and drugs dispensed by the

  17. Effects of aging and rifampicin pretreatment on the pharmacokinetics of human cytochrome P450 probes caffeine, warfarin, omeprazole, metoprolol and midazolam in common marmosets genotyped for cytochrome P450 2C19.

    PubMed

    Toda, Akiko; Uehara, Shotaro; Inoue, Takashi; Utoh, Masahiro; Kusama, Takashi; Shimizu, Makiko; Uno, Yasuhiro; Mogi, Masayuki; Sasaki, Erika; Yamazaki, Hiroshi

    2018-07-01

    1. The pharmacokinetics were investigated for human cytochrome P450 probes after single intravenous and oral administrations of 0.20 and 1.0 mg/kg, respectively, of caffeine, warfarin, omeprazole, metoprolol and midazolam to aged (10-14 years old, n = 4) or rifampicin-treated/young (3 years old, n = 3) male common marmosets all genotyped as heterozygous for a cytochrome P450 2C19 variant. 2. Slopes of the plasma concentration-time curves after intravenous administration of warfarin and midazolam were slightly, but significantly (two-way analysis of variance), decreased in aged marmosets compared with young marmosets. The mean hepatic clearances determined by in silico fitting for individual pharmacokinetic models of warfarin and midazolam in the aged group were, respectively, 23% and 56% smaller than those for the young group. 3. Significantly enhanced plasma clearances of caffeine, warfarin, omeprazole and midazolam were evident in young marmosets pretreated with rifampicin (25 mg/kg daily for 4 days). Two- to three-fold increases in hepatic intrinsic clearance values were observed in the individual pharmacokinetic models. 4. The in vivo dispositions of multiple simultaneously administered drugs in old, young and P450-enzyme-induced marmosets were elucidated. The results suggest that common marmosets could be experimental models for aged, induced or polymorphic P450 enzymes in P450-dependent drug metabolism studies.

  18. In vitro metabolism of an estrogen-related receptor γ modulator, GSK5182, by human liver microsomes and recombinant cytochrome P450s.

    PubMed

    Joo, Jeongmin; Wu, Zhexue; Lee, Boram; Shon, Jong Cheol; Lee, Taeho; Lee, In-Kyu; Sim, Taebo; Kim, Kyung-Hee; Kim, Nam Doo; Kim, Seong Heon; Liu, Kwang-Hyeon

    2015-04-01

    GSK5182 (4-[(Z)-1-[4-(2-dimethylaminoethyloxy)phenyl]-hydroxy-2-phenylpent-1-enyl]phenol) is a specific inverse agonist for estrogen-related receptor γ, a member of the orphan nuclear receptor family that has important functions in development and homeostasis. This study was performed to elucidate the metabolites of GSK5182 and to characterize the enzymes involved in its metabolism. Incubation of human liver microsomes with GSK5182 in the presence of NADPH resulted in the formation of three metabolites, M1, M2 and M3. M1 and M3 were identified as N-desmethyl-GSK5182 and GSK5182 N-oxide, respectively, on the basis of liquid chromatography-tandem mass spectrometric (LC-MS/MS) analysis. M2 was suggested to be hydroxy-GSK5182 through interpretation of its MS/MS fragmentation pattern. In addition, the specific cytochrome P450 (P450) and flavin-containing monooxygenase (FMO) isoforms responsible for GSK5182 oxidation to the three metabolites were identified using a combination of correlation analysis, chemical inhibition in human liver microsomes and metabolism by expressed recombinant P450 and FMO isoforms. GSK5182 N-demethylation and hydroxylation is mainly mediated by CYP3A4, whereas FMO1 and FMO3 contribute to the formation of GSK5182 N-oxide from GSK5182. The present data will be useful for understanding the pharmacokinetics and drug interactions of GSK5182 in vivo. Copyright © 2014 John Wiley & Sons, Ltd.

  19. Co-treatment with grapefruit juice inhibits while chronic administration activates intestinal P-glycoprotein-mediated drug efflux.

    PubMed

    Panchagnula, R; Bansal, T; Varma, M V S; Kaul, C L

    2005-12-01

    P-Glycoprotein (P-gp) mediated efflux is recognized as a significant biochemical barrier affecting oral absorption for a number of drugs. Various conflicting reports have been published regarding the effects of grapefruit juice (GFJ) on P-gp-mediated drug efflux, in which GFJ has been shown both to inhibit and activate it. Hence, the present study adopted a two-way approach, involving both co-treatment and chronic administration. Bi-directional transport of paclitaxel (PCL) was carried out in the absence and presence of GFJ extract, in rat everted ileum sac. Further, the effect of chronic administration of GFJ to rats was characterized by permeability studies with indinavir (INDI). Co-treatment of GFJ extract at 100% concentration reduced the asymmetric transport of PCL (efflux ratio = 20.8) by increasing absorptive (A --> B) transport by 921% and reducing secretory (B --> A) transport by 41%. Further, GFJ showed a concentration dependent effect on PCL permeability. Imipramine, a passive permeability marker with absorptive permeability of 15.33 +/- 4.26 x 10(-6) cm/s showed no asymmetric transport and also no significant (P < 0.05) change in permeability in the presence of GFJ. Chronic administration of GFJ resulted in a significant decrease in absorptive transport of indinavir, which was even greater than that produced by rifampicin pretreatment. No change in permeability of propranolol, a passive permeability marker, was observed. Further, the decrease in absorptive transport of INDI was reversed by the P-gp inhibitor verapamil. In conclusion, GFJ extract inhibited P-gp-mediated efflux in co-treatment, whereas chronic administration led to increased levels of P-gp expression, thus having a profound effect on intestinal absorption and GFJ-drug interactions in vivo.

  20. Structural Characterization and Ligand/Inhibitor Identification Provide Functional Insights into the Mycobacterium tuberculosis Cytochrome P450 CYP126A1*

    PubMed Central

    Chenge, Jude T.; Duyet, Le Van; Swami, Shalini; McLean, Kirsty J.; Kavanagh, Madeline E.; Coyne, Anthony G.; Rigby, Stephen E. J.; Cheesman, Myles R.; Girvan, Hazel M.; Levy, Colin W.; Rupp, Bernd; von Kries, Jens P.; Abell, Chris; Leys, David; Munro, Andrew W.

    2017-01-01

    The Mycobacterium tuberculosis H37Rv genome encodes 20 cytochromes P450, including P450s crucial to infection and bacterial viability. Many M. tuberculosis P450s remain uncharacterized, suggesting that their further analysis may provide new insights into M. tuberculosis metabolic processes and new targets for drug discovery. CYP126A1 is representative of a P450 family widely distributed in mycobacteria and other bacteria. Here we explore the biochemical and structural properties of CYP126A1, including its interactions with new chemical ligands. A survey of azole antifungal drugs showed that CYP126A1 is inhibited strongly by azoles containing an imidazole ring but not by those tested containing a triazole ring. To further explore the molecular preferences of CYP126A1 and search for probes of enzyme function, we conducted a high throughput screen. Compounds containing three or more ring structures dominated the screening hits, including nitroaromatic compounds that induce substrate-like shifts in the heme spectrum of CYP126A1. Spectroelectrochemical measurements revealed a 155-mV increase in heme iron potential when bound to one of the newly identified nitroaromatic drugs. CYP126A1 dimers were observed in crystal structures of ligand-free CYP126A1 and for CYP126A1 bound to compounds discovered in the screen. However, ketoconazole binds in an orientation that disrupts the BC-loop regions at the P450 dimer interface and results in a CYP126A1 monomeric crystal form. Structural data also reveal that nitroaromatic ligands “moonlight” as substrates by displacing the CYP126A1 distal water but inhibit enzyme activity. The relatively polar active site of CYP126A1 distinguishes it from its most closely related sterol-binding P450s in M. tuberculosis, suggesting that further investigations will reveal its diverse substrate selectivity. PMID:27932461

  1. Drug Interactions with Clinafloxacin

    PubMed Central

    Randinitis, Edward J.; Alvey, Christine W.; Koup, Jeffery R.; Rausch, George; Abel, Robert; Bron, Nicola J.; Hounslow, Neil J.; Vassos, Artemios B.; Sedman, Allen J.

    2001-01-01

    Many fluoroquinolone antibiotics are inhibitors of cytochrome P450 enzyme systems and may produce potentially important drug interactions when administered with other drugs. Studies were conducted to determine the effect of clinafloxacin on the pharmacokinetics of theophylline, caffeine, warfarin, and phenytoin, as well as the effect of phenytoin on the pharmacokinetics of clinafloxacin. Concomitant administration of 200 or 400 mg of clinafloxacin reduces mean theophylline clearance by approximately 50 and 70%, respectively, and reduces mean caffeine clearance by 84%. (R)-Warfarin concentrations in plasma during clinafloxacin administration are 32% higher and (S)-warfarin concentrations do not change during clinafloxacin treatment. An observed late pharmacodynamic effect was most likely due to gut flora changes. Phenytoin has no effect on clinafloxacin pharmacokinetics, while phenytoin clearance is 15% lower during clinafloxacin administration. PMID:11502527

  2. Cytochrome P450-derived eicosanoids: the neglected pathway in cancer

    PubMed Central

    Kaipainen, Arja; Greene, Emily R.; Huang, Sui

    2010-01-01

    Endogenously produced lipid autacoids are locally acting small molecule mediators that play a central role in the regulation of inflammation and tissue homeostasis. A well-studied group of autacoids are the products of arachidonic acid metabolism, among which the prostaglandins and leukotrienes are the best known. They are generated by two pathways controlled by the enzyme systems cyclooxygenase and lipoxygenase, respectively. However, arachidonic acid is also substrate for a third enzymatic pathway, the cytochrome P450 (CYP) system. This third eicosanoid pathway consists of two main branches: ω-hydroxylases convert arachidonic acid to hydroxyeicosatetraenoic acids (HETEs) and epoxygenases convert it to epoxyeicosatrienoic acids (EETs). This third CYP pathway was originally studied in conjunction with inflammatory and cardiovascular disease. Arachidonic acid and its metabolites have recently stimulated great interest in cancer biology; but, unlike prostaglandins and leukotrienes the link between cytochome P450 metabolites and cancer has received little attention. In this review, the emerging role in cancer of cytochrome P450 metabolites, notably 20-HETE and EETs, are discussed. PMID:20941528

  3. Marmoset Cytochrome P450 3A4 Ortholog Expressed in Liver and Small-Intestine Tissues Efficiently Metabolizes Midazolam, Alprazolam, Nifedipine, and Testosterone.

    PubMed

    Uehara, Shotaro; Uno, Yasuhiro; Nakanishi, Kazuyuki; Ishii, Sakura; Inoue, Takashi; Sasaki, Erika; Yamazaki, Hiroshi

    2017-05-01

    Common marmosets ( Callithrix jacchus ), small New World primates, are increasingly attracting attention as potentially useful animal models for drug development. However, characterization of cytochrome P450 (P450) 3A enzymes involved in the metabolism of a wide variety of drugs has not investigated in marmosets. In this study, sequence homology, tissue distribution, and enzymatic properties of marmoset P450 3A4 ortholog, 3A5 ortholog, and 3A90 were investigated. Marmoset P450 3A forms exhibited high amino acid sequence identities (88-90%) to the human and cynomolgus monkey P450 3A orthologs and evolutionary closeness to human and cynomolgus monkey P450 3A orthologs compared with other P450 3A enzymes. Among the five marmoset tissues examined, P450 3A4 ortholog mRNA was abundant in livers and small intestines where P450 3A4 ortholog proteins were immunologically detected. Three marmoset P450 3A proteins heterologously expressed in Escherichia coli membranes catalyzed midazolam 1'- and 4-hydroxylation, alprazolam 4-hydroxylation, nifedipine oxidation, and testosterone 6 β -hydroxylation, similar to cynomolgus monkey and human P450 3A enzymes. Among the marmoset P450 3A enzymes, P450 3A4 ortholog effectively catalyzed midazolam 1'-hydroxylation, comparable to microsomes from marmoset livers and small intestines. Correlation analyses with 23 individual marmoset liver microsomes suggested contributions of P450 3A enzymes to 1'-hydroxylation of both midazolam (human P450 3A probe) and bufuralol (human P450 2D6 probe), similar to cynomolgus monkey P450 3A enzymes. These results indicated that marmoset P450 3A forms had functional characteristics roughly similar to cynomolgus monkeys and humans in terms of tissue expression patterns and catalytic activities, suggesting marmosets as suitable animal models for P450 3A-dependent drug metabolism. Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.

  4. Muscle symptoms in statin users, associations with cytochrome P450, and membrane transporter inhibitor use: a subanalysis of the USAGE study.

    PubMed

    Ito, Matthew K; Maki, Kevin C; Brinton, Eliot A; Cohen, Jerome D; Jacobson, Terry A

    2014-01-01

    Drug interactions have been identified as a risk factor for muscle-related side effects in statin users. The aim was to assess whether use of medications that inhibit cytochrome P450 (CYP450) isozymes, organic anion transporting polypeptide 1B1 (OATP1B1), or P-glycoprotein (P-gp) are associated with muscle-related symptoms among current and former statin users. Persons (n = 10,138) from the Understanding Statin Use in America and Gaps in Education (USAGE) internet survey were categorized about whether they ever reported new or worsening muscle pain while taking a statin (n = 2935) or ever stopped a statin because of muscle pain (n = 1516). Univariate and multivariate logistic regression models were used to assess associations between use of concomitant therapies that inhibit CYP450 isozymes, OATP1B1, P-gp, or a combination and muscle-related outcomes. In multivariate analyses, concomitant use of a CYP450 inhibitor was associated with increased odds for new or worse muscle pain (odds ratio [OR] = 1.42; P < .001) or ever having stopped a statin because of muscle pain (OR = 1.28; P = .037). Concomitant use of medication known to inhibit both OATP1B1 and P-gp was also associated with increased odds (OR = 1.80; P = .030) of ever having stopped a statin because of muscle pain. Concomitant use of medication(s) that inhibit statin metabolism was associated with increased odds of new or worse muscle pain while taking a statin and having previously stopped a statin because of muscle symptoms. These data emphasize the importance of enhancing the capabilities of clinicians and health systems for identifying and reducing statin drug interactions. Copyright © 2014 National Lipid Association. All rights reserved.

  5. The Interactions of P-Glycoprotein with Antimalarial Drugs, Including Substrate Affinity, Inhibition and Regulation

    PubMed Central

    Senarathna, S M D K Ganga; Page-Sharp, Madhu; Crowe, Andrew

    2016-01-01

    The combination of passive drug permeability, affinity for uptake and efflux transporters as well as gastrointestinal metabolism defines net drug absorption. Efflux mechanisms are often overlooked when examining the absorption phase of drug bioavailability. Knowing the affinity of antimalarials for efflux transporters such as P-glycoprotein (P-gp) may assist in the determination of drug absorption and pharmacokinetic drug interactions during oral absorption in drug combination therapies. Concurrent administration of P-gp inhibitors and P-gp substrate drugs may also result in alterations in the bioavailability of some antimalarials. In-vitro Caco-2 cell monolayers were used here as a model for potential drug absorption related problems and P-gp mediated transport of drugs. Artemisone had the highest permeability at around 50 x 10−6 cm/sec, followed by amodiaquine around 20 x 10−6 cm/sec; both mefloquine and artesunate were around 10 x 10−6 cm/sec. Methylene blue was between 2 and 6 x 10−6 cm/sec depending on the direction of transport. This 3 fold difference was able to be halved by use of P-gp inhibition. MRP inhibition also assisted the consolidation of the methylene blue transport. Mefloquine was shown to be a P-gp inhibitor affecting our P-gp substrate, Rhodamine 123, although none of the other drugs impacted upon rhodamine123 transport rates. In conclusion, mefloquine is a P-gp inhibitor and methylene blue is a partial substrate; methylene blue may have increased absorption if co-administered with such P-gp inhibitors. An upregulation of P-gp was observed when artemisone and dihydroartemisinin were co-incubated with mefloquine and amodiaquine. PMID:27045516

  6. Biotechnological synthesis of drug metabolites using human cytochrome P450 isozymes heterologously expressed in fission yeast.

    PubMed

    Peters, Frank T; Bureik, Matthias; Maurer, Hans H

    2009-07-01

    Cytochrome P450 mono-oxygenases (CYPs) are the major enzymes involved in the metabolism of drugs and poisons in humans. The variation of their activity - due to genetic polymorphisms or enzyme inhibition/induction - potentially increases the risk of side effects or toxicity. Studies on CYP-dependent metabolism are important in drug-development or toxicity studies. Reference standards of drug metabolites required for such studies, especially in the context of metabolites in safety testing (MIST), are often not commercially available and their classical chemical synthesis can be cumbersome. Recently, a biotechnological approach using human CYP isozymes heterologously expressed in fission yeast was developed for the synthesis of drug metabolites. Among other aspects, this approach has the distinct advantages that the reactions run under mild conditions and that only the final product must be isolated and characterized. This review overviews the first practical applications of this new approach and discusses the selection of substrates, metabolites and fission yeast strains as well as important aspects of incubation, product isolation and clean-up.

  7. Genomewide annotation and comparative genomics of cytochrome P450 monooxygenases (P450s) in the polypore species Bjerkandera adusta, Ganoderma sp. and Phlebia brevispora.

    PubMed

    Syed, Khajamohiddin; Nelson, David R; Riley, Robert; Yadav, Jagjit S

    2013-01-01

    Genomewide annotation of cytochrome P450 monooxygenases (P450s) in three white-rot species of the fungal order Polyporales, namely Bjerkandera adusta, Ganoderma sp. and Phlebia brevispora, revealed a large contingent of P450 genes (P450ome) in their genomes. A total of 199 P450 genes in B. adusta and 209 P450 genes each in Ganoderma sp. and P. brevispora were identified. These P450omes were classified into families and subfamilies as follows: B. adusta (39 families, 86 subfamilies), Ganoderma sp. (41 families, 105 subfamilies) and P. brevispora (42 families, 111 subfamilies). Of note, the B. adusta genome lacked the CYP505 family (P450foxy), a group of P450-CPR fusion proteins. The three polypore species revealed differential enrichment of individual P450 families in their genomes. The largest CYP families in the three genomes were CYP5144 (67 P450s), CYP5359 (46 P450s) and CYP5344 (43 P450s) in B. adusta, Ganoderma sp. and P. brevispora, respectively. Our analyses showed that tandem gene duplications led to expansions in certain P450 families. An estimated 33% (72 P450s), 28% (55 P450s) and 23% (49 P450s) of P450ome genes were duplicated in P. brevispora, B. adusta and Ganoderma sp., respectively. Family-wise comparative analysis revealed that 22 CYP families are common across the three Polypore species. Comparative P450ome analysis with Ganoderma lucidum revealed the presence of 143 orthologs and 56 paralogs in Ganoderma sp. Multiple P450s were found near the characteristic biosynthetic genes for secondary metabolites, namely polyketide synthase (PKS), non-ribosomal peptide synthetase (NRPS), terpene cyclase and terpene synthase in the three genomes, suggesting a likely role of these P450s in secondary metabolism in these Polyporales. Overall, the three species had a richer P450 diversity both in terms of the P450 genes and P450 subfamilies as compared to the model white-rot and brown-rot polypore species Phanerochaete chrysosporium and Postia placenta.

  8. Defining the in Vivo Role for cytochrome b5 in cytochrome P450 function through the conditional hepatic deletion of microsomal cytochrome b5.

    PubMed

    Finn, Robert D; McLaughlin, Lesley A; Ronseaux, Sebastien; Rosewell, Ian; Houston, J Brian; Henderson, Colin J; Wolf, C Roland

    2008-11-14

    In vitro, cytochrome b5 modulates the rate of cytochrome P450-dependent mono-oxygenation reactions. However, the role of this enzyme in determining drug pharmacokinetics in vivo and the consequential effects on drug absorption distribution, metabolism, excretion, and toxicity are unclear. In order to resolve this issue, we have carried out the conditional deletion of microsomal cytochrome b5 in the liver to create the hepatic microsomal cytochrome b5 null mouse. These mice develop and breed normally and have no overt phenotype. In vitro studies using a range of substrates for different P450 enzymes showed that in hepatic microsomal cytochrome b5 null NADH-mediated metabolism was essentially abolished for most substrates, and the NADPH-dependent metabolism of many substrates was reduced by 50-90%. This reduction in metabolism was also reflected in the in vivo elimination profiles of several drugs, including midazolam, metoprolol, and tolbutamide. In the case of chlorzoxazone, elimination was essentially unchanged. For some drugs, the pharmacokinetics were also markedly altered; for example, when administered orally, the maximum plasma concentration for midazolam was increased by 2.5-fold, and the clearance decreased by 3.6-fold in hepatic microsomal cytochrome b5 null mice. These data indicate that microsomal cytochrome b5 can play a major role in the in vivo metabolism of certain drugs and chemicals but in a P450- and substrate-dependent manner.

  9. Identification of the rat liver cytochrome P450 enzymes involved in the metabolism of the calcium channel blocker dipfluzine hydrochloride.

    PubMed

    Guo, Wei; Shi, Xiaowei; Wang, Wei; Zhang, Weili; Li, Junxia

    2014-11-01

    This study aimed to identify the specific cytochrome P450 (CYP450) enzymes involved in the metabolism of dipfluzine hydrochloride using the combination of a chemical inhibition study, a correlation analysis and a panel of recombinant rat CYP450 enzymes. The incubation of Dip with rat liver microsomes yielded four metabolites, which were identified by liquid chromatography-coupled tandem mass spectrometry (LC/MS/MS). The results from the assays involving eight selective inhibitors indicated that CYP3A and CYP2A1 contributed most to the metabolism of Dip, followed by CYP2C11, CYP2E1 and CYP1A2; however, CYP2B1, CYP2C6 and CYP2D1 did not contribute to the formation of the metabolites. The results of the correlation analysis and the assays involving the recombinant CYP450 enzymes further confirmed the above results and concluded that CYP3A2 contributed more than CYP3A1. The results will be valuable in understanding drug-drug interactions when Dip is coadministered with other drugs. Copyright © 2014 Elsevier B.V. All rights reserved.

  10. Indolealkylamines: biotransformations and potential drug-drug interactions.

    PubMed

    Yu, Ai-Ming

    2008-06-01

    Indolealkylamine (IAA) drugs are 5-hydroxytryptamine (5-HT or serotonin) analogs that mainly act on the serotonin system. Some IAAs are clinically utilized for antimigraine therapy, whereas other substances are notable as drugs of abuse. In the clinical evaluation of antimigraine triptan drugs, studies on their biotransformations and pharmacokinetics would facilitate the understanding and prevention of unwanted drug-drug interactions (DDIs). A stable, principal metabolite of an IAA drug of abuse could serve as a useful biomarker in assessing intoxication of the IAA substance. Studies on the metabolism of IAA drugs of abuse including lysergic acid amides, tryptamine derivatives and beta-carbolines are therefore emerging. An important role for polymorphic cytochrome P450 2D6 (CYP2D6) in the metabolism of IAA drugs of abuse has been revealed by recent studies, suggesting that variations in IAA metabolism, pharmaco- or toxicokinetics and dynamics can arise from distinct CYP2D6 status, and CYP2D6 polymorphism may represent an additional risk factor in the use of these IAA drugs. Furthermore, DDIs with IAA agents could occur additively at the pharmaco/toxicokinetic and dynamic levels, leading to severe or even fatal serotonin toxicity. In this review, the metabolism and potential DDIs of these therapeutic and abused IAA drugs are described.

  11. In Silico Prediction of Cytochrome P450-Mediated Biotransformations of Xenobiotics: A Case Study of Epoxidation.

    PubMed

    Zhang, Jing; Ji, Li; Liu, Weiping

    2015-08-17

    Predicting the biotransformation of xenobiotics is important in toxicology; however, as more compounds are synthesized than can be investigated experimentally, powerful computational methods are urgently needed to prescreen potentially useful candidates. Cytochrome P450 enzymes (P450s) are the major enzymes involved in xenobiotic metabolism, and many substances are bioactivated by P450s to form active compounds. An example is the conversion of olefinic substrates to epoxides, which are intermediates in the metabolic activation of many known or suspected carcinogens. We have calculated the activation energies for epoxidation by the active species of P450 enzymes (an iron-oxo porphyrin cation radical oxidant, compound I) for a diverse set of 36 olefinic substrates with state-of-the-art density functional theory (DFT) methods. Activation energies can be estimated by the computationally less demanding method of calculating the ionization potentials of the substrates, which provides a useful and simple predictive model based on the reaction mechanism; however, the preclassification of these diverse substrates into weakly polar and strongly polar groups is a prerequisite for the construction of specific predictive models with good predictability for P450 epoxidation. This approach has been supported by both internal and external validations. Furthermore, the relation between the activation energies for the regioselective epoxidation and hydroxylation reactions of P450s and experimental data has been investigated. The results show that the computational method used in this work, single-point energy calculations with the B3LYP functional including zero-point energy and solvation and dispersion corrections based on B3LYP-optimized geometries, performs well in reproducing the experimental trends of the epoxidation and hydroxylation reactions.

  12. Hypericum perforatum: pharmacokinetic, mechanism of action, tolerability, and clinical drug-drug interactions.

    PubMed

    Russo, Emilio; Scicchitano, Francesca; Whalley, Benjamin J; Mazzitello, Carmela; Ciriaco, Miriam; Esposito, Stefania; Patanè, Marinella; Upton, Roy; Pugliese, Michela; Chimirri, Serafina; Mammì, Maria; Palleria, Caterina; De Sarro, Giovambattista

    2014-05-01

    Hypericum perforatum (HP) belongs to the Hypericaceae family and is one of the oldest used and most extensively investigated medicinal herbs. The medicinal form comprises the leaves and flowering tops of which the primary ingredients of interest are naphthodianthrones, xanthones, flavonoids, phloroglucinols (e.g. hyperforin), and hypericin. Although several constituents elicit pharmacological effects that are consistent with HP's antidepressant activity, no single mechanism of action underlying these effects has thus far been found. Various clinical trials have shown that HP has a comparable antidepressant efficacy as some currently used antidepressant drugs in the treatment of mild/moderate depression. Interestingly, low-hyperforin-content preparations are effective in the treatment of depression. Moreover, HP is also used to treat certain forms of anxiety. However, HP can induce various cytochrome P450s isozymes and/or P-glycoprotein, of which many drugs are substrates and which are the main origin of HP-drug interactions. Here, we analyse the existing evidence describing the clinical consequence of HP-drug interactions. Although some of the reported interactions are based on findings from in vitro studies, the clinical importance of which remain to be demonstrated, others are based on case reports where causality can, in some cases, be determined to reveal clinically significant interactions that suggest caution, consideration, and disclosure of potential interactions prior to informed use of HP. Copyright © 2013 John Wiley & Sons, Ltd.

  13. Physiologically-Based Pharmacokinetic Modeling of Macitentan: Prediction of Drug-Drug Interactions.

    PubMed

    de Kanter, Ruben; Sidharta, Patricia N; Delahaye, Stéphane; Gnerre, Carmela; Segrestaa, Jerome; Buchmann, Stephan; Kohl, Christopher; Treiber, Alexander

    2016-03-01

    Macitentan is a novel dual endothelin receptor antagonist for the treatment of pulmonary arterial hypertension (PAH). It is metabolized by cytochrome P450 (CYP) enzymes, mainly CYP3A4, to its active metabolite ACT-132577. A physiological-based pharmacokinetic (PBPK) model was developed by combining observations from clinical studies and physicochemical parameters as well as absorption, distribution, metabolism and excretion parameters determined in vitro. The model predicted the observed pharmacokinetics of macitentan and its active metabolite ACT-132577 after single and multiple dosing. It performed well in recovering the observed effect of the CYP3A4 inhibitors ketoconazole and cyclosporine, and the CYP3A4 inducer rifampicin, as well as in predicting interactions with S-warfarin and sildenafil. The model was robust enough to allow prospective predictions of macitentan-drug combinations not studied, including an alternative dosing regimen of ketoconazole and nine other CYP3A4-interacting drugs. Among these were the HIV drugs ritonavir and saquinavir, which were included because HIV infection is a known risk factor for the development of PAH. This example of the application of PBPK modeling to predict drug-drug interactions was used to support the labeling of macitentan (Opsumit).

  14. Purification, Reconstitution, and Inhibition of Cytochrome P-450 Sterol Δ22-Desaturase from the Pathogenic Fungus Candida glabrata

    PubMed Central

    Lamb, David C.; Maspahy, Segula; Kelly, Diane E.; Manning, Nigel J.; Geber, Antonia; Bennett, John E.; Kelly, Steven L.

    1999-01-01

    Sterol Δ22-desaturase has been purified from a strain of Candida glabrata with a disruption in the gene encoding sterol 14α-demethylase (cytochrome P-45051; CYP51). The purified cytochrome P-450 exhibited sterol Δ22-desaturase activity in a reconstituted system with NADPH–cytochrome P-450 reductase in dilaurylphosphatidylcholine, with the enzyme kinetic studies revealing a Km for ergosta-5,7-dienol of 12.5 μM and a Vmax of 0.59 nmol of this substrate metabolized/min/nmol of P-450. This enzyme is encoded by CYP61 (ERG5) in Saccharomyces cerevisiae, and homologues have been shown in the Candida albicans and Schizosaccharomyces pombe genome projects. Ketoconazole, itraconazole, and fluconazole formed low-spin complexes with the ferric cytochrome and exhibited type II spectra, which are indicative of an interaction between the azole moiety and the cytochrome heme. The azole antifungal compounds inhibited reconstituted sterol Δ22-desaturase activity by binding to the cytochrome with a one-to-one stoichiometry, with total inhibition of enzyme activity occurring when equimolar amounts of azole and cytochrome P-450 were added. These results reveal the potential for sterol Δ22-desaturase to be an antifungal target and to contribute to the binding of drugs within the fungal cell. PMID:10390230

  15. Validation of a microdose probe drug cocktail for clinical drug interaction assessments for drug transporters and CYP3A.

    PubMed

    Prueksaritanont, T; Tatosian, D A; Chu, X; Railkar, R; Evers, R; Chavez-Eng, C; Lutz, R; Zeng, W; Yabut, J; Chan, G H; Cai, X; Latham, A H; Hehman, J; Stypinski, D; Brejda, J; Zhou, C; Thornton, B; Bateman, K P; Fraser, I; Stoch, S A

    2017-04-01

    A microdose cocktail containing midazolam, dabigatran etexilate, pitavastatin, rosuvastatin, and atorvastatin has been established to allow simultaneous assessment of a perpetrator impact on the most common drug metabolizing enzyme, cytochrome P450 (CYP)3A, and the major transporters organic anion-transporting polypeptides (OATP)1B, breast cancer resistance protein (BCRP), and MDR1 P-glycoprotein (P-gp). The clinical utility of these microdose cocktail probe substrates was qualified by conducting clinical drug interaction studies with three inhibitors with different in vitro inhibitory profiles (rifampin, itraconazole, and clarithromycin). Generally, the pharmacokinetic profiles of the probe substrates, in the absence and presence of the inhibitors, were comparable to their reported corresponding pharmacological doses, and/or in agreement with theoretical expectations. The exception was dabigatran, which resulted in an approximately twofold higher magnitude for microdose compared to conventional dosing, and, thus, can be used to flag a worst-case scenario for P-gp. Broader application of the microdose cocktail will facilitate a more comprehensive understanding of the roles of drug transporters in drug disposition and drug interactions. © 2016 American Society for Clinical Pharmacology and Therapeutics.

  16. The interaction of representative members from two classes of antimycotics--the azoles and the allylamines--with cytochromes P-450 in steroidogenic tissues and liver.

    PubMed

    Schuster, I

    1985-06-01

    Spectrophotometric studies with ketoconazole, clotrimazole and miconazole show strong type-II interactions with several cytochromes P-450, particularly (Ks greater than 10(7)M-1; pH7.4; 25 degrees C) with the 11 beta-hydroxylase of adrenal mitochondria, with the 17 alpha/20 lyase of testis microsomes and with some forms of cytochromes P-450 of liver. A tight binding of the azoles also occurs to the reduced cytochromes, giving rise to an impeded CO binding to the haem iron. The binding of the azoles to 11 beta-hydroxylase and 17 alpha/20 lyase is much tighter than the binding of endogenous substrates, and consequently inhibition of steroidogenesis will occur at these sites. The metabolism of xenobiotic substrates by the cytochromes P-450 of liver will also be severely impeded. In contrast, the allylamines naftifine and SF 86-327 are type-I substrates for a small portion of cytochromes P-450 of liver microsomes only and there is no spectral evidence for binding to the cytochromes P-450 involved in steroid biosynthesis.

  17. [Screen potential CYP450 2E1 inhibitors from Chinese herbal medicine based on support vector regression and molecular docking method].

    PubMed

    Chen, Xi; Lu, Fang; Jiang, Lu-di; Cai, Yi-Lian; Li, Gong-Yu; Zhang, Yan-Ling

    2016-07-01

    Inhibition of cytochrome P450 (CYP450) enzymes is the most common reasons for drug interactions, so the study on early prediction of CYPs inhibitors can help to decrease the incidence of adverse reactions caused by drug interactions.CYP450 2E1(CYP2E1), as a key role in drug metabolism process, has broad spectrum of drug metabolism substrate. In this study, 32 CYP2E1 inhibitors were collected for the construction of support vector regression (SVR) model. The test set data were used to verify CYP2E1 quantitative models and obtain the optimal prediction model of CYP2E1 inhibitor. Meanwhile, one molecular docking program, CDOCKER, was utilized to analyze the interaction pattern between positive compounds and active pocket to establish the optimal screening model of CYP2E1 inhibitors.SVR model and molecular docking prediction model were combined to screen traditional Chinese medicine database (TCMD), which could improve the calculation efficiency and prediction accuracy. 6 376 traditional Chinese medicine (TCM) compounds predicted by SVR model were obtained, and in further verification by using molecular docking model, 247 TCM compounds with potential inhibitory activities against CYP2E1 were finally retained. Some of them have been verified by experiments. The results demonstrated that this study could provide guidance for the virtual screening of CYP450 inhibitors and the prediction of CYPs-mediated DDIs, and also provide references for clinical rational drug use. Copyright© by the Chinese Pharmaceutical Association.

  18. Identification of novel cytochrome P450s in the Acari

    USDA-ARS?s Scientific Manuscript database

    Cytochrome P450s are the major phase I drug metabolising enzymes found in most organisms, including arthropods. Much of the work within the area of xenobiotic metabolism in this group of animals has centered around mosquito species, e.g. Anopheles gambiae and Culex quinquefasciatus, due to their rol...

  19. How is a metabolic intermediate formed in the mechanism-based inactivation of cytochrome P450 by using 1,1-dimethylhydrazine: hydrogen abstraction or nitrogen oxidation?

    PubMed

    Hirao, Hajime; Chuanprasit, Pratanphorn; Cheong, Ying Yi; Wang, Xiaoqing

    2013-06-03

    A precise understanding of the mechanism-based inactivation of cytochrome P450 enzymes (P450s) at the quantum mechanical level should allow more reliable predictions of drug-drug interactions than those currently available. Hydrazines are among the molecules that act as mechanism-based inactivators to terminate the function of P450s, which are essential heme enzymes responsible for drug metabolism in the human body. Despite its importance, the mechanism explaining how a metabolic intermediate (MI) is formed from hydrazine is not fully understood. We used density functional theory (DFT) calculations to compare four possible mechanisms underlying the reaction between 1,1-dimethylhydrazine (or unsymmetrical dimethylhydrazine, UDMH) and the reactive compound I (Cpd I) intermediate of P450. Our DFT calculations provided a clear view on how an aminonitrene-type MI is formed from UDMH. In the most favorable pathway, hydrogen is spontaneously abstracted from the N2 atom of UDMH by Cpd I, followed by a second hydrogen abstraction from the N2 atom by Cpd II. Nitrogen oxidation of nitrogen atoms and hydrogen abstraction from the C-H bond of the methyl group were found to be less favorable than the hydrogen abstraction from the N-H bond. We also found that the reaction of protonated UDMH with Cpd I is rather sluggish. The aminonitrene-type MI binds to the ferric heme more strongly than a water molecule. This is consistent with the notion that the catalytic cycle of P450 is impeded when such an MI is produced through the P450-catalyzed reaction. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. Plant Expression of a Bacterial Cytochrome P450 That Catalyzes Activation of a Sulfonylurea Pro-Herbicide.

    PubMed Central

    O'Keefe, D. P.; Tepperman, J. M.; Dean, C.; Leto, K. J.; Erbes, D. L.; Odell, J. T.

    1994-01-01

    The Streptomyces griseolus gene encoding herbicide-metabolizing cytochrome P450SU1 (CYP105A1) was expressed in transgenic tobacco (Nicotiana tabacum). Because this P450 can be reduced by plant chloroplast ferredoxin in vitro, chloroplast-targeted and nontargeted expression were compared. Whereas P450SU1 antigen was found in the transgenic plants regardless of the targeting, only those with chloroplast-directed enzyme performed P450SU1-mediated N-dealkylation of the sulfonylurea 2-methylethyl-2,3-dihydro-N-[(4,6-dimethoxypyrimidin-2-yl)aminocarbonyl]-1, 2-benzoisothiazole- 7-sulfonamide-1,1-dioxide (R7402). Chloroplast targeting appears to be essential for the bacterial P450 to function in the plant. Because the R7402 metabolite has greater phytotoxicity than R7402 itself, plants bearing active P450SU1 are susceptible to injury from R7402 treatment that is harmless to plants without P450SU1. Thus, P450SU1 expression and R7402 treatment can be used as a negative selection system in plants. Furthermore, expression of P450SU1 from a tissue-specific promoter can sequester production of the phytotoxic R7402 metabolite to a single plant tissue. In tobacco expressing P450SU1 from a tapetum-specific promoter, treatment of immature flower buds with R7402 caused dramatically lowered pollen viability. Such treatment could be the basis for a chemical hybridizing agent. PMID:12232216

  1. Enhanced hepatic and kidney cytochrome p-450 activities in nandrolone decanoate treated albino mice.

    PubMed

    Acharjee, B K; Mahanta, R

    2009-04-01

    Anabolic androgenic steroids are the xenobiotic substrates that are metabolized in the body by the protective enzyme systems. Mixed function oxygenase enzymes include a group of enzymes which play an essential role in the metabolism of a broad range of xenobiotics including endogenous and exogenous substrates. Cytochrome P-450, a member of mixed function oxygenase enzymes, plays an important role in oxidative metabolism of drugs and xenobiotics entering human body. Various anabolic steroids are found either to increase or decrease the activity of cytochrome P-450. However, effect of nandrolone decanoate, most commonly abused anabolic steroid, on cytochrome P-450 activity is still fragmentary. In the present study, albino mice were administered intramuscular 2.5 mg of nandrolone decanoate injection at 15 days interval. Cytochrome P-450 activity is determined by following the method of Omura and Sato (1964) in liver and kidney tissues of both normal and experimental groups upto 90 days. Investigation shows a significant (p <0.01) increase of cytochrome P-450 (nmol/mg) activity in liver tissue as compared to that of kidney tissues. A tissue specific and dose specific increase of cytochrome P-450 activity is observed. Mean cytochrome P-450 is found highest in liver tissue on 45(th) day whereas the activity in kidney tissue is noticed on 90(th) day of treatment. From the above observation, nandrolone decanoate can be suggested as a potent inducer of cytochrome P-450 activity like other anabolic steroids.

  2. Antibodies against human cytochrome P-450db1 in autoimmune hepatitis type II.

    PubMed Central

    Zanger, U M; Hauri, H P; Loeper, J; Homberg, J C; Meyer, U A

    1988-01-01

    In a subgroup of children with chronic active hepatitis, circulating autoantibodies occur that bind to liver and kidney endoplasmic reticulum (anti-liver/kidney microsome antibody type I or anti-LKM1). Anti-LKM1 titers follow the severity of the disease and the presence of these antibodies serves as a diagnostic marker for this autoimmune hepatitis type II. We demonstrate that anti-LKM1 IgGs specifically inhibit the hydroxylation of bufuralol in human liver microsomes. Using two assay systems with different selectivity for the two cytochrome P-450 isozymes catalyzing bufuralol metabolism in human liver, we show that anti-LKM1 exclusively recognizes cytochrome P-450db1. Immunopurification of the LKM1 antigen from solubilized human liver microsomes resulted in an electrophoretically homogenous protein that had the same molecular mass (50 kDa) as purified P-450db1 and an identical N-terminal amino acid sequence. Recognition of both purified P-450db1 and the immunoisolated protein on western blots by several monoclonal antibodies confirmed the identity of the LKM1 antigen with cytochrome P-450db1. Cytochrome P-450db1 has been identified as the target of a common genetic polymorphism of drug oxidation. However, the relationship between the polymorphic cytochrome P-450db1 and the appearance of anti-LKM1 autoantibodies as well as their role in the pathogenesis of chronic active hepatitis remains speculative. Images PMID:3186722

  3. Antibodies against human cytochrome P-450db1 in autoimmune hepatitis type II.

    PubMed

    Zanger, U M; Hauri, H P; Loeper, J; Homberg, J C; Meyer, U A

    1988-11-01

    In a subgroup of children with chronic active hepatitis, circulating autoantibodies occur that bind to liver and kidney endoplasmic reticulum (anti-liver/kidney microsome antibody type I or anti-LKM1). Anti-LKM1 titers follow the severity of the disease and the presence of these antibodies serves as a diagnostic marker for this autoimmune hepatitis type II. We demonstrate that anti-LKM1 IgGs specifically inhibit the hydroxylation of bufuralol in human liver microsomes. Using two assay systems with different selectivity for the two cytochrome P-450 isozymes catalyzing bufuralol metabolism in human liver, we show that anti-LKM1 exclusively recognizes cytochrome P-450db1. Immunopurification of the LKM1 antigen from solubilized human liver microsomes resulted in an electrophoretically homogenous protein that had the same molecular mass (50 kDa) as purified P-450db1 and an identical N-terminal amino acid sequence. Recognition of both purified P-450db1 and the immunoisolated protein on western blots by several monoclonal antibodies confirmed the identity of the LKM1 antigen with cytochrome P-450db1. Cytochrome P-450db1 has been identified as the target of a common genetic polymorphism of drug oxidation. However, the relationship between the polymorphic cytochrome P-450db1 and the appearance of anti-LKM1 autoantibodies as well as their role in the pathogenesis of chronic active hepatitis remains speculative.

  4. Thermodynamics of interactions between mammalian cytochromes P450 and b5.

    PubMed

    Yablokov, Evgeny; Florinskaya, Anna; Medvedev, Alexei; Sergeev, Gennady; Strushkevich, Natallia; Luschik, Alexander; Shkel, Tatsiana; Haidukevich, Irina; Gilep, Andrei; Usanov, Sergey; Ivanov, Alexis

    2017-04-01

    Cytochromes P450 (CYPs) play an important role in the metabolism of xenobiotics and various endogenous substrates. Being a crucial component of the microsomal monooxygenase system, CYPs are involved in numerous protein-protein interactions. However, mechanisms underlying molecular interactions between components of the monooxygenase system still need better characterization. In this study thermodynamic parameters of paired interactions between mammalian CYPs and cytochromes b5 (CYB5) have been evaluated using a Surface Plasmon Resonance (SPR) based biosensor Biacore 3000. Analysis of 18 pairs of CYB5-CYP complexes formed by nine different isoforms of mammalian CYPs and two isoforms of human CYB5 has shown that thermodynamically these complexes can be subdivided into enthalpy-driven and entropy-driven groups. Formation of the enthalpy-driven complexes was observed in the case of microsomal CYPs allosterically regulated by CYB5 (CYB5A-CYP3A4, CYB5A-CYP3A5, CYB5A-CYP17A1). The entropy-driven complexes were formed when CYB5 had no effect on the CYP activity (CYB5A-CYP51A1, CYB5A-CYP1B1, CYB5B-CYP11A1). Results of this study suggest that such interactions determining protein clustering are indirectly linked to the monooxygenase functioning. Positive ΔH values typical for such interactions may be associated with displacement of the solvation shells of proteins upon clustering. CYB5-CYP complex formation accompanied by allosteric regulation of CYP activity by CYB5 is enthalpy-dependent. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. Potential risks resulting from fruit/vegetable-drug interactions: effects on drug-metabolizing enzymes and drug transporters.

    PubMed

    Rodríguez-Fragoso, Lourdes; Martínez-Arismendi, José Luis; Orozco-Bustos, Danae; Reyes-Esparza, Jorge; Torres, Eliseo; Burchiel, Scott W

    2011-05-01

    It has been well established that complex mixtures of phytochemicals in fruits and vegetables can be beneficial for human health. Moreover, it is becoming increasingly apparent that phytochemicals can influence the pharmacological activity of drugs by modifying their absorption characteristics through interactions with drug transporters as well as drug-metabolizing enzyme systems. Such effects are more likely to occur in the intestine and liver, where high concentrations of phytochemicals may occur. Alterations in cytochrome P450 and other enzyme activities may influence the fate of drugs subject to extensive first-pass metabolism. Although numerous studies of nutrient-drug interactions have been published and systematic reviews and meta-analyses of these studies are available, no generalizations on the effect of nutrient-drug interactions on drug bioavailability are currently available. Several publications have highlighted the unintended consequences of the combined use of nutrients and drugs. Many phytochemicals have been shown to have pharmacokinetic interactions with drugs. The present review is limited to commonly consumed fruits and vegetables with significant beneficial effects as nutrients and components in folk medicine. Here, we discuss the phytochemistry and pharmacokinetic interactions of the following fruit and vegetables: grapefruit, orange, tangerine, grapes, cranberry, pomegranate, mango, guava, black raspberry, black mulberry, apple, broccoli, cauliflower, watercress, spinach, tomato, carrot, and avocado. We conclude that our knowledge of the potential risk of nutrient-drug interactions is still limited. Therefore, efforts to elucidate potential risks resulting from food-drug interactions should be intensified in order to prevent undesired and harmful clinical consequences. © 2011 Institute of Food Technologists®

  6. Metabolism of a 5HT6 antagonist, 2-methyl-1-(phenylsulfonyl)-4-(piperazin-1-yl)-1H-benzo[d]imidazole (SAM-760): impact of sulfonamide metabolism on diminution of a ketoconazole mediated clinical drug-drug interaction.

    PubMed

    Sawant-Basak, Aarti; Obach, R Scott; Doran, Angela C; Lockwood, Peter; Schildknegt, Klaas; Gao, Hongying; Mancuso, Jessica; Tse, Susanna; Comery, Tom

    2018-04-25

    SAM-760, (2-methyl-1-(phenylsulfonyl)-4-(piperazin-1-yl)-1H-benzo[d]imidazole), a 5HT 6 antagonist, was investigated in humans for the treatment of Alzheimer's dementia. In liver microsomes and recombinant CYP450 isozymes, SAM-760 was predominantly metabolized by CYP3A (~85%). Based on these observations and an expectation of 5-fold magnitude of interaction with moderate to strong CYP3A inhibitors, a clinical DDI study was performed. In presence of ketoconazole, mean C max and AUC 0-inf of SAM-760 showed only a modest increase by 30% and 38%, respectively. In vitro investigation of this unexpectedly low interaction was undertaken using [ 14 C]SAM-760. Radiometric profiling in human hepatocytes, confirmed all oxidative metabolites observed previously with unlabeled SAM-760; however the pre-dominant radiometric peak was an unexpected polar metabolite which was insensitive to pan-CYP inhibitor, 1-aminobenzotriazole. In human hepatocytes, radiometric integration attributed 43% of total metabolism of SAM-760 to this non-CYP pathway. Using an authentic standard, this predominant metabolite was confirmed as benzenesulfinic acid. Additional investigation revealed that the benzenesulfinic acid metabolite may be a novel, non-enzymatic, thiol mediated reductive cleavage of aryl sulfonamide group of SAM-760. We also determined the relative contribution of P450 to metabolism of SAM-760 in human hepatocytes, by following the rate of formation of oxidative metabolites in presence and absence of P450 isoform specific inhibitors. P450 mediated oxidative metabolism of SAM-760 was still primarily attributed to CYP3A (33%), with minor contributions from CYP isoforms 2C19 and 2D6. Thus, disposition of [ 14 C]SAM-760 in human hepatocytes via novel sulfonamide metabolism and CYP3A verified the lower than expected clinical DDI when SAM-760 was co-administered with ketoconazole. The American Society for Pharmacology and Experimental Therapeutics.

  7. Model studies in cytochrome P-450-mediated toxicity of halogenated compounds: radical processes involving iron porphyrins.

    PubMed Central

    Brault, D

    1985-01-01

    Haloalkane toxicity originates from attack on biological targets by reactive intermediates derived from haloalkane metabolism by a hemoprotein, cytochrome P-450. Carbon-centered radicals and their peroxyl derivatives are most likely involved. The reactions of iron porphyrin--a model for cytochrome P-450--with various carbon-centered and peroxyl radicals generated by pulse radiolysis are examined. Competition between iron porphyrin and unsaturated fatty acids for attack by peroxyl radicals is pointed out. These kinetic data are used to derive a model for toxicity of haloalkanes with particular attention to carbon tetrachloride and halothane. The importance of local oxygen concentration and structural arrangement of fatty acids around cytochrome P-450 is emphasized. PMID:3007100

  8. Individual variability in clinical effect and tolerability of opioid analgesics - Importance of drug interactions and pharmacogenetics.

    PubMed

    Solhaug, Vigdis; Molden, Espen

    2017-10-01

    As pain is often a comorbid condition, many patients use opioid analgesics in combination with several other drugs. This implies a generally increased risk of drug interactions, which along with inherent pharmacogenetic variability and other factors may cause differences in therapeutic response of opioids. To provide an overview of interactions and pharmacogenetic variability of relevance for individual differences in effect and tolerability of opioid analgesics, which physicians and other healthcare professionals should be aware of in clinical practice. The article was based on unsystematic searches in PubMed to identify literature highlighting the clinical impact of drug interactions and pharmacogenetics as sources of variable response of opioid analgesics. Cytochrome P450 (CYP)-mediated metabolism is an important process for both clinically relevant interactions and pharmacogenetic variability of several opioids. Concomitant use of CYP inhibitors (e.g. paroxetine, fluoxetine and bupropion) or inducers (e.g. carbamazepine, phenobarbital and phenytoin) could counteract the clinical effect or trigger side effects of analgesics in the same manner as genetically determined differences in CYP2D6-mediated metabolism of many opioids. Moreover, combination treatment with drugs that inhibit or induce P-glycoprotein (ABCB1), a blood-brain barrier efflux transporter, may alter the amount ('dose') of opioids distributed to the brain. At the pharmacodynamic level, it is crucial to be aware of the potential risk of interaction causing serotonergic syndrome when combining opioids and serotonergic drugs, in particular antidepressants inhibiting serotonin reuptake (SSRIs and SNRIs). Regarding pharmacogenetics at the receptor level of pain treatment, the knowledge is currently scarce, but an allelic variant of the μ1 opioid receptor (OPRM1) gene has been associated with higher dosage requirement to achieve analgesia. Drug interactions and pharmacogenetic differences may lead to

  9. Stereoselective metabolism of endosulfan by human liver microsomes and human cytochrome P450 isoforms.

    PubMed

    Lee, Hwa-Kyung; Moon, Joon-Kwan; Chang, Chul-Hee; Choi, Hoon; Park, Hee-Won; Park, Byeoung-Soo; Lee, Hye-Suk; Hwang, Eul-Chul; Lee, Young-Deuk; Liu, Kwang-Hyeon; Kim, Jeong-Han

    2006-07-01

    Endosulfan (6,7,8,9,10,10-hexachloro-1,5,5a,6,9,9a-hexahydro-6,9-methano-2,3,4-benzo(e)dioxathiepin-3-oxide) is a broad-spectrum chlorinated cyclodiene insecticide. This study was performed to elucidate the stereoselective metabolism of endosulfan in human liver microsomes and to characterize the cytochrome P450 (P450) enzymes that are involved in the metabolism of endosulfan. Human liver microsomal incubation of endosulfan in the presence of NADPH resulted in the formation of the toxic metabolite, endosulfan sulfate. The intrinsic clearances (CL(int)) of endosulfan sulfate from beta-endosulfan were 3.5-fold higher than those from alpha-endosulfan, suggesting that beta-endosulfan would be cleared more rapidly than alpha-endosulfan. Correlation analysis between the known P450 enzyme activities and the rate of the formation of endosulfan sulfate in the 14 human liver microsomes showed that alpha-endosulfan metabolism is significantly correlated with CYP2B6-mediated bupropion hydroxylation and CYP3A-mediated midazolam hydroxylation, and that beta-endosulfan metabolism is correlated with CYP3A activity. The P450 isoform-selective inhibition study in human liver microsomes and the incubation study of cDNA-expressed enzymes also demonstrated that the stereoselective sulfonation of alpha-endosulfan is mediated by CYP2B6, CYP3A4, and CYP3A5, and that that of beta-endosulfan is transformed by CYP3A4 and CYP3A5. The total CL(int) values of endosulfan sulfate formation catalyzed by CYP3A4 and CYP3A5 were consistently higher for beta-endosulfan than for the alpha-form (CL(int) of 0.67 versus 10.46 microl/min/pmol P450, respectively). CYP2B6 enantioselectively metabolizes alpha-endosulfan, but not beta-endosulfan. These findings suggest that the CYP2B6 and CYP3A enzymes are major enzymes contributing to the stereoselective disposition of endosulfan.

  10. Improvement of Transmembrane Transport Mechanism Study of Imperatorin on P-Glycoprotein-Mediated Drug Transport.

    PubMed

    Liao, Zheng-Gen; Tang, Tao; Guan, Xue-Jing; Dong, Wei; Zhang, Jing; Zhao, Guo-Wei; Yang, Ming; Liang, Xin-Li

    2016-11-24

    P-glycoprotein (P-gp) affects the transport of many drugs; including puerarin and vincristine. Our previous study demonstrated that imperatorin increased the intestinal absorption of puerarin and vincristine by inhibiting P-gp-mediated drug efflux. However; the underlying mechanism was not known. The present study investigated the mechanism by which imperatorin promotes P-gp-mediated drug transport. We used molecular docking to predict the binding force between imperatorin and P-gp and the effect of imperatorin on P-gp activity. P-gp efflux activity and P-gp ATPase activity were measured using a rhodamine 123 (Rh-123) accumulation assay and a Pgp-Glo™ assay; respectively. The fluorescent probe 1,6-diphenyl-1,3,5-hexatriene (DPH) was used to assess cellular membrane fluidity in MDCK-MDR1 cells. Western blotting was used to analyze the effect of imperatorin on P-gp expression; and P-gp mRNA levels were assessed by qRT-PCR. Molecular docking results demonstrated that the binding force between imperatorin and P-gp was much weaker than the force between P-gp and verapamil (a P-gp substrate). Imperatorin activated P-gp ATPase activity; which had a role in the inhibition of P-gp activity. Imperatorin promoted Rh-123 accumulation in MDCK-MDR1 cells and decreased cellular membrane fluidity. Western blotting demonstrated that imperatorin inhibited P-gp expression; and qRT-PCR revealed that imperatorin down-regulated P-gp (MDR1) gene expression. Imperatorin decreased P-gp-mediated drug efflux by inhibiting P-gp activity and the expression of P-gp mRNA and protein. Our results suggest that imperatorin could down-regulate P-gp expression to overcome multidrug resistance in tumors.

  11. Hyperforin in St. John's wort drug interactions.

    PubMed

    Madabushi, Rajanikanth; Frank, Bruno; Drewelow, Bernd; Derendorf, Hartmut; Butterweck, Veronika

    2006-03-01

    Recently, interactions of herbal medicines with synthetic drugs came into focus of particular interest. In the past 3 years, more than 50 papers were published regarding interactions between St. John's wort (Hypericum perforatum L.; SJW) and prescription drugs. Co-medication with SJW resulted in decreased plasma concentrations of a number of drugs including amitriptyline, cyclosporine, digoxin, indinavir, irinotecan, warfarin, phenprocoumon, alprazolam, dextrometorphane, simvastatin, and oral contraceptives. Sufficient evidence from interaction studies and case reports indicate that SJW is a potent inducer of cytochrome P450 enzymes (particularly CYP3A4) and/or P-glycoprotein. Recent studies could show that the degree of enzyme induction by SJW correlates strongly with the amount of hyperforin found in the product. Products that do not contain substantial amounts of hyperforin (<1%) have not been shown to produce clinically relevant enzyme induction. On the other hand, some evidence suggests that hyperforin may also contribute to the antidepressant activity of SJW. However, clinical studies using SJW preparations with a low hyperforin amount (<1%) clearly demonstrated the superiority of this plant extract over placebo and its equivalence to imipramine and fluoxetine in the treatment of mild to moderate forms of depression. In the present paper clinical significant SJW interactions are critically evaluated against the background of hyperforin.

  12. Identification of the cytochrome P450 enzymes involved in the metabolism of cisapride: in vitro studies of potential co-medication interactions

    PubMed Central

    Bohets, H; Lavrijsen, K; Hendrickx, J; van Houdt, J; van Genechten, V; Verboven, P; Meuldermans, W; Heykants, J

    2000-01-01

    Cisapride is a prokinetic drug that is widely used to facilitate gastrointestinal tract motility.Structurally, cisapride is a substituted piperidinyl benzamide that interacts with 5-hydroxytryptamine-4 receptors and which is largely without central depressant or antidopaminergic side-effects.The aims of this study were to investigate the metabolism of cisapride in human liver microsomes and to determine which cytochrome P-450 (CYP) isoenzyme(s) are involved in cisapride biotransformation. Additionally, the effects of various drugs on the metabolism of cisapride were investigated.The major in vitro metabolite of cisapride was formed by oxidative N-dealkylation at the piperidine nitrogen, leading to the production of norcisapride.By using competitive inhibition data, correlation studies and heterologous expression systems, it was demonstrated that CYP3A4 was the major CYP involved. CYP2A6 also contributed to the metabolism of cisapride, albeit to a much lesser extent.The mean apparent Km against cisapride was 8.6±3.5 μM (n=3). The peak plasma levels of cisapride under normal clinical practice are approximately 0.17 μM; therefore it is unlikely that cisapride would inhibit the metabolism of co-administered drugs.In this in vitro study the inhibitory effects of 44 drugs were tested for any effect on cisapride biotransformation. In conclusion, 34 of the drugs are unlikely to have a clinically relevant interaction; however, the antidepressant nefazodone, the macrolide antibiotic troleandomycin, the HIV-1 protease inhibitors ritonavir and indinavir and the calcium channel blocker mibefradil inhibited the metabolism of cisapride and these interactions are likely to be of clinical relevance. Furthermore, the antimycotics ketoconazole, miconazole, hydroxy-itraconazole, itraconazole and fluconazole, when administered orally or intravenously, would inhibit cisapride metabolism. PMID:10780971

  13. Inhibitory effects of herbal constituents on P-glycoprotein in vitro and in vivo: Herb–drug interactions mediated via P-gp

    SciTech Connect

    Li, Xue, E-mail: lixue@imm.ac.cn; Hu, Jinping, E-mail: hujp@imm.ac.cn; Wang, Baolian, E-mail: wangbaolian@imm.ac.cn

    Modulation of drug transporters via herbal medicines which have been widely used in combination with conventional prescription drugs may result in herb–drug interactions in clinical practice. The present study was designed to investigate the inhibitory effects of 50 major herbal constituents on P-glycoprotein (P-gp) in vitro and in vivo as well as related inhibitory mechanisms. Among these herbal medicines, four constituents, including emodin, 18β-glycyrrhetic acid (18β-GA), dehydroandrographolide (DAG), and 20(S)-ginsenoside F{sub 1} [20(S)-GF{sub 1}] exhibited significant inhibition (> 50%) on P-gp in MDR1-MDCKII and Caco-2 cells. Emodin was the strongest inhibitor of P-gp (IC{sub 50} = 9.42 μM), followed bymore » 18β-GA (IC{sub 50} = 21.78 μM), 20(S)-GF{sub 1} (IC{sub 50} = 76.08 μM) and DAG (IC{sub 50} = 77.80 μM). P-gp ATPase activity, which was used to evaluate the affinity of substrates to P-gp, was stimulated by emodin and DAG with K{sub m} and V{sub max} values of 48.61, 29.09 μM and 71.29, 38.45 nmol/min/mg protein, respectively. However, 18β-GA and 20(S)-GF{sub 1} exhibited significant inhibition on both basal and verapamil-stimulated P-gp ATPase activities at high concentration. Molecular docking analysis (CDOCKER) further elucidated the mechanism for structure–inhibition relationships of herbal constituents with P-gp. When digoxin was co-administered to male SD rats with emodin or 18β-GA, the AUC{sub 0−t} and Cmax of digoxin were increased by approximately 51% and 58%, respectively. Furthermore, 18β-GA, DAG, 20(S)-GF{sub 1} and Rh{sub 1} at 10 μM significantly inhibited CYP3A4/5 activity, while emodin activated the metabolism of midazolam in human liver microsomes. In conclusion, four herbal constituents demonstrated inhibition of P-gp to specific extents in vitro and in vivo. Taken together, our findings provided the basis for the reliable assessment of the potential risks of herb–drug interactions in humans. - Highlights:

  14. Evaluation of a New Molecular Entity as a Victim of Metabolic Drug-Drug Interactions-an Industry Perspective.

    PubMed

    Bohnert, Tonika; Patel, Aarti; Templeton, Ian; Chen, Yuan; Lu, Chuang; Lai, George; Leung, Louis; Tse, Susanna; Einolf, Heidi J; Wang, Ying-Hong; Sinz, Michael; Stearns, Ralph; Walsky, Robert; Geng, Wanping; Sudsakorn, Sirimas; Moore, David; He, Ling; Wahlstrom, Jan; Keirns, Jim; Narayanan, Rangaraj; Lang, Dieter; Yang, Xiaoqing

    2016-08-01

    Under the guidance of the International Consortium for Innovation and Quality in Pharmaceutical Development (IQ), scientists from 20 pharmaceutical companies formed a Victim Drug-Drug Interactions Working Group. This working group has conducted a review of the literature and the practices of each company on the approaches to clearance pathway identification (fCL), estimation of fractional contribution of metabolizing enzyme toward metabolism (fm), along with modeling and simulation-aided strategy in predicting the victim drug-drug interaction (DDI) liability due to modulation of drug metabolizing enzymes. Presented in this perspective are the recommendations from this working group on: 1) strategic and experimental approaches to identify fCL and fm, 2) whether those assessments may be quantitative for certain enzymes (e.g., cytochrome P450, P450, and limited uridine diphosphoglucuronosyltransferase, UGT enzymes) or qualitative (for most of other drug metabolism enzymes), and the impact due to the lack of quantitative information on the latter. Multiple decision trees are presented with stepwise approaches to identify specific enzymes that are involved in the metabolism of a given drug and to aid the prediction and risk assessment of drug as a victim in DDI. Modeling and simulation approaches are also discussed to better predict DDI risk in humans. Variability and parameter sensitivity analysis were emphasized when applying modeling and simulation to capture the differences within the population used and to characterize the parameters that have the most influence on the prediction outcome. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

  15. NAD(P)H-dependent quinone oxidoreductase 1 (NQO1) and cytochrome P450 oxidoreductase (CYP450OR) differentially regulate menadione-mediated alterations in redox status, survival and metabolism in pancreatic β-cells.

    PubMed

    Gray, Joshua P; Karandrea, Shpetim; Burgos, Delaine Zayasbazan; Jaiswal, Anil A; Heart, Emma A

    2016-11-16

    NQO1 (NAD(P)H-quinone oxidoreductase 1) reduces quinones and xenobiotics to less-reactive compounds via 2-electron reduction, one feature responsible for the role of NQO1 in antioxidant defense in several tissues. In contrast, NADPH cytochrome P450 oxidoreductase (CYP450OR), catalyzes the 1-electron reduction of quinones and xenobiotics, resulting in enhanced superoxide formation. However, to date, the roles of NQO1 and CYP450OR in pancreatic β-cell metabolism under basal conditions and oxidant challenge have not been characterized. Using NQO1 inhibition, over-expression and knock out, we have demonstrated that, in addition to protection of β-cells from toxic concentrations of the redox cycling quinone menadione, NQO1 also regulates the basal level of reduced-to-oxidized nucleotides, suggesting other role(s) beside that of an antioxidant enzyme. In contrast, over-expression of NADPH cytochrome P450 oxidoreductase (CYP450OR) resulted in enhanced redox cycling activity and decreased cellular viability, consistent with the enhanced generation of superoxide and H 2 O 2 . Basal expression of NQO1 and CYP450OR was comparable in isolated islets and liver. However, NQO1, but not CYP450OR, was strongly induced in β-cells exposed to menadione. NQO1 and CYP450OR exhibited a reciprocal preference for reducing equivalents in β-cells: while CYP450OR preferentially utilized NADPH, NQO1 primarily utilized NADH. Together, these results demonstrate that NQO1 and CYP450OR reciprocally regulate oxidant metabolism in pancreatic β-cells. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  16. NAD(P)H-dependent Quinone Oxidoreductase 1 (NQO1) and Cytochrome P450 Oxidoreductase (CYP450OR) differentially regulate menadione-mediated alterations in redox status, survival and metabolism in pancreatic β-cells

    PubMed Central

    Gray, Joshua P.; Karandrea, Shpetim; Burgos, Delaine Zayasbazan; Jaiswal, Anil A; Heart, Emma A.

    2017-01-01

    NQO1 (NAD(P)H-quinone oxidoreductase 1) reduces quinones and xenobiotics to less-reactive compounds via 2-electron reduction, one feature responsible for the role of NQO1 in antioxidant defense in several tissues. In contrast, NADPH cytochrome P450 oxidoreductase (CYP450OR), catalyzes the 1-electron reduction of quinones and xenobiotics, resulting in enhanced superoxide formation. However, to date, the roles of NQO1 and CYP450OR in pancreatic β-cell metabolism under basal conditions and oxidant challenge have not been characterized. Using NQO1 inhibition, over-expression and knock out, we have demonstrated that, in addition to protection of β-cells from toxic concentrations of the redox cycling quinone menadione, NQO1 also regulates the basal level of reduced-to-oxidized nucleotides, suggesting other role(s) beside that of an antioxidant enzyme. In contrast, over-expression of NADPH cytochrome P450 oxidoreductase (CYP450OR) resulted in enhanced redox cycling activity and decreased cellular viability, consistent with the enhanced generation of superoxide and H2O2. Basal expression of NQO1 and CYP450OR was comparable in isolated islets and liver. However, NQO1, but not CYP450OR, was strongly induced in β-cells exposed to menadione. NQO1 and CYP450OR exhibited a reciprocal preference for reducing equivalents in β-cells: while CYP450OR preferentially utilized NADPH, NQO1 primarily utilized NADH. Together, these results demonstrate that NQO1 and CYP450OR reciprocally regulate oxidant metabolism in pancreatic β-cells. PMID:27558805

  17. Inhibitory effects of cytostatically active 6-aminobenzo[c]phenanthridines on cytochrome P450 enzymes in human hepatic microsomes.

    PubMed

    Zebothsen, Inga; Kunze, Thomas; Clement, Bernd

    2006-07-01

    Besides assays for the evaluation of efficacy new drug candidates have to undergo extensive testings for enhancement of pharmaceutical drug safety and optimization of application. The objective of the present work was to investigate the pharmacokinetic drug drug interaction potential for the cytostatically active 6-aminobenzo[c]phenanthridines BP-11 (6-amino-11,12-dihydro-11-(4-hydroxy-3,5-dimethoxyphenyl)benzo[c]phenanthridine) and BP-D7 (6-amino-11-(3,4,5-trimethoxyphenyl)benzo[c]phenanthridine) in vitro through incubation with human hepatic microsomes and marker substrates. For these studies the cytochrome P-450 isoenzymes and corresponding marker substrates recommended by the EMEA (The European Agency for the Evaluation of Medicinal Products) were chosen. In detail these selective substrates were caffeine (CYP1A2), coumarin (CYP2A6), tolbutamide (CYP2C9), S-(+)-mephenytoin (CYP2C19), dextromethorphane (CYP2D6), chlorzoxazone (CYP2E1) and testosterone (CYP3A4). Incubations with each substrate were carried out without a possible inhibitor and in the presence of a benzo[c]phenanthridine or a selective inhibitor at varying concentrations. Marker activities were determined by HPLC (high performance liquid chromatography). For the isoenzymes showing more than 50% inhibition by the addition of 20 microM BP-11 or BP-D7 additional concentrations of substrate and inhibitor were tested for a characterization of the inhibition. The studies showed a moderate risk for BP-11 for interactions with the cytochrome P-450 isoenzymes CYP1A2, CYP2C9, CYP2D6 and CYP3A4. BP-D7, the compound with the highest cytotstatic efficacy, showed only a moderate risk for interactions with drugs, also metabolized by CYP3A4.

  18. Evidence for induction of cytochrome P-450I in patients with tropical chronic pancreatitis.

    PubMed

    Chaloner, C; Sandle, L N; Mohan, V; Snehalatha, C; Viswanathan, M; Braganza, J M

    1990-06-01

    Theophylline kinetics, as an in vivo probe for the potentially toxic cytochrome P-450I pathway of drug metabolism, were studied in 11 healthy volunteers and 11 patients with calcific chronic pancreatitis at Madras, South India. Theophylline clearance was faster in the patients than controls [median 69 (range 39-114) vs 45 (33-56) ml h-1 kg-1, p = 0.003]. In keeping with this finding, detailed social histories identified a higher exposure level in the patients to xenobiotics that are inducers of cytochrome P-450I and/or yield reactive metabolites upon processing thereby (score 7, 4-11 vs 3, 2-9, p = 0.002). However, the concentration of D-glucaric acid in urine, as a marker of phase II conjugating pathways of drug metabolism, was similar in patients and controls. This pattern of drug metabolism could predispose to oxidant stress: hence micronutrient antioxidant supplements may have therapeutic (or even prophylactic) value in tropical chronic pancreatitis.

  19. An enlarged, adaptable active site in CYP164 family P450 enzymes, the sole P450 in Mycobacterium leprae.

    PubMed

    Agnew, Christopher R J; Warrilow, Andrew G S; Burton, Nicholas M; Lamb, David C; Kelly, Steven L; Brady, R Leo

    2012-01-01

    CYP164 family P450 enzymes are found in only a subset of mycobacteria and include CYP164A1, which is the sole P450 found in Mycobacterium leprae, the causative agent of leprosy. This has previously led to interest in this enzyme as a potential drug target. Here we describe the first crystal structure of a CYP164 enzyme, CYP164A2 from Mycobacterium smegmatis. CYP164A2 has a distinctive, enlarged hydrophobic active site that extends above the porphyrin ring toward the access channels. Unusually, we find that CYP164A2 can simultaneously bind two econazole molecules in different regions of the enlarged active site and is accompanied by the rearrangement and ordering of the BC loop. The primary location is through a classic interaction of the azole group with the porphyrin iron. The second econazole molecule is bound to a unique site and is linked to a tetracoordinated metal ion complexed to one of the heme carboxylates and to the side chains of His 105 and His 364. All of these features are preserved in the closely homologous M. leprae CYP164A1. The computational docking of azole compounds to a homology model of CYP164A1 suggests that these compounds will form effective inhibitors and is supported by the correlation of parallel docking with experimental binding studies of CYP164A2. The binding of econazole to CYP164A2 occurs primarily through the high-spin "open" conformation of the enzyme (K(d) [dissociation constant] of 0.1 μM), with binding to the low-spin "closed" form being significantly hindered (K(d) of 338 μM). These studies support previous suggestions that azole derivatives may provide an effective strategy to improve the treatment of leprosy.

  20. An Enlarged, Adaptable Active Site in CYP164 Family P450 Enzymes, the Sole P450 in Mycobacterium leprae

    PubMed Central

    Agnew, Christopher R. J.; Warrilow, Andrew G. S.; Burton, Nicholas M.; Lamb, David C.; Kelly, Steven L.

    2012-01-01

    CYP164 family P450 enzymes are found in only a subset of mycobacteria and include CYP164A1, which is the sole P450 found in Mycobacterium leprae, the causative agent of leprosy. This has previously led to interest in this enzyme as a potential drug target. Here we describe the first crystal structure of a CYP164 enzyme, CYP164A2 from Mycobacterium smegmatis. CYP164A2 has a distinctive, enlarged hydrophobic active site that extends above the porphyrin ring toward the access channels. Unusually, we find that CYP164A2 can simultaneously bind two econazole molecules in different regions of the enlarged active site and is accompanied by the rearrangement and ordering of the BC loop. The primary location is through a classic interaction of the azole group with the porphyrin iron. The second econazole molecule is bound to a unique site and is linked to a tetracoordinated metal ion complexed to one of the heme carboxylates and to the side chains of His 105 and His 364. All of these features are preserved in the closely homologous M. leprae CYP164A1. The computational docking of azole compounds to a homology model of CYP164A1 suggests that these compounds will form effective inhibitors and is supported by the correlation of parallel docking with experimental binding studies of CYP164A2. The binding of econazole to CYP164A2 occurs primarily through the high-spin “open” conformation of the enzyme (Kd [dissociation constant] of 0.1 μM), with binding to the low-spin “closed” form being significantly hindered (Kd of 338 μM). These studies support previous suggestions that azole derivatives may provide an effective strategy to improve the treatment of leprosy. PMID:22037849

  1. Ecologically Appropriate Xenobiotics Induce Cytochrome P450s in Apis mellifera

    PubMed Central

    Johnson, Reed M.; Mao, Wenfu; Pollock, Henry S.; Niu, Guodong; Schuler, Mary A.; Berenbaum, May R.

    2012-01-01

    Background Honey bees are exposed to phytochemicals through the nectar, pollen and propolis consumed to sustain the colony. They may also encounter mycotoxins produced by Aspergillus fungi infesting pollen in beebread. Moreover, bees are exposed to agricultural pesticides, particularly in-hive acaricides used against the parasite Varroa destructor. They cope with these and other xenobiotics primarily through enzymatic detoxificative processes, but the regulation of detoxificative enzymes in honey bees remains largely unexplored. Methodology/Principal Findings We used several approaches to ascertain effects of dietary toxins on bee susceptibility to synthetic and natural xenobiotics, including the acaricide tau-fluvalinate, the agricultural pesticide imidacloprid, and the naturally occurring mycotoxin aflatoxin. We administered potential inducers of cytochrome P450 enzymes, the principal biochemical system for Phase 1 detoxification in insects, to investigate how detoxification is regulated. The drug phenobarbital induces P450s in many insects, yet feeding bees with phenobarbital had no effect on the toxicity of tau-fluvalinate, a pesticide known to be detoxified by bee P450s. Similarly, no P450 induction, as measured by tau-fluvalinate tolerance, occurred in bees fed xanthotoxin, salicylic acid, or indole-3-carbinol, all of which induce P450s in other insects. Only quercetin, a common pollen and honey constituent, reduced tau-fluvalinate toxicity. In microarray comparisons no change in detoxificative gene expression was detected in phenobarbital-treated bees. However, northern blot analyses of guts of bees fed extracts of honey, pollen and propolis showed elevated expression of three CYP6AS P450 genes. Diet did not influence tau-fluvalinate or imidacloprid toxicity in bioassays; however, aflatoxin toxicity was higher in bees consuming sucrose or high-fructose corn syrup than in bees consuming honey. Conclusions/Significance These results suggest that regulation of

  2. Methoxyflurane acts at the substrate binding site of cytochrome P450 LM2 to induce a dependence on cytochrome b5.

    PubMed

    Lipka, J J; Waskell, L A

    1989-01-01

    Rabbit cytochrome P450 isozyme 2 requires cytochrome b5 to metabolize the volatile anesthetic methoxyflurane but not the substrate benzphetamine [E. Canova-Davis and L. Waskell (1984) J. Biol. Chem. 259, 2541-2546]. To determine whether the requirement for cytochrome b5 for methoxyflurane oxidation is mediated by an allosteric effect on cytochrome P450 LM2 or cytochrome P450 reductase, we have investigated whether this anesthetic can induce a role for cytochrome b5 in benzphetamine metabolism. Using rabbit liver microsomes and antibodies raised in guinea pigs against rabbit cytochrome b5, we found that methoxyflurane did not create a cytochrome b5 requirement for benzphetamine metabolism. Methoxyflurane also failed to induce a role for cytochrome b5 in benzphetamine metabolism in the purified, reconstituted mixed function oxidase system. Studies of the reaction kinetics established that in the absence of cytochrome b5, methoxyflurane and benzphetamine are competitive inhibitors, and that in the presence of cytochrome b5, benzphetamine and methoxyflurane are two alternate substrates in competition for a single site on the same enzyme. These results all indicate that the methoxyflurane-induced cytochrome b5 dependence of the mixed function oxidase cytochrome P450 LM2 system is a direct result of the interaction between methoxyflurane and the substrate binding site of cytochrome P450 LM2 and suggest the focus of future studies of this question.

  3. Contextualization of drug-mediator relations using evidence networks.

    PubMed

    Tran, Hai Joey; Speyer, Gil; Kiefer, Jeff; Kim, Seungchan

    2017-05-31

    Genomic analysis of drug response can provide unique insights into therapies that can be used to match the "right drug to the right patient." However, the process of discovering such therapeutic insights using genomic data is not straightforward and represents an area of active investigation. EDDY (Evaluation of Differential DependencY), a statistical test to detect differential statistical dependencies, is one method that leverages genomic data to identify differential genetic dependencies. EDDY has been used in conjunction with the Cancer Therapeutics Response Portal (CTRP), a dataset with drug-response measurements for more than 400 small molecules, and RNAseq data of cell lines in the Cancer Cell Line Encyclopedia (CCLE) to find potential drug-mediator pairs. Mediators were identified as genes that showed significant change in genetic statistical dependencies within annotated pathways between drug sensitive and drug non-sensitive cell lines, and the results are presented as a public web-portal (EDDY-CTRP). However, the interpretability of drug-mediator pairs currently hinders further exploration of these potentially valuable results. In this study, we address this challenge by constructing evidence networks built with protein and drug interactions from the STITCH and STRING interaction databases. STITCH and STRING are sister databases that catalog known and predicted drug-protein interactions and protein-protein interactions, respectively. Using these two databases, we have developed a method to construct evidence networks to "explain" the relation between a drug and a mediator.  RESULTS: We applied this approach to drug-mediator relations discovered in EDDY-CTRP analysis and identified evidence networks for ~70% of drug-mediator pairs where most mediators were not known direct targets for the drug. Constructed evidence networks enable researchers to contextualize the drug-mediator pair with current research and knowledge. Using evidence networks, we were

  4. Phenobarbital induction of cytochromes P-450. High-level long-term responsiveness of primary rat hepatocyte cultures to drug induction, and glucocorticoid dependence of the phenobarbital response.

    PubMed Central

    Waxman, D J; Morrissey, J J; Naik, S; Jauregui, H O

    1990-01-01

    The induction of hepatic cytochromes P-450 by phenobarbital (PB) was studied in rat hepatocytes cultured for up to 5 weeks on Vitrogen-coated plates in serum-free modified Chee's medium then exposed to PB (0.75 mM) for an additional 4 days. Immunoblotting analysis indicated that P-450 forms PB4 (IIB1) and PB5 (IIB2) were induced dramatically (greater than 50-fold increase), up to levels nearly as high as those achieved in PB-induced rat liver in vivo. The newly synthesized cytochrome P-450 was enzymically active, as shown by the major induction of the P-450 PB4-dependent steroid 16 beta-hydroxylase and pentoxyresorufin O-dealkylase activities in the PB-induced hepatocyte microsomes (up to 90-fold increase). PB induction of these P-450s was markedly enhanced by the presence of dexamethasone (50 nM-1 microM), which alone was not an affective inducing agent, and was inhibited by greater than 90% by 10% fetal bovine serum. The PB response was also inhibited (greater than 85%) by growth hormone (250 ng/ml), indicating that this hormone probably acts directly on the hepatocyte when it antagonizes the induction of P-450 PB4 in intact rats. In untreated hepatocytes, P-450 RLM2 (IIA2), P-450 3 (IIA1) and NADPH P-450 reductase levels were substantially maintained in the cultures for 10-20 days. The latter two enzymes were also inducible by PB to an extent (3-4 fold elevation) that is comparable with that observed in the liver in vivo. Moreover, P-450c (IA1) and P-450 3 (IIA1) were highly inducible by 3-methylcholanthrene (5 microM; 48 h exposure) even after 3 weeks in culture. In contrast, the male-specific pituitary-regulated P-450 form 2c (IIC11) was rapidly lost upon culturing the hepatocytes, suggesting that supplementation of appropriate hormonal factors may be necessary for its expression. The present hepatocyte culture system exhibits a responsiveness to drug inducers that is qualitatively and quantitatively comparable with that observed in vivo, and should prove

  5. Modulation of Cytochrome P450 Metabolism and Transport across Intestinal Epithelial Barrier by Ginger Biophenolics

    PubMed Central

    Yang, Chunhua; Donthamsetty, Shashikiran; Cantuaria, Guilherme; Jadhav, Gajanan R.; Vangala, Subrahmanyam; Reid, Michelle D.; Aneja, Ritu

    2014-01-01

    Natural and complementary therapies in conjunction with mainstream cancer care are steadily gaining popularity. Ginger extract (GE) confers significant health-promoting benefits owing to complex additive and/or synergistic interactions between its bioactive constituents. Recently, we showed that preservation of natural “milieu” confers superior anticancer activity on GE over its constituent phytochemicals, 6-gingerol (6G), 8-gingerol (8G), 10-gingerol (10G) and 6-shogaol (6S), through enterohepatic recirculation. Here we further evaluate and compare the effects of GE and its major bioactive constituents on cytochrome P450 (CYP) enzyme activity in human liver microsomes by monitoring metabolites of CYP-specific substrates using LC/MS/MS detection methods. Our data demonstrate that individual gingerols are potent inhibitors of CYP isozymes, whereas GE exhibits a much higher half-maximal inhibition value, indicating no possible herb-drug interactions. However, GE's inhibition of CYP1A2 and CYP2C8 reflects additive interactions among the constituents. In addition, studies performed to evaluate transporter-mediated intestinal efflux using Caco-2 cells revealed that GE and its phenolics are not substrates of P-glycoprotein (Pgp). Intriguingly, however, 10G and 6S were not detected in the receiver compartment, indicating possible biotransformation across the Caco-2 monolayer. These data strengthen the notion that an interplay of complex interactions among ginger phytochemicals when fed as whole extract dictates its bioactivity highlighting the importance of consuming whole foods over single agents. Our study substantiates the need for an in-depth analysis of hepatic biotransformation events and distribution profiles of GE and its active phenolics for the design of safe regimens. PMID:25251219

  6. The Ontogeny of Cytochrome P450 Enzyme Activity and Protein Abundance in Conventional Pigs in Support of Preclinical Pediatric Drug Research.

    PubMed

    Millecam, Joske; De Clerck, Laura; Govaert, Elisabeth; Devreese, Mathias; Gasthuys, Elke; Schelstraete, Wim; Deforce, Dieter; De Bock, Lies; Van Bocxlaer, Jan; Sys, Stanislas; Croubels, Siska

    2018-01-01

    Since the implementation of several legislations to improve pediatric drug research, more pediatric clinical trials are being performed. In order to optimize these pediatric trials, adequate preclinical data are necessary, which are usually obtained by juvenile animal models. The growing piglet has been increasingly suggested as a potential animal model due to a high degree of anatomical and physiological similarities with humans. However, physiological data in pigs on the ontogeny of major organs involved in absorption, distribution, metabolism, and excretion of drugs are largely lacking. The aim of this study was to unravel the ontogeny of porcine hepatic drug metabolizing cytochrome P450 enzyme (CYP450) activities as well as protein abundances. Liver microsomes from 16 conventional pigs (8 males and 8 females) per age group: 2 days, 4 weeks, 8 weeks, and 6-7 months were prepared. Activity measurements were performed with substrates of major human CYP450 enzymes: midazolam (CYP3A), tolbutamide (CYP2C), and chlorzoxazone (CYP2E). Next, the hepatic scaling factor, microsomal protein per gram liver (MPPGL), was determined to correct for enzyme losses during the fractionation process. Finally, protein abundance was determined using proteomics and correlated with enzyme activity. No significant sex differences within each age category were observed in enzyme activity or MPPGL. The biotransformation rate of all three substrates increased with age, comparable with human maturation of CYP450 enzymes. The MPPGL decreased from birth till 8 weeks of age followed by an increase till 6-7 months of age. Significant sex differences in protein abundance were observed for CYP1A2, CYP2A19, CYP3A22, CYP4V2, CYP2C36, CYP2E_1, and CYP2E_2. Midazolam and tolbutamide are considered good substrates to evaluate porcine CYP3A/2C enzymes, respectively. However, chlorzoxazone is not advised to evaluate porcine CYP2E enzyme activity. The increase in biotransformation rate with age can be

  7. Marketed Drugs Can Inhibit Cytochrome P450 27A1, a Potential New Target for Breast Cancer Adjuvant Therapy

    PubMed Central

    Mast, Natalia; Lin, Joseph B.

    2015-01-01

    Cytochrome P450 CYP27A1 is the only enzyme in humans converting cholesterol to 27-hydroxycholesterol, an oxysterol of multiple functions, including tissue-specific modulation of estrogen and liver X receptors. Both receptors seem to mediate adverse effects of 27-hydroxycholesterol in breast cancer when the levels of this oxysterol are elevated. The present work assessed druggability of CYP27A1 as a potential antibreast cancer target. We selected 26 anticancer and noncancer medications, most approved by the Food and Drug Administration, and evaluated them first in vitro for inhibition of purified recombinant CYP27A1 and binding to the enzyme active site. Six strong CYP27A1 inhibitors/binders were identified. These were the two antibreast cancer pharmaceuticals anastrozole and fadrozole, antiprostate cancer drug bicalutamide, sedative dexmedetomidine, and two antifungals ravuconazole and posaconazole. Anastrozole was then tested in vivo on mice, which received subcutaneous drug injections for 1 week. Mouse plasma and hepatic 27-hydroxycholesterol levels were decreased 2.6- and 1.6-fold, respectively, whereas plasma and hepatic cholesterol content remained unchanged. Thus, pharmacologic CYP27A1 inhibition is possible in the whole body and individual organs, but does not negatively affect cholesterol elimination. Our results enhance the potential of CYP27A1 as an antibreast cancer target, could be of importance for the interpretation of Femara versus Anastrozole Clinical Evaluation Trial, and bring attention to posaconazole as a potential complementary anti-breast cancer medication. More medications on the US market may have unanticipated off-target inhibition of CYP27A1, and we propose strategies for their identification. PMID:26082378

  8. Cytochrome P450 Activity in Ex Vivo Cornea Models and a Human Cornea Construct.

    PubMed

    Kölln, Christian; Reichl, Stephan

    2016-07-01

    The pharmacokinetic behaviors of novel ophthalmic drugs are often preliminarily investigated in preclinical studies using ex vivo animal cornea or corneal cell culture models. During transcorneal passage, topically applied drugs may be affected by drug metabolizing enzymes. The knowledge regarding the functional expression of metabolic enzymes in corneal tissue is marginal; thus, the aim of this study was to investigate cytochrome P450 activity in an organotypic three-dimensional human cornea construct and to compare it with porcine and rabbit corneas, which are commonly used ex vivo cornea models. The total cytochrome P450 activity was determined by measuring the transformation of 7-ethoxycoumarin. Furthermore, the expression of the cytochrome P450 enzyme 2D6 (CYP2D6) was investigated at the protein level using immunohistochemistry and western blotting. CYP2D6 activity measurements were performed using a d-luciferin-based assay. In summary, similar levels of the total cytochrome P450 activity were identified in all 3 cornea models. The protein expression of CYP2D6 was confirmed in the human cornea construct and porcine cornea, whereas the signals in the rabbit cornea were weak. The analysis of the CYP2D6 activity indicated similar values for the human cornea construct and porcine cornea; however, a distinctly lower activity was observed in the rabbit cornea. Copyright © 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

  9. Cytochrome P450-mediated hepatic metabolism of new fluorescent substrates in cats and dogs.

    PubMed

    van Beusekom, C D; Schipper, L; Fink-Gremmels, J

    2010-12-01

    This study aimed to investigate the biotransformation of cat liver microsomes in comparison to dogs and humans using a high throughput method with fluorescent substrates and classical inhibitors specific for certain isozymes of the human cytochrome P450 (CYP) enzyme family. The metabolic activities associated with CYP1A, CYP2B, CYP2C, CYP2D, CYP2E and CYP3A were measured. Cat liver microsomes metabolized all substrates selected for the assessment of cytochrome P450 activity. The activities associated with CYP3A and CYP2B were higher than the activities of the other measured CYPs. Substrate selectivity could be demonstrated by inhibition studies with α-naphthoflavone (CYP1A), tranylcypromine/quercetine (CYP2C), quinidine (CYP2D), diethyldithiocarbamic acid (CYP2E) and ketoconazole (CYP3A) respectively. Other prototypical inhibitors used for characterization of human CYP activities such as furafylline (CYP1A), tranylcypromine (CYP2B) and sulfaphenazole (CYP2C) did not show significant effects in cat and dog liver microsomes. Moreover, IC50-values of cat CYPs differed from dog and human CYPs underlining the interspecies differences. Gender differences were observed in the oxidation of 7-ethoxy-4-trifluoromethylcoumarin (CYP2B) and 3-[2-(N, N-diethyl-N-methylamino)ethyl]-7-methoxy-4-methylcoumarin (CYP2D), which were significantly higher in male cats than in females. Conversely, oxidation of the substrates dibenzylfluorescein (CYP2C) and 7-methoxy-4-trifluoromethylcoumarin (CYP2E) showed significant higher activities in females than in male cats. Overall CYP-activities in cat liver microsomes were lower than in those from dogs or humans, except for CYP2B. The presented difference between feline and canine CYP-activities are useful to establish dose corrections for feline patients of intensively metabolized drugs licensed for dogs or humans. © 2010 Blackwell Publishing Ltd.

  10. Isolation of the alkane inducible cytochrome P450 (P450alk) gene from the yeast Candida tropicalis

    EPA Science Inventory

    The gene for the alkane-inducible cytochrome P450, P450alk, has been isolated from the yeast Candida tropicalis by immunoscreening a λgt11 library. Isolation of the gene has been identified on the basis of its inducibility and partial DNA sequence. Transcripts of this gene were i...

  11. P-gp, MRP2 and OAT1/OAT3 mediate the drug-drug interaction between resveratrol and methotrexate

    SciTech Connect

    Jia, Yongming

    The purpose of present study was to investigate the effect of resveratrol (Res) on altering methotrexate (MTX) pharmacokinetics and clarify the related molecular mechanism. Res significantly increased rat intestinal absorption of MTX in vivo and in vitro. Simultaneously, Res inhibited MTX efflux transport in MDR1-MDCK and MRP2-MDCK cell monolayers, suggesting that the target of drug interaction was MDR1 and MRP2 in the intestine during the absorption process. Furthermore, there was a significant decrease in renal clearance of MTX after simultaneous intravenous administration. Similarly, MTX uptake was markedly inhibited by Res in rat kidney slices and hOAT1/3-HEK293 cell, indicating that OAT1more » and OAT3 were involved in the drug interaction in the kidney. Additionally, concomitant administration of Res decreased cytotoxic effects of MTX in hOAT1/3-HEK293 cells, and ameliorated nephrotoxicity caused by MTX in rats. Conversely, intestinal damage caused by MTX was not exacerbated after Res treatment. In conclusion, Res enhanced MTX absorption in intestine and decreased MTX renal elimination by inhibiting P-gp, MRP2, OAT1 and OAT3 in vivo and in vitro. Res improved MTX-induced renal damage without increasing intestinal toxicity. - Highlights: • DDI between MTX and Res will occur when they are co-administered. • The first targets of the DDI are P-gp and MRP2 located in intestine. • The second targets of the DDI are OAT1 and OAT3 in kidney. • Res improved MTX-induced renal damage without increasing intestinal toxicity.« less

  12. P450 Pharmacogenetics in Indigenous North American Populations

    PubMed Central

    Claw, Katrina G.; Robinson, Renee F.; Boyer, Bert B.; Burke, Wylie; Thummel, Kenneth E.

    2018-01-01

    Indigenous North American populations, including American Indian and Alaska Native peoples in the United States, the First Nations, Métis and Inuit peoples in Canada and Amerindians in Mexico, are historically under-represented in biomedical research, including genomic research on drug disposition and response. Without adequate representation in pharmacogenetic studies establishing genotype-phenotype relationships, Indigenous populations may not benefit fully from new innovations in precision medicine testing to tailor and improve the safety and efficacy of drug treatment, resulting in health care disparities. The purpose of this review is to summarize and evaluate what is currently known about cytochrome P450 genetic variation in Indigenous populations in North America and to highlight the importance of including these groups in future pharmacogenetic studies for implementation of personalized drug therapy. PMID:29389890

  13. CYP2J2 and CYP2C19 Are the Major Enzymes Responsible for Metabolism of Albendazole and Fenbendazole in Human Liver Microsomes and Recombinant P450 Assay Systems

    PubMed Central

    Wu, Zhexue; Lee, Doohyun; Joo, Jeongmin; Shin, Jung-Hoon; Kang, Wonku; Oh, Sangtaek; Lee, Do Yup; Lee, Su-Jun; Yea, Sung Su; Lee, Hye Suk

    2013-01-01

    Albendazole and fenbendazole are broad-spectrum anthelmintics that undergo extensive metabolism to form hydroxyl and sulfoxide metabolites. Although CYP3A and flavin-containing monooxygenase have been implicated in sulfoxide metabolite formation, the enzymes responsible for hydroxyl metabolite formation have not been identified. In this study, we used human liver microsomes and recombinant cytochrome P450s (P450s) to characterize the enzymes involved in the formation of hydroxyalbendazole and hydroxyfenbendazole from albendazole and fenbendazole, respectively. Of the 10 recombinant P450s, CYP2J2 and/or CYP2C19 was the predominant enzyme catalyzing the hydroxylation of albendazole and fenbendazole. Albendazole hydroxylation to hydroxyalbendazole is primarily mediated by CYP2J2 (0.34 μl/min/pmol P450, which is a rate 3.9- and 8.1-fold higher than the rates for CYP2C19 and CYP2E1, respectively), whereas CYP2C19 and CYP2J2 contributed to the formation of hydroxyfenbendazole from fenbendazole (2.68 and 1.94 μl/min/pmol P450 for CYP2C19 and CYP2J2, respectively, which are rates 11.7- and 8.4-fold higher than the rate for CYP2D6). Correlation analysis between the known P450 enzyme activities and the rate of hydroxyalbendazole and hydroxyfenbendazole formation in samples from 14 human liver microsomes showed that albendazole hydroxylation correlates with CYP2J2 activity and fenbendazole hydroxylation correlates with CYP2C19 and CYP2J2 activities. These findings were supported by a P450 isoform-selective inhibition study in human liver microsomes. In conclusion, our data for the first time suggest that albendazole hydroxylation is primarily catalyzed by CYP2J2, whereas fenbendazole hydroxylation is preferentially catalyzed by CYP2C19 and CYP2J2. The present data will be useful in understanding the pharmacokinetics and drug interactions of albendazole and fenbendazole in vivo. PMID:23959307

  14. CYP2J2 and CYP2C19 are the major enzymes responsible for metabolism of albendazole and fenbendazole in human liver microsomes and recombinant P450 assay systems.

    PubMed

    Wu, Zhexue; Lee, Doohyun; Joo, Jeongmin; Shin, Jung-Hoon; Kang, Wonku; Oh, Sangtaek; Lee, Do Yup; Lee, Su-Jun; Yea, Sung Su; Lee, Hye Suk; Lee, Taeho; Liu, Kwang-Hyeon

    2013-11-01

    Albendazole and fenbendazole are broad-spectrum anthelmintics that undergo extensive metabolism to form hydroxyl and sulfoxide metabolites. Although CYP3A and flavin-containing monooxygenase have been implicated in sulfoxide metabolite formation, the enzymes responsible for hydroxyl metabolite formation have not been identified. In this study, we used human liver microsomes and recombinant cytochrome P450s (P450s) to characterize the enzymes involved in the formation of hydroxyalbendazole and hydroxyfenbendazole from albendazole and fenbendazole, respectively. Of the 10 recombinant P450s, CYP2J2 and/or CYP2C19 was the predominant enzyme catalyzing the hydroxylation of albendazole and fenbendazole. Albendazole hydroxylation to hydroxyalbendazole is primarily mediated by CYP2J2 (0.34 μl/min/pmol P450, which is a rate 3.9- and 8.1-fold higher than the rates for CYP2C19 and CYP2E1, respectively), whereas CYP2C19 and CYP2J2 contributed to the formation of hydroxyfenbendazole from fenbendazole (2.68 and 1.94 μl/min/pmol P450 for CYP2C19 and CYP2J2, respectively, which are rates 11.7- and 8.4-fold higher than the rate for CYP2D6). Correlation analysis between the known P450 enzyme activities and the rate of hydroxyalbendazole and hydroxyfenbendazole formation in samples from 14 human liver microsomes showed that albendazole hydroxylation correlates with CYP2J2 activity and fenbendazole hydroxylation correlates with CYP2C19 and CYP2J2 activities. These findings were supported by a P450 isoform-selective inhibition study in human liver microsomes. In conclusion, our data for the first time suggest that albendazole hydroxylation is primarily catalyzed by CYP2J2, whereas fenbendazole hydroxylation is preferentially catalyzed by CYP2C19 and CYP2J2. The present data will be useful in understanding the pharmacokinetics and drug interactions of albendazole and fenbendazole in vivo.

  15. Co-up-regulation of three P450 genes in response to permethrin exposure in permethrin resistant house flies, Musca domestica.

    PubMed

    Zhu, Fang; Li, Ting; Zhang, Lee; Liu, Nannan

    2008-09-25

    Insects may use various biochemical pathways to enable them to tolerate the lethal action of insecticides. For example, increased cytochrome P450 detoxification is known to play an important role in many insect species. Both constitutively increased expression (overexpression) and induction of P450s are thought to be responsible for increased levels of detoxification of insecticides. However, unlike constitutively overexpressed P450 genes, whose expression association with insecticide resistance has been extensively studied, the induction of P450s is less well characterized in insecticide resistance. The current study focuses on the characterization of individual P450 genes that are induced in response to permethrin treatment in permethrin resistant house flies. The expression of 3 P450 genes, CYP4D4v2, CYP4G2, and CYP6A38, was co-up-regulated by permethrin treatment in permethrin resistant ALHF house flies in a time and dose-dependent manner. Comparison of the deduced protein sequences of these three P450s from resistant ALHF and susceptible aabys and CS house flies revealed identical protein sequences. Genetic linkage analysis located CYP4D4v2 and CYP6A38 on autosome 5, corresponding to the linkage of P450-mediated resistance in ALHF, whereas CYP4G2 was located on autosome 3, where the major insecticide resistance factor(s) for ALHF had been mapped but no P450 genes reported prior to this study. Our study provides the first direct evidence that multiple P450 genes are co-up-regulated in permethrin resistant house flies through the induction mechanism, which increases overall expression levels of P450 genes in resistant house flies. Taken together with the significant induction of CYP4D4v2, CYP4G2, and CYP6A38 expression by permethrin only in permethrin resistant house flies and the correlation of the linkage of the genes with resistance and/or P450-mediated resistance in resistant ALHF house flies, this study sheds new light on the functional importance of P450

  16. A role for cytochrome b5 in the in vivo disposition of anti-cancer and cytochrome P450 probe drugs in mice

    PubMed Central

    Henderson, Colin J.; McLaughlin, Lesley A.; Finn, Robert D.; Ronseaux, Sebastien; Kapelyukh, Yury; Wolf, C. Roland

    2014-01-01

    The role of microsomal cytochrome b5 (Cyb5) in defining the rate of drug metabolism and disposition has been intensely debated for several decades. Recently we described mouse models involving the hepatic or global deletion of Cyb5, demonstrating its central role in in vivo drug disposition. We have now used the cytochrome b5 complete null (BCN) model to determine the role of Cyb5 in the metabolism of ten pharmaceuticals metabolised by a range of cytochrome P450s, including five anti-cancer drugs, in vivo and in vitro. The extent to which metabolism was significantly affected by the absence of Cyb5 was substrate-dependent, with AUC increased (75-245%), and clearance decreased (35-72%), for phenacetin, metoprolol and chlorzoxazone. Tolbutamide disposition was not significantly altered by Cyb5 deletion, while for midazolam clearance was decreased by 66%. The absence of Cyb5 had no effect on gefitinib and paclitaxel disposition, while significant changes in the in vivo pharmacokinetics of cyclophosphamide were measured (Cmax and terminal half-life increased 55% and 40%, respectively), tamoxifen (AUClast and Cmax increased 370% and 233%, respectively) and anastrozole (AUC and terminal half-life increased 125% and 62%, respectively; clearance down 80%). These data from provide strong evidence that both hepatic and extra-hepatic Cyb5 levels are an important determinant of in vivo drug disposition catalysed by a range of cytochrome P450s, including currently-prescribed anti-cancer agents, and that individuality in Cyb5 expression could be a significant determinant in rates of drug disposition in man. PMID:24115751

  17. Interactions between clinically used drugs and oral contraceptives.

    PubMed Central

    Bolt, H M

    1994-01-01

    Metabolism of contraceptive compounds may be influenced by various drugs. Of clinical importance is induction by barbiturates, by diphenylhydantoin, and especially by rifampicin, of enzymes that are responsible for degradation of estrogens. The major target is the hepatic microsomal estrogen-2-hydroxylase (cytochrome P450 3A4). Another type of interaction of drugs with disposition and effectiveness of estrogens is impairment of their enterohepatic circulation. This may be due to absorption of biliary estrogen conjugates (e.g., by cholestyramine) or to insufficient cleavage of the conjugate by intestinal bacteria, the latter being observed after administration of antibiotics (e.g., ampicillin, neomycin). PMID:7698081

  18. Conformational change of cytochrome P450 1A2 induced by phospholipids and detergents.

    PubMed

    Yun, C H; Song, M; Kim, H

    1997-08-08

    . It was found that the activity increase of rabbit P450 1A2 coincides with the conformational change including raised alpha-helix content. It is proposed that the interaction with the phospholipid molecules surrounding P450 1A2 in the endoplasmic reticulum is important for a functional conformation of P450 1A2 in a monooxygenase system including NADPH-P450 reductase.

  19. Membrane microparticles mediate transfer of P-glycoprotein to drug sensitive cancer cells.

    PubMed

    Bebawy, M; Combes, V; Lee, E; Jaiswal, R; Gong, J; Bonhoure, A; Grau, G E R

    2009-09-01

    Multidrug resistance (MDR), a significant impediment to the successful treatment of cancer clinically, has been attributed to the overexpression of P-glycoprotein (P-gp), a plasma membrane multidrug efflux transporter. P-gp maintains sublethal intracellular drug concentrations by virtue of its drug efflux capacity. The cellular regulation of P-gp expression is currently known to occur at either pre- or post-transcriptional levels. In this study, we identify a 'non-genetic' mechanism whereby microparticles (MPs) serve as vectors in the acquisition and spread of MDR. MPs isolated from drug-resistant cancer cells (VLB(100)) were co-cultured with drug sensitive cells (CCRF-CEM) over a 4 h period to allow for MP binding and P-gp transfer. Presence of P-gp on MPs was established using flow cytometry (FCM) and western blotting. Whole-cell drug accumulation assays using rhodamine 123 and daunorubicin (DNR) were carried out to validate the transfer of functional P-gp after co-culture. We establish that MPs shed in vitro from drug-resistant cancer cells incorporate cell surface P-gp from their donor cells, effectively bind to drug-sensitive recipient cells and transfer functional P-gp to the latter. These findings serve to substantially advance our understanding of the molecular basis for the emergence of MDR in cancer clinically and lead to new treatment strategies which target and inhibit MP mediated transfer of P-gp during the course of treatment.

  20. Prediction of Cytochrome P450 Profiles of Environmental Chemicals with QSAR Models Built from Drug-like Molecules

    EPA Science Inventory

    The human cytochrome P450 (CYP450) enzyme family is involved in the biotransformation of many environmental chemicals. As part of the U.S. Tox21 effort, we profiled the CYP450 activity of ~2800 chemicals predominantly of environmental concern against CYP1A2, CYP2C19, CYP2C9, CYP2...

  1. Analysis of the complex formation, interaction and electron transfer pathway between the "open" conformation of NADPH-cytochrome P450 reductase and aromatase.

    PubMed

    Dai, Yuejie; Zhen, Jing; Zhang, Xiuli; Zhong, Yonghui; Liu, Shaodan; Sun, Ziyue; Guo, Yue; Wu, Qingli

    2015-09-01

    The complex structure of human aromatase (CYP19) and the open form of ΔTGEE mutant NADPH-cytochrome P450 reductase (mCPR) was constructed using template-based protein alignment method. Dynamic simulation of formed complex was performed on NAMD 2.9, in which CHARMm all 27_prot_lipid_na force field and an explicit TIP3P water solvent model were applied. The result showed mCPR in its open conformation could steadily combine with aromatase from the proximal face. Data analysis indicates hydrogen bonds and four salt bridges on the binding surface enhance the interaction between the two protein molecules. Amino acid, Lys108 plays a key role in aromatase activity through the formation of a salt bridge with Asp147 and two hydrogen bonds with Asp147 and Gln150 in mCPR. The optimal pathway for the first electron transfer from CPR to aromatase was revealed and calculated using HARLEM software. The rates for solvent mediated and non-solvent mediated electron transfer from FMNH2 to heme were determined as 1.04×10(6)s(-)(1) and 4.86×10(5)s(-)(1) respectively, which indicates the solvent water can facilitate the electron transfer from FMNH2 to heme. This study presents a novel strategy for the study of the protein-protein interactions based on the template-based protein alignment, which may help new aromtase development targeting the electron transfer between mCPR and aromatase. Copyright © 2015 Elsevier Inc. All rights reserved.

  2. Molecular LEGO by domain-imprinting of cytochrome P450 BM3.

    PubMed

    Jetzschmann, K J; Yarman, A; Rustam, L; Kielb, P; Urlacher, V B; Fischer, A; Weidinger, I M; Wollenberger, U; Scheller, F W

    2018-04-01

    Electrosynthesis of the MIP nano-film after binding of the separated domains or holo-cytochrome BM3 via an engineered anchor should result in domain-specific cavities in the polymer layer. Both the two domains and the holo P450 BM3 have been bound prior polymer deposition via a N-terminal engineered his6-anchor to the electrode surface. Each step of MIP preparation was characterized by cyclic voltammetry of the redox-marker ferricyanide. Rebinding after template removal was evaluated by quantifying the suppression of the diffusive permeability of the signal for ferricyanide and by the NADH-dependent reduction of cytochrome c by the reductase domain (BMR). The working hypothesis is verified by the discrimination of the two domains by the respective MIPs: The holoenzyme P450 BM3 was ca. 5.5 times more effectively recognized by the film imprinted with the oxidase domain (BMO) as compared to the BMR-MIP or the non-imprinted polymer (NIP). Obviously, a cavity is formed during the imprinting process around the his 6 -tag-anchored BMR which cannot accommodate the broader BMO or the P450 BM3. The affinity of the MIP towards P450 BM3 is comparable with that to the monomer in solution. The his 6 -tagged P450 BM3 binds (30 percent) stronger which shows the additive effect of the interaction with the MIP and the binding to the electrode. Copyright © 2018. Published by Elsevier B.V.

  3. Interaction between S100P and the anti-allergy drug cromolyn

    SciTech Connect

    Penumutchu, Srinivasa R.; Chou, Ruey-Hwang; Department of Biotechnology, Asia University, Taichung 413, Taiwan

    2014-11-21

    Highlights: • The interaction between S100P–cromolyn was investigated by fluorescence spectroscopy. • The interfacial residues on S100P and cromolyn contact surface were mapped by {sup 1}H-{sup 15}N HSQC experiments. • S100P–cromolyn complex model was generated from NMR restraints using HADDOCK program. • The stability of the S100P–cromolyn complex was studied using molecular dynamics simulations. - Abstract: The S100P protein has been known to mediate cell proliferation by binding the receptor for advanced glycation end products (RAGE) to activate signaling pathways, such as the extracellular regulated kinase (ERK) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathways. S100P/RAGE signaling ismore » involved in a variety of diseases, such as cancer, metastasis, and diabetes. Cromolyn is an anti-allergy drug that binds S100P to block the interaction between S100P and RAGE. In the present study, we characterized the properties of the binding between cromolyn and calcium-bound S100P using various biophysical techniques. The binding affinity for S100P and cromolyn was measured to be in the millimolar range by fluorescence spectroscopy. NMR-HSQC titration experiments and HADDOCK modeling was employed to determine the spatial structure of the proposed heterotetramer model of the S100P–cromolyn complex. Additional MD simulation results revealed the important properties in the complex stability and conformational flexibility of the S100P–cromolyn complex. This proposed model has provided an understanding of the molecular level interactions of S100P–cromolyn complex.« less

  4. [Effect of bemethyl on cytochrome P-450-dependent monoxygenases in the human liver and lymphocytes].

    PubMed

    Sorokina, E A; Sibiriak, S V; Sergeeva, S A

    2002-01-01

    Effects of the actoprotector bemithyl (50 mg/kg, p.o.) upon a single or five-fold administration on the cytochrome P-450 and b5 content and the isoform-specific and nonspecific monooxygenase activity [aminopyrine-N-demethylase, aniline-p-hydroxylase, 4-nitroanisole-o-demethylase,2,5-diphenyloxazole-p-hydroxylase, 7-ethoxyresorufin-o-deethylase (EROD), benzyloxyresorufin-o-debenzylase (BROD)] in rat liver were evaluated. In addition, the influence of bemithyl (0.(1)-100 microM) on the development of EROD and BROD activity was studied on the mitogen-stimulated human lymphocytes in vitro. Administered in rats, bemithyl exhibited the properties of a cytochrome P-450 inductor of the mixed type, which was manifested by an increase in the total cytochrome P-450 content in liver microsomes and in the monooxygenase activity related to both Ah-receptor-dependent and -independent isoforms (except for the aniline-p-hydroxylase activity). The induction of the monooxygenase activity realized by Ah-receptor-dependent isoforms (4-nitroanisole-o-demethylase, 2,5-diphenyloxazole-p-hydroxylase, and EROD activity) was more pronounced, reaching maximum upon a single drug administration. Acting upon the human lymphocytes in vitro, high concentrations of bemithyl increased expression of the EROD activity, while low drug concentrations stimulated the BROD activity.

  5. Label-free genotyping of cytochrome P450 2D6*10 using ligation-mediated strand displacement amplification with DNAzyme-based chemiluminescence detection.

    PubMed

    Wang, Hong-Qi; Wu, Zhan; Zhang, Yan; Tang, Li-Juan; Yu, Ru-Qin; Jiang, Jian-Hui

    2012-01-13

    Genotyping of cytochrome P450 monooxygenase 2D6*10 (CYP2D6*10) plays an important role in pharmacogenomics, especially in clinical drug therapy of Asian populations. This work reported a novel label-free technique for genotyping of CYP2D6*10 based on ligation-mediated strand displacement amplification (SDA) with DNAzyme-based chemiluminescence detection. Discrimination of single-base mismatch is firstly accomplished using DNA ligase to generate a ligation product. The ligated product then initiates a SDA reaction to produce aptamer sequences against hemin, which can be probed by chemiluminescence detection. The proposed strategy is used for the assay of CYP2D6*10 target and the genomic DNA. The results reveal that the proposed technique displays chemiluminescence responses in linear correlation to the concentrations of DNA target within the range from 1 pM to 1 nM. A detection limit of 0.1 pM and a signal-to-background ratio of 57 are achieved. Besides such high sensitivity, the proposed CYP2D6*10 genotyping strategy also offers superb selectivity, great robustness, low cost and simplified operations due to its label-free, homogeneous, and chemiluminescence-based detection format. These advantages suggest this technique may hold considerable potential for clinical CYP2D6*10 genotyping and association studies. Copyright © 2011 Elsevier B.V. All rights reserved.

  6. Single-molecule height measurements on microsomal cytochrome P450 in nanometer-scale phospholipid bilayer disks

    NASA Astrophysics Data System (ADS)

    Bayburt, Timothy H.; Sligar, Stephen G.

    2002-05-01

    The architecture of membrane proteins in their native environment of the phospholipid bilayer is critical for understanding physiological function, but has been difficult to realize experimentally. In this communication we describe the incorporation of a membrane-anchored protein into a supported phospholipid bilayer. Cytochrome P450 2B4 solubilized and purified from the hepatic endoplasmic reticulum was incorporated into phospholipid bilayer nanostructures and oriented on a surface for visualization by atomic force microscopy. Individual P450 molecules were observed protruding from the bilayer surface. Problems associated with deformation of the protein by the atomic force microscopy probe were avoided by analyzing force-dependent height measurements to quantitate the height of the protein above the bilayer surface. Measurements of the atomic force microscopy cantilever deflection as a function of probe-sample separation reveal that the top of the P450 opposite the N-terminal membrane anchor region sits 3.5 nanometers above the phospholipid-water boundary. Models of the orientation of the enzyme are presented and discussed in relation to membrane interactions and interaction with cytochrome P450 reductase.

  7. In Vitro-In Vivo Extrapolation of Metabolism- and Transporter-Mediated Drug-Drug Interactions-Overview of Basic Prediction Methods.

    PubMed

    Yoshida, Kenta; Zhao, Ping; Zhang, Lei; Abernethy, Darrell R; Rekić, Dinko; Reynolds, Kellie S; Galetin, Aleksandra; Huang, Shiew-Mei

    2017-09-01

    Evaluation of drug-drug interaction (DDI) risk is vital to establish benefit-risk profiles of investigational new drugs during drug development. In vitro experiments are routinely conducted as an important first step to assess metabolism- and transporter-mediated DDI potential of investigational new drugs. Results from these experiments are interpreted, often with the aid of in vitro-in vivo extrapolation methods, to determine whether and how DDI should be evaluated clinically to provide the basis for proper DDI management strategies, including dosing recommendations, alternative therapies, or contraindications under various DDI scenarios and in different patient population. This article provides an overview of currently available in vitro experimental systems and basic in vitro-in vivo extrapolation methodologies for metabolism- and transporter-mediated DDIs. Published by Elsevier Inc.

  8. Hydrogen Peroxide Inhibits Cytochrome P450 Epoxygenases

    PubMed Central

    Larsen, Brandon T.; Gutterman, David D.; Sato, Atsushi; Toyama, Kazuyoshi; Campbell, William B.; Zeldin, Darryl C.; Manthati, Vijay L.; Falck, John R.; Miura, Hiroto

    2008-01-01

    The cytochrome P450 epoxygenase (CYP)-derived metabolites of arachidonic acid the epoxyeicosatrienoic acids (EETs) and hydrogen peroxide (H2O2) both function as endothelium-derived hyperpolarizing factors (EDHFs) in the human coronary microcirculation. However, the relative importance of and potential interactions between these 2 vasodilators remain unexplored. We identified a novel inhibitory interaction between CYPs and H2O2 in human coronary arterioles, where EDHF-mediated vasodilatory mechanisms are prominent. Bradykinin induced vascular superoxide and H2O2 production in an endothelium-dependent manner and elicited a concentration-dependent dilation that was reduced by catalase but not by 14,15-epoxyeicosa-5(Z)-enoic acid (EEZE), 6-(2-propargyloxyphenyl)hexanoic acid, sulfaphenazole, or iberiotoxin. However, in the presence of catalase, an inhibitory effect of these compounds was unmasked. In a tandem-bioassay preparation, application of bradykinin to endothelium-intact donor vessels elicited dilation of downstream endothelium-denuded detectors that was partially inhibited by donor-applied catalase but not by detector-applied EEZE; however, EEZE significantly inhibited dilation in the presence of catalase. EET production by human recombinant CYP 2C9 and 2J2, 2 major epoxygenase isozymes expressed in human coronary arterioles, was directly inhibited in a concentration-dependent fashion by H2O2 in vitro, as observed by high-performance liquid chromatography (HPLC); however, EETs were not directly sensitive to oxidative modification. H2O2 inhibited dilation to arachidonic acid but not to 11,12-EET. These findings suggest that an inhibitory interaction exists between 2 EDHFs in the human coronary microcirculation. CYP epoxygenases are directly inhibited by H2O2, and this interaction may modulate vascular EET bioavailability. PMID:17975109

  9. A world of cytochrome P450s

    PubMed Central

    Nelson, David R.

    2013-01-01

    The world we live in is a biosphere influenced by all organisms who inhabit it. It is also an ecology of genes, with some having rather startling effects. The premise put forth in this issue is cytochrome P450 is a significant player in the world around us. Life and the Earth itself would be visibly different and diminished without cytochrome P450s. The contributions to this issue range from evolution on the billion year scale to the colour of roses, from Darwin to Rachel Carson; all as seen through the lens of cytochrome P450. PMID:23297353

  10. Transcriptome analysis and identification of P450 genes relevant to imidacloprid detoxification in Bradysia odoriphaga.

    PubMed

    Chen, Chengyu; Wang, Cuicui; Liu, Ying; Shi, Xueyan; Gao, Xiwu

    2018-02-07

    Pesticide tolerance poses many challenges for pest control, particularly for destructive pests such as Bradysia odoriphaga. Imidacloprid has been used to control B. odoriphaga since 2013, however, imidacloprid resistance in B. odoriphaga has developed in recent years. Identifying actual and potential genes involved in detoxification metabolism of imidacloprid could offer solutions for controlling this insect. In this study, RNA-seq was used to explore differentially expressed genes in B. odoriphaga that respond to imidacloprid treatment. Differential expression data between imidacloprid treatment and the control revealed 281 transcripts (176 with annotations) showing upregulation and 394 transcripts (235 with annotations) showing downregulation. Among them, differential expression levels of seven P450 unigenes were associated with imidacloprid detoxification mechanism, with 4 unigenes that were upregulated and 3 unigenes that were downregulated. The qRT-PCR results of the seven differential expression P450 unigenes after imidacloprid treatment were consistent with RNA-Seq data. Furthermore, oral delivery mediated RNA interference of these four upregulated P450 unigenes followed by an insecticide bioassay significantly increased the mortality of imidacloprid-treated B. odoriphaga. This result indicated that the four upregulated P450s are involved in detoxification of imidacloprid. This study provides a genetic basis for further exploring P450 genes for imidacloprid detoxification in B. odoriphaga.

  11. Species-related exposure of phase II metabolite gemfibrozil 1-O-β-glucuronide between human and mice: A net induction of mouse P450 activity was revealed.

    PubMed

    Luo, Min; Dai, Manyun; Lin, Hante; Xie, Minzhu; Lin, Jiao; Liu, Aiming; Yang, Julin

    2017-12-01

    Gemfibrozil is a fibrate drug used widely for dyslipidemia associated with atherosclerosis. Clinically, both gemfibrozil and its phase II metabolite gemfibrozil 1-O-β-glucuronide (gem-glu) are involved in drug-drug interaction (DDI). But the DDI risk caused by gem-glu between human and mice has not been compared. In this study, six volunteers were recruited and took a therapeutic dose of gemfibrozil for 3 days for examination of the gemfibrozil and gem-glu level in human. Male mice were fed a gemfibrozil diet (0.75%) for 7 days, following which a cocktail-based inhibitory DDI experiment was performed. Plasma samples and liver tissues from mice were collected for determination of gemfibrozil, gem-glu concentration and cytochrome p450 enzyme (P450) induction analysis. In human, the molar ratio of gem-glu/gemfibrozil was 15% and 10% at the trough concentration and the concentration at 1.5 h after the 6th dose. In contrast, this molar ratio at steady state in mice was 91%, demonstrating a 6- to 9-fold difference compared with that in human. Interestingly, a net induction of P450 activity and in vivo inductive DDI potential in mice was revealed. The P450 activity was not inhibited although the gem-glu concentration was high. These data suggested species difference of relative gem-glu exposure between human and mice, as well as a net inductive DDI potential of gemfibrozil in mouse model. Copyright © 2017 John Wiley & Sons, Ltd.

  12. Reduction of aromatic and heterocyclic aromatic N-hydroxylamines by human cytochrome P450 2S1.

    PubMed

    Wang, Kai; Guengerich, F Peter

    2013-06-17

    Many aromatic amines and heterocyclic aromatic amines (HAAs) are known carcinogens for animals, and there is also strong evidence of some in human cancer. The activation of these compounds, including some arylamine drugs, involves N-hydroxylation, usually by cytochrome P450 enzymes (P450) in Family 1 (1A2, 1A1, and 1B1). We previously demonstrated that the bioactivation product of the anticancer agent 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole (5F 203), an N-hydroxylamine, can be reduced by P450 2S1 to its amine precursor under anaerobic conditions and, to a lesser extent, under aerobic conditions [Wang, K., and Guengerich, F. P. (2012) Chem. Res. Toxicol. 25, 1740-1751]. In the study presented here, we tested the hypothesis that P450 2S1 is involved in the reductive biotransformation of known carcinogenic aromatic amines and HAAs. The N-hydroxylamines of 4-aminobiphenyl (4-ABP), 2-naphthylamine (2-NA), and 2-aminofluorene (2-AF) were synthesized and found to be reduced by P450 2S1 under both anaerobic and aerobic conditions. The formation of amines due to P450 2S1 reduction also occurred under aerobic conditions but was less apparent because the competitive disproportionation reactions (of the N-hydroxylamines) also yielded amines. Further, some nitroso and nitro derivatives of the arylamines could also be reduced by P450 2S1. None of the amines tested were oxidized by P450 2S1. These results suggest that P450 2S1 may be involved in the reductive detoxication of several of the activated products of carcinogenic aromatic amines and HAAs.

  13. Reduction of Aromatic and Heterocyclic Aromatic N-Hydroxylamines by Human Cytochrome P450 2S1

    PubMed Central

    Wang, Kai; Guengerich, F. Peter

    2013-01-01

    Many aromatic amines and heterocyclic aromatic amines (HAAs) are known carcinogens for animals and there is also strong evidence for some in human cancer. The activation of these compounds, including some arylamine drugs, involves N-hydroxylation, usually by cytochrome P450 enzymes (P450) in Family 1 (1A2, 1A1, and 1B1). We previously demonstrated that the bioactivation product of the anti-cancer agent 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole (5F 203), an N-hydroxylamine, can be reduced by P450 2S1 to its amine precursor under anaerobic conditions and, to a lesser extent, under aerobic conditions (Wang, K., and Guengerich, F. P. (2012) Chem. Res. Toxicol. 25, 1740–1751). In the present study, we tested the hypothesis that P450 2S1 is involved in the reductive biotransformation of known carcinogenic aromatic amines and HAAs. The N-hydroxylamines of 4-aminobiphenyl (4-ABP), 2-naphthylamine (2-NA), and 2-aminofluorene (2-AF) were synthesized and found to be reduced by P450 2S1 under both anaerobic and aerobic conditions. The formation of amines due to P450 2S1 reduction also occurred under aerobic conditions but was less apparent because the competitive disproportionation reactions (of the N-hydroxylamines) also yielded amines. Further, some nitroso and nitro derivatives of the arylamines could also be reduced by P450 2S1. None of the amines tested were oxidized by P450 2S1. These results suggest that P450 2S1 may be involved in the reductive detoxication of several of the activated products of carcinogenic aromatic amines and HAAs. PMID:23682735

  14. The relevance of cytochrome P450 polymorphism in forensic medicine and akathisia-related violence and suicide.

    PubMed

    Eikelenboom-Schieveld, Selma J M; Lucire, Yolande; Fogleman, James C

    2016-07-01

    Adverse drug reactions and interactions are among the major causes of death in the United States. Antidepressants have been reported as causing suicide and homicide and share the class attribute of frequently producing akathisia, a state of severe restlessness associated with thoughts of death and violence. Medical examiners can now identify some pharmacogenetic interactions that cause drugs, deemed safe for most, to be lethal to others. Such deaths do not yet include medication-induced, akathisia-related suicides and homicides. An extrapyramidal side effect, akathisia is a manifestation of drug toxicity whose causes lie, inter alia, in drugs, doses, and co-prescribed medications that inhibit and compete for metabolizing enzymes, which may themselves be defective. In this paper, we report our investigation into adverse drug reactions/interactions in three persons who committed homicide, two also intending suicide, while on antidepressants prescribed for stressful life events. Their histories of medication use, adverse reactions and reasons for changes in medications are presented. DNA samples were screened for variants in the cytochrome P450 gene family; that produce drug metabolizing enzymes. All three cases exhibit genotype-based diminished metabolic capability that, in combination with their enzyme inhibiting/competing medications, decreased metabolism further and are the likely cause of these catastrophic events. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

  15. Resistance to lambda-cyhalothrin in Spanish field populations of Ceratitis capitata and metabolic resistance mediated by P450 in a resistant strain.

    PubMed

    Arouri, Rabeh; Le Goff, Gaelle; Hemden, Hiethem; Navarro-Llopis, Vicente; M'saad, Mariem; Castañera, Pedro; Feyereisen, René; Hernández-Crespo, Pedro; Ortego, Félix

    2015-09-01

    The withdrawal of malathion in the European Union in 2009 resulted in a large increase in lambda-cyhalothrin applications for the control of the Mediterranean fruit fly, Ceratitis capitata, in Spanish citrus crops. Spanish field populations of C. capitata have developed resistance to lambda-cyhalothrin (6-14-fold), achieving LC50 values (129-287 ppm) higher than the recommended concentration for field treatments (125 ppm). These results contrast with the high susceptibility to lambda-cyhalothrin found in three Tunisian field populations. We have studied the mechanism of resistance in the laboratory-selected resistant strain W-1Kλ (205-fold resistance). Bioassays with synergists showed that resistance was almost completely suppressed by the P450 inhibitor PBO. The study of the expression of 53 P450 genes belonging to the CYP4, CYP6, CYP9 and CYP12 families in C. capitata revealed that CYP6A51 was overexpressed (13-18-fold) in the resistant strain. The W-1Kλ strain also showed high levels of cross-resistance to etofenprox (240-fold) and deltamethrin (150-fold). Field-evolved resistance to lambda-cyhalothrin has been found in C. capitata. Metabolic resistance mediated by P450 appears to be the main resistance mechanism in the resistant strain W-1Kλ. The levels of cross-resistance found may compromise the effectiveness of other pyrethroids for the control of this species. © 2014 Society of Chemical Industry. © 2014 Society of Chemical Industry.

  16. Target mediated drug disposition with drug–drug interaction, Part II: competitive and uncompetitive cases

    PubMed Central

    Jusko, William J.; Schropp, Johannes

    2017-01-01

    We present competitive and uncompetitive drug–drug interaction (DDI) with target mediated drug disposition (TMDD) equations and investigate their pharmacokinetic DDI properties. For application of TMDD models, quasi-equilibrium (QE) or quasi-steady state (QSS) approximations are necessary to reduce the number of parameters. To realize those approximations of DDI TMDD models, we derive an ordinary differential equation (ODE) representation formulated in free concentration and free receptor variables. This ODE formulation can be straightforward implemented in typical PKPD software without solving any non-linear equation system arising from the QE or QSS approximation of the rapid binding assumptions. This manuscript is the second in a series to introduce and investigate DDI TMDD models and to apply the QE or QSS approximation. PMID:28074396

  17. Mechanism-based pharmacokinetic modeling to evaluate transporter-enzyme interplay in drug interactions and pharmacogenetics of glyburide.

    PubMed

    Varma, Manthena V S; Scialis, Renato J; Lin, Jian; Bi, Yi-An; Rotter, Charles J; Goosen, Theunis C; Yang, Xin

    2014-07-01

    The purpose of this study is to characterize the involvement of hepato-biliary transport and cytochrome-P450 (CYP)-mediated metabolism in the disposition of glyburide and predict its pharmacokinetic variability due to drug interactions and genetic variations. Comprehensive in vitro studies suggested that glyburide is a highly permeable drug with substrate affinity to multiple efflux pumps and to organic anion transporting polypeptide (OATP)1B1 and OATP2B1. Active hepatic uptake was found to be significantly higher than the passive uptake clearance (15.8 versus 5.3 μL/min/10(6)-hepatocytes), using the sandwich-cultured hepatocyte model. In vitro, glyburide is metabolized (intrinsic clearance, 52.9 μL/min/mg-microsomal protein) by CYP3A4, CYP2C9, and CYP2C8 with fraction metabolism of 0.53, 0.36, and 0.11, respectively. Using these in vitro data, physiologically based pharmacokinetic models, assuming rapid-equilibrium between blood and liver compartments or permeability-limited hepatic disposition, were built to describe pharmacokinetics and evaluate drug interactions. Permeability-limited model successfully predicted glyburide interactions with rifampicin and other perpetrator drugs. Conversely, model assuming rapid-equilibrium mispredicted glyburide interactions, overall, suggesting hepatic uptake as the primary rate-determining process in the systemic clearance of glyburide. Further modeling and simulations indicated that the impairment of CYP2C9 function has a minimal effect on the systemic exposure, implying discrepancy in the contribution of CYP2C9 to glyburide clearance.

  18. Preparation and characterization of monoclonal antibodies recognizing unique epitopes on sexually differentiated rat liver cytochrome P-450 isozymes.

    PubMed

    Morgan, E T; Rönnholm, M; Gustafsson, J A

    1987-07-14

    Cytochrome P-450 isozymes P-450(16 alpha), P-450(15 beta), and P-450DEa are immunochemically related, as indicated by mutual cross-reactivity with polyclonal antibody preparations. We have isolated five monoclonal antibodies to P-450(15 beta) and one antibody to P-450(16 alpha) that show selectivity for the respective antigens. High frequencies of cross-reactivity were observed, indicating a high degree of homology among P-450(16 alpha), P-450(15 beta), and P-450DEa. All of the P-450(15 beta-specific antibodies bound to the same epitope, or closely grouped epitopes, supporting this conclusion. The specificity of each monoclonal antibody was characterized by enzyme-linked immunosorbent assay. Western immunoblotting, and antibody-Sepharose immunoadsorption of solubilized rat liver microsomes. Antibodies F22 and F23, which were apparently identical, were specific for P-450(15 beta) by these criteria. However, the apparent specificities of antibodies F3 and F20 for P-450(15 beta), and of M16 for P-450(16 alpha), were highly dependent on the analytical technique used. The five anti-P-450(15 beta) antibodies all inhibited the catalytic activity of microsomal P-450(15 beta), by a maximum of 70%. However, they also produced a similar inhibition of microsomal P-450(16 alpha-specific antibody M16 and F23 have a low-affinity interaction with an epitope on P-450(16 alpha). The P-450(16 alpha)-specific antibody M16 was not inhibitory. The results indicate that the apparent specificity of a monoclonal antibody for an antigen determined by, e.g., Western blotting does not allow the conclusive identification of a protein in another system, e.g., immunoprecipitation of in vitro translation reaction products.(ABSTRACT TRUNCATED AT 250 WORDS)

  19. Mangifera indica L. extract and mangiferin modulate cytochrome P450 and UDP-glucuronosyltransferase enzymes in primary cultures of human hepatocytes.

    PubMed

    Rodeiro, Idania; José Gómez-Lechón, M; Perez, Gabriela; Hernandez, Ivones; Herrera, José Alfredo; Delgado, Rene; Castell, José V; Teresa Donato, M

    2013-05-01

    The aqueous stem bark extract of Mangifera indica L. (MSBE) has been reported to have antioxidant, anti-inflammatory and analgesic properties. In previous studies, we showed that MSBE and mangiferin, its main component, lower the activity of some cytochrome P-450 (P450) enzymes in rat hepatocytes and human liver microsomes. In the present study, the effects of MSBE and mangiferin on several P450 enzymes and UDP-glucuronosyltransferases (UGTs) in human-cultured hepatocytes have been examined. After hepatocytes underwent a 48-h treatment with sub-cytotoxic concentrations of the products (50-250 µg/mL), a concentration-dependent decrease of the activity of the five P450 enzymes measured (CYP1A2, 2A6, 2C9, 2D6 and 3A4) was observed. For all the activities, a reduction of at least 50% at the highest concentration (250 µg/mL) was observed. In addition, UGT activities diminished. MSBE considerably reduced UGT1A9 activity (about 60% at 250 µg/mL) and lesser effects on the other UGTs. In contrast, 250 µg/mL mangiferin had greater effects on UGT1A1 and 2B7 than on UGT1A9 (about 55% vs. 35% reduction, respectively). Quantification of specific mRNAs revealed reduced CYP3A4 and 3A5 mRNAs content, and an increase in CYP1A1, CYP1A2, UGT1A1 and UGT1A9 mRNAs. No remarkable effects on the CYP2A6, 2B6, 2C9, 2C19, 2D6 and 2E1 levels were observed. Our results suggest that the activity and/or expression of major P450 and UGT enzymes is modulated by MSBE and that potential herb-drugs interactions could arise after a combined intake of this extract with conventional medicines. Therefore, the potential safety risks of this natural product derived by altering the ADMET properties of co-administered drugs should be examined. Copyright © 2012 John Wiley & Sons, Ltd.

  20. Cytochrome P-450 isoforms involved in carboxylic acid ester cleavage of Hantzsch pyridine ester of pranidipine.

    PubMed

    Kudo, S; Okumura, H; Miyamoto, G; Ishizaki, T

    1999-02-01

    Cytochrome P-450 (CYP) isoforms responsible for the cleavage of Hantzsch pyridine ester at the 3-position of pranidipine were studied in vitro using cDNA-expressed human CYP enzymes. CYP1A1, 1A2, 2D6, and 3A4 cleaved the ester with a catalytic activity of 5.5, 0. 93, 13.1, and 22.4 nmol/30 min/nmol P-450, respectively. CYP2A6, 2B6, 2C8, 2C9, 2C19, and 2E1 were not involved in the de-esterification. The Km and Vmax values for the de-esterification were 11.8 microM and 0.47 nmol/min/nmol P-450 in the CYP2D6-catalyzed reaction and 8. 7 microM and 0.84 nmol/min/nmol P-450 in the CYP3A4-catalyzed reaction. The intrinsic clearance (Vmax/Km) of the de-esterification by CYP3A4 was 2-fold greater than that by CYP2D6. Quinidine almost completely inhibited the CYP2D6-mediated de-esterification at the concentration of 1 x 10(-6) M. Ketoconazole and troleandomycin inhibited the CYP3A4-mediated reaction in a dose-related manner. The results indicate that although the multiple CYP isoforms can catalyze the de-esterification, CYP3A4 and 2D6 are the major isoforms.

  1. Drug Modulation of Water–Heme Interactions in Low-Spin P450 Complexes of CYP2C9d and CYP125A1

    PubMed Central

    Conner, Kip P.; Cruce, Alex A.; Krzyaniak, Matthew D.; Schimpf, Alina M.; Frank, Daniel J.; de Montellano, Paul Ortiz; Atkins, William M.; Bowman, Michael K.

    2015-01-01

    Azoles and pyridines are commonly incorporated into small molecule inhibitor scaffolds that target cytochromes P450 (CYPs) as a strategy to increase drug binding affinity, impart isoform-dependent selectivity, and improve metabolic stability. Optical absorbance spectra of the CYP–inhibitor complex are widely used to infer whether these inhibitors are ligated directly to the heme iron as catalytically inert, low-spin (type II) complexes. Here, we show that the low-spin complex between a drug-metabolizing CYP2C9 variant and 4-(3-phenyl-propyl)-1H-1,2,3-triazole (PPT) retains an axial water ligand despite exhibiting elements of “classic” type II optical behavior. Hydrogens of the axial water ligand are observed by pulsed electron paramagnetic resonance (EPR) spectroscopy for both inhibitor-free and inhibitor-bound species and show that inhibitor binding does not displace the axial water. A 15N label incorporated into PPT is 0.444 nm from the heme iron, showing that PPT is also in the active site. The reverse type I inhibitor, LP10, of CYP125A1 from Mycobacterium tuberculosis, known from X-ray crystal structures to form a low-spin water-bridged complex, is found by EPR and by visible and near-infrared magnetic circular dichroism spectroscopy to retain the axial water ligand in the complex in solution. PMID:25591012

  2. Venetoclax (ABT-199) Might Act as a Perpetrator in Pharmacokinetic Drug–Drug Interactions

    PubMed Central

    Weiss, Johanna; Gajek, Thomas; Köhler, Bruno Christian; Haefeli, Walter Emil

    2016-01-01

    Venetoclax (ABT-199) represents a specific B-cell lymphoma 2 (Bcl-2) inhibitor that is currently under development for the treatment of lymphoid malignancies. So far, there is no published information on its interaction potential with important drug metabolizing enzymes and drug transporters, or its efficacy in multidrug resistant (MDR) cells. We therefore scrutinized its drug–drug interaction potential in vitro. Inhibition of cytochrome P450 enzymes (CYPs) was quantified by commercial kits. Inhibition of drug transporters (P-glycoprotein (P-gp, ABCB1), breast cancer resistance protein (BCRP), and organic anion transporting polypeptides (OATPs)) was evaluated by the use of fluorescent probe substrates. Induction of drug transporters and drug metabolizing enzymes was quantified by real-time RT-PCR. The efficacy of venetoclax in MDR cells lines was evaluated with proliferation assays. Venetoclax moderately inhibited P-gp, BCRP, OATP1B1, OATP1B3, CYP3A4, and CYP2C19, whereas CYP2B6 activity was increased. Venetoclax induced the mRNA expression of CYP1A1, CYP1A2, UGT1A3, and UGT1A9. In contrast, expression of ABCB1 was suppressed, which might revert tumor resistance towards antineoplastic P-gp substrates. P-gp over-expression led to reduced antiproliferative effects of venetoclax. Effective concentrations for inhibition and induction lay in the range of maximum plasma concentrations of venetoclax, indicating that it might act as a perpetrator drug in pharmacokinetic drug–drug interactions. PMID:26927160

  3. Physiogenomic analysis of CYP450 drug metabolism correlates dyslipidemia with pharmacogenetic functional status in psychiatric patients

    PubMed Central

    Ruaño, Gualberto; Villagra, David; Szarek, Bonnie; Windemuth, Andreas; Kocherla, Mohan; Gorowski, Krystyna; Berrezueta, Christopher; Schwartz, Harold I; Goethe, John

    2011-01-01

    Aims To investigate associations between novel human cytochrome P450 (CYP450) combinatory (multigene) and substrate-specific drug metabolism indices, and elements of metabolic syndrome, such as low density lipoprotein cholesterol (LDLc), high density lipoprotein cholesterol (HDLc), triglycerides and BMI, using physiogenomic analysis. Methods CYP2C9, CYP2C19 and CYP2D6 genotypes and clinical data were obtained for 150 consecutive, consenting hospital admissions with a diagnosis of major depressive disorder and who were treated with psychotropic medications. Data analysis compared clinical measures of LDLc, HDLc, triglyceride and BMI with novel combinatory and substrate-specific CYP450 drug metabolism indices. Results We found that a greater metabolic reserve index score is related to lower LDLc and higher HDLc, and that a greater metabolic alteration index score corresponds with higher LDLc and lower HLDc values. We also discovered that the sertraline drug-specific indices correlated with cholesterol and triglyceride values. Conclusions Overall, we demonstrated how a multigene approach to CYP450 genotype analysis yields more accurate and significant results than single-gene analyses. Ranking the individual with respect to the population represents a potential tool for assessing risk of dyslipidemia in major depressive disorder patients who are being treated with psychotropics. In addition, the drug-specific indices appear useful for modeling a variable of potential relevance to an individual’s risk of drug-related dyslipidemia. PMID:21861666

  4. Physiogenomic analysis of CYP450 drug metabolism correlates dyslipidemia with pharmacogenetic functional status in psychiatric patients.

    PubMed

    Ruaño, Gualberto; Villagra, David; Szarek, Bonnie; Windemuth, Andreas; Kocherla, Mohan; Gorowski, Krystyna; Berrezueta, Christopher; Schwartz, Harold I; Goethe, John

    2011-08-01

    To investigate associations between novel human cytochrome P450 (CYP450) combinatory (multigene) and substrate-specific drug metabolism indices, and elements of metabolic syndrome, such as low density lipoprotein cholesterol (LDLc), high density lipoprotein cholesterol (HDLc), triglycerides and BMI, using physiogenomic analysis. CYP2C9, CYP2C19 and CYP2D6 genotypes and clinical data were obtained for 150 consecutive, consenting hospital admissions with a diagnosis of major depressive disorder and who were treated with psychotropic medications. Data analysis compared clinical measures of LDLc, HDLc, triglyceride and BMI with novel combinatory and substrate-specific CYP450 drug metabolism indices. We found that a greater metabolic reserve index score is related to lower LDLc and higher HDLc, and that a greater metabolic alteration index score corresponds with higher LDLc and lower HLDc values. We also discovered that the sertraline drug-specific indices correlated with cholesterol and triglyceride values. Overall, we demonstrated how a multigene approach to CYP450 genotype analysis yields more accurate and significant results than single-gene analyses. Ranking the individual with respect to the population represents a potential tool for assessing risk of dyslipidemia in major depressive disorder patients who are being treated with psychotropics. In addition, the drug-specific indices appear useful for modeling a variable of potential relevance to an individual's risk of drug-related dyslipidemia.

  5. p110α Inhibition Overcomes Stromal Cell-Mediated Ibrutinib Resistance in Mantle Cell Lymphoma.

    PubMed

    Guan, Jiyu; Huang, Dan; Yakimchuk, Konstantin; Okret, Sam

    2018-05-01

    Acquired resistance to cancer drugs is common, also for modern targeted drugs like the Bruton tyrosine kinase (BTK) inhibitor ibrutinib, a new drug approved for the treatment of the highly aggressive and relapsing mantle cell lymphoma (MCL). The tumor microenvironment often impacts negatively on drug response. Here, we demonstrate that stromal cells protect MCL cells from ibrutinib-induced apoptosis and support MCL cell regrowth after drug removal by impairing ibrutinib-mediated downregulation of PI3K/AKT signaling. Importantly, the stromal cell-mediated ibrutinib resistance was overcome in vitro by inhibiting AKT activity using the PI3K catalytic p110α subunit-specific inhibitor BYL719. This was seen both for MCL cell lines and primary MCL cells. Furthermore, inhibition of p110α activity by BYL719 potentiated the ability of ibrutinib to inhibit MCL tumor growth in vivo in a mouse xenograft model. The stromal cell-mediated ibrutinib resistance was found to be due to a direct interaction with MCL cells and involves the integrin VLA-4, as disrupting stromal cell-MCL cell interaction using a VLA-4 blocking antibody abrogated the ibrutinib resistance. This suggests that combined treatment with ibrutinib and a p110α inhibitor, alternatively by disrupting stromal cell-MCL cell interaction, may be a promising therapeutic strategy to overcome stromal cell-mediated ibrutinib resistance in MCL. Mol Cancer Ther; 17(5); 1090-100. ©2018 AACR . ©2018 American Association for Cancer Research.

  6. Cytochromes P450 Catalyze the Reduction of α,β-Unsaturated Aldehydes

    PubMed Central

    Amunom, Immaculate; Dieter, Laura J.; Tamasi, Viola; Cai, Jan; Conklin, Daniel J.; Srivastava, Sanjay; Martin, Martha V.; Guengerich, F. Peter; Prough, Russell A.

    2011-01-01

    The metabolism of α,β-unsaturated aldehydes, e.g. 4-hydroxynonenal, involves oxidation to carboxylic acids, reduction to alcohols, and glutathionylation to eventually form mercapturide conjugates. Recently we demonstrated that P450s can oxidize aldehydes to carboxylic acids, a reaction previously thought to involve aldehyde dehydrogenase. When recombinant cytochrome P450 3A4 was incubated with 4-hydroxynonenal, O2, and NADPH, several products were produced, including 1,4-dihydroxynonene (DHN), 4-hydroxy-2-nonenoic acid (HNA), and an unknown metabolite. Several P450s catalyzed the reduction reaction in the order (human) P450 2B6 ≅ P450 3A4 > P450 1A2 > P450 2J2 > (mouse) P450 2c29. Other P450s did not catalyze the reduction reaction (human P450 2E1 & rabbit P450 2B4). Metabolism by isolated rat hepatocytes showed that HNA formation was inhibited by cyanamide, while DHN formation was not affected. Troleandomycin increased HNA production 1.6-fold while inhibiting DHN formation, suggesting that P450 3A11 is a major enzyme involved in rat hepatic clearance of 4-HNE. A fluorescent assay was developed using 9-anthracenealdehyde to measure both reactions. Feeding mice diet containing t-butylated hydroxyanisole increased the level of both activities with hepatic microsomal fractions, but not proportionally. Miconazole (0.5 mM) was a potent inhibitor of these microsomal reduction reactions, while phenytoin and α-naphthoflavone (both at 0.5 mM) were partial inhibitors, suggesting the role of multiple P450 enzymes. The oxidative metabolism of these aldehydes was inhibited >90% in an Ar or CO atmosphere, while the reductive reactions were not greatly affected. These results suggest that P450s are significant catalysts of reduction of α,β-unsaturated aldehydes in liver. PMID:21766881

  7. Psychedelic 5-methoxy-N,N-dimethyltryptamine: metabolism, pharmacokinetics, drug interactions, and pharmacological actions.

    PubMed

    Shen, Hong-Wu; Jiang, Xi-Ling; Winter, Jerrold C; Yu, Ai-Ming

    2010-10-01

    5-methoxy-N,N-dimethyltryptamine (5-MeO-DMT) belongs to a group of naturally-occurring psychoactive indolealkylamine drugs. It acts as a nonselective serotonin (5-HT) agonist and causes many physiological and behavioral changes. 5-MeO-DMT is O-demethylated by polymorphic cytochrome P450 2D6 (CYP2D6) to an active metabolite, bufotenine, while it is mainly inactivated through the deamination pathway mediated by monoamine oxidase A (MAO-A). 5-MeO-DMT is often used with MAO-A inhibitors such as harmaline. Concurrent use of harmaline reduces 5-MeO-DMT deamination metabolism and leads to a prolonged and increased exposure to the parent drug 5-MeO-DMT, as well as the active metabolite bufotenine. Harmaline, 5-MeO-DMT and bufotenine act agonistically on serotonergic systems and may result in hyperserotonergic effects or serotonin toxicity. Interestingly, CYP2D6 also has important contribution to harmaline metabolism, and CYP2D6 genetic polymorphism may cause considerable variability in the metabolism, pharmacokinetics and dynamics of harmaline and its interaction with 5-MeO-DMT. Therefore, this review summarizes recent findings on biotransformation, pharmacokinetics, and pharmacological actions of 5-MeO-DMT. In addition, the pharmacokinetic and pharmacodynamic drug-drug interactions between harmaline and 5-MeO-DMT, potential involvement of CYP2D6 pharmacogenetics, and risks of 5-MeO-DMT intoxication are discussed.

  8. Psychedelic 5-Methoxy-N,N-dimethyltryptamine: Metabolism, Pharmacokinetics, Drug Interactions, and Pharmacological Actions

    PubMed Central

    Shen, Hong-Wu; Jiang, Xi-Ling; Winter, Jerrold C.; Yu, Ai-Ming

    2011-01-01

    5-Methoxy-N,N-dimethyltryptamine (5-MeO-DMT) belongs to a group of naturally-occurring psychoactive indolealkylamine drugs. It acts as a nonselective serotonin (5-HT) agonist and causes many physiological and behavioral changes. 5-MeO-DMT is O-demethylated by polymorphic cytochrome P450 2D6 (CYP2D6) to an active metabolite, bufotenine, while it is mainly inactivated through the deamination pathway mediated by monoamine oxidase A (MAO-A). 5-MeO-DMT is often used with MAO-A inhibitors such as harmaline. Concurrent use of harmaline reduces 5-MeO-DMT deamination metabolism and leads to a prolonged and increased exposure to the parent drug 5-MeO-DMT, as well as the active metabolite bufotenine. Harmaline, 5-MeO-DMT and bufotenine act agonistically on serotonergic systems and may result in hyperserotonergic effects or serotonin toxicity. Interestingly, CYP2D6 also has important contribution to harmaline metabolism, and CYP2D6 genetic polymorphism may cause considerable variability in the metabolism, pharmacokinetics and dynamics of harmaline and its interaction with 5-MeO-DMT. Therefore, this review summarizes recent findings on biotransformation, pharmacokinetics, and pharmacological actions of 5-MeO-DMT. In addition, the pharmacokinetic and pharmacodynamic drug-drug interactions between harmaline and 5-MeO-DMT, potential involvement of CYP2D6 pharmacogenetics, and risks of 5-MeO-DMT intoxication are discussed. PMID:20942780

  9. Metabolic mechanisms of drug-nutrient interactions.

    PubMed

    Hathcock, J N

    1985-01-01

    Metabolic mechanisms of nutrition and drug interactions include 1) the effects of diet on drug metabolism and action and 2) the effects of drugs on nutritional processes. The type, amount, and timing of foods consumed influence drug dissolution, absorption, distribution, metabolism, and excretion. High-fat meals enhance the absorption of griseofulvin and some other drugs. Milk and other sources of calcium inhibit absorption of tetracycline. High-fat meals increase plasma concentrations of free fatty acids and thereby displace many drugs from binding sites on plasma albumin. High-protein diets increase the activity of the mixed-function oxidase system and enhance the metabolism of numerous drugs. High-electrolyte intakes increase excretion of lithium and also diminish the action of diuretic agents. Bile acid sequestrants and some laxatives decrease lipid digestion and absorption, as well as absorption of the fat-soluble vitamins. Numerous drugs, including tetracycline and cholestyramine, bind iron and decrease its absorption. Coumarins inhibit the function of vitamin K. Phenobarbital and other anticonvulsants are inducers of cytochrome P-450 and the mixed-function oxidase system. Long-term treatment with these inducers can cause excessive metabolism and deficiency of vitamin D. Prooxidant drugs such as chloroquine, drugs detoxified by conjugation with glutathione, and alcohol can deplete reduced glutathione with consequent effects on amino acid transport and the redox status of cells. Acid-forming foods acidify the urine and increase the loss of alkaline drugs such as the amphetamines. Base-forming drugs increase the loss of acidic drugs such as barbiturates. The range of metabolic interactions of drugs and nutrients includes the full scope of physiological processes to which drugs and nutrients are subject.

  10. Cytochrome P450 Bioconjugate as a Nanovehicle for Improved Chemotherapy Treatment.

    PubMed

    Quester, Katrin; Juarez-Moreno, Karla; Secundino, Isamel; Roseinstein, Yvonne; Alejo, Karla P; Huerta-Saquero, Alejandro; Vazquez-Duhalt, Rafael

    2017-05-01

    Cancer is still a growing public health problem, especially breast cancer that is one of the most important cancers in women. Chemotherapy, even though a successful treatment, is accompanied by severe side effects. Moreover, most of the drugs used for chemotherapy are administered as prodrugs and need to be transformed to the active form by cytochromes P450 (CYPs). In addition, increasing numbers of cancer tissues show lower CYP activity than the surrounding healthy tissues in which prodrugs are preferentially activated causing cytotoxicity. Here, the design of a functionalized cytochrome P450 bioconjugate is reported as nanovehicle for the enzyme direct delivery to the tumor tissue in order to improve the local drug activation. MCF-7 breast cancer cells are treated with CYP-polyethylene glycol bioconjugate functionalized folic acid, where it activates the prodrug tamoxifen and significantly reduces the dose of tamoxifen needed to kill the tumor cells. The CYP bioconjugate covered with polyethylene glycol shows no immunogenic activity. The advantages of increasing the site-specific CYP activity in tumor tissues are discussed. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. A double transgenic mouse model expressing human pregnane X receptor and cytochrome P450 3A4

    PubMed Central

    Ma, Xiaochao; Cheung, Connie; Krausz, Kristopher W.; Shah, Yatrik M.; Wang, Ting; Idle, Jeffrey R.; Gonzalez, Frank J.

    2008-01-01

    Cytochrome P450 3A4 (CYP3A4), the most abundant human P450 in liver, participates in the metabolism of ∼50% of clinically used drugs. The pregnane X receptor (PXR), a member of the nuclear receptor superfamily, is the major activator of CYP3A4 transcription. However, due to species differences in response to PXR ligands, it is problematic to use rodents to assess CYP3A4 regulation and function. The generation of double transgenic mice expressing human PXR and CYP3A4 (TgCYP3A4/hPXR) would provide a means to this problem. In the current study, a TgCYP3A4/hPXR mouse model was generated by bacterial artificial chromosome transgenesis in Pxr-null mice. In TgCYP3A4/hPXR mice, CYP3A4 was strongly induced by rifampicin, a human-specific PXR ligand, but not by pregnenolone 16α-carbonitrile, a rodent-specific PXR ligand. Consistent with CYP3A expression, hepatic CYP3A activity increased ∼five-fold in TgCYP3A4/hPXR mice pretreated with rifampicin. Most anti-human immunodeficiency virus protease inhibitors are CYP3A substrates and their interactions with rifamycins are a source of major concern in patients co-infected with human immunodeficiency virus and Mycobacterium tuberculosis. By using TgCYP3A4/hPXR mice, human PXR-CYP3A4 mediated rifampicin-protease inhibitor interactions were recapitulated, as the metabolic stability of amprenavir, nelfinavir, and saquinavir decreased 52%, 53%, and 99% respectively in the liver microsomes of TgCYP3A4/hPXR mice pretreated with rifampicin. In vivo, rifampicin pretreatment resulted in ∼80% decrease in the area under serum amprenavir concentration-time curve in TgCYP3A4/hPXR mice. These results suggest that the TgCYP3A4/hPXR mouse model could serve as a useful tool for studies on CYP3A4 transcription and function in vivo. PMID:18799805

  12. In vivo assessment of botanical supplementation on human cytochrome P450 phenotypes: Citrus aurantium, Echinacea purpurea, milk thistle, and saw palmetto.

    PubMed

    Gurley, Bill J; Gardner, Stephanie F; Hubbard, Martha A; Williams, D Keith; Gentry, W Brooks; Carrier, Julie; Khan, Ikhlas A; Edwards, David J; Shah, Amit

    2004-11-01

    Phytochemical-mediated modulation of cytochrome P450 (CYP) activity may underlie many herb-drug interactions. Single-time point phenotypic metabolic ratios were used to determine whether long-term supplementation of Citrus aurantium , Echinacea purpurea , milk thistle (Silybum marianum), or saw palmetto (Serenoa repens) extracts affected CYP1A2, CYP2D6, CYP2E1, or CYP3A4 activity. Twelve healthy volunteers (6 women, 6 men) were randomly assigned to receive C aurantium , E purpurea , milk thistle, or saw palmetto for 28 days. For each subject, a 30-day washout period was interposed between each supplementation phase. Probe drug cocktails of midazolam and caffeine, followed 24 hours later by chlorzoxazone and debrisoquin (INN, debrisoquine), were administered before (baseline) and at the end of supplementation. Presupplementation and postsupplementation phenotypic trait measurements were determined for CYP3A4, CYP1A2, CYP2E1, and CYP2D6 by use of 1-hydroxymidazolam/midazolam serum ratios (1-hour sample), paraxanthine/caffeine serum ratios (6-hour sample), 6-hydroxychlorzoxazone/chlorzoxazone serum ratios (2-hour sample), and debrisoquin urinary recovery ratios (8-hour collection), respectively. The content of purported "active" phytochemicals was determined for each supplement. Comparisons of presupplementation and postsupplementation phenotypic ratios suggested that these particular supplements had no significant effect on CYP1A2, CYP2D6, CYP2E1, or CYP3A4 activity. Phytochemical profiles indicated that C aurantium was devoid of the CYP3A4 inhibitor 6',7'-dihydroxybergamottin. Quantities of fatty acids, flavonolignans, and cichoric acid were consistent with label claims for saw palmetto, milk thistle, and E purpurea , respectively. Botanical supplements containing C aurantium , milk thistle, or saw palmetto extracts appear to pose a minimal risk for CYP-mediated herb-drug interactions in humans. Although the effects of E purpurea on CYP activity were minor, further

  13. Olfactory cytochrome P-450. Studies with suicide substrates of the haemoprotein.

    PubMed Central

    Reed, C J; Lock, E A; De Matteis, F

    1988-01-01

    1. The olfactory epithelium of male hamsters has been found to be extremely active in the cumene hydroperoxide-supported oxidation of tetramethylphenylenediamine, and this peroxidase activity has been shown to be cytochrome P-450-dependent. 2. The interaction of a series of suicide substrates of cytochrome P-450 with the hepatic and olfactory mono-oxygenase systems has been assessed by determination of peroxidase, 7-ethoxycoumarin O-de-ethylase (ECOD) and 7-ethoxyresorufin O-de-ethylase (EROD) activities after treatment in vivo with these compounds. Chloramphenicol, OOS-trimethylphosphorothiolate and two dihydropyridines [DDC (3,5-diethoxycarbonyl-1,4-dihydrocollidine) and 4-ethyl DDC (3,5-diethoxycarbonyl-4-ethyl-1,4-dihydro-2,6-dimethylpyridine)] all caused similar percentage inhibitions of hepatic and olfactory activities, but the absolute amounts of enzymic activity lost were considerably greater in the latter tissue. In contrast, halothane had little effect upon hepatic cytochrome P-450-dependent reactions, whereas it severely inhibited those of the olfactory epithelium. 3. The time course of loss and recovery of hepatic and olfactory peroxidase, ECOD and EROD activities after a single dose of 4-ethyl DDC was studied. The rates of loss of activity observed were very similar, irrespective of tissue or reaction examined. In the olfactory epithelium, all three activities recovered concurrently and at a rate similar to that of the hepatic peroxidase activity. In contrast, the hepatic de-ethylation of 7-ethoxycoumarin and 7-ethoxy-resorufin recovered significantly more rapidly. 4. It is suggested that this behaviour is due to 4-ethyl DDC acting not only as a suicidal inhibitor but also as an inducer of certain forms of cytochrome P-450 in the liver; in the olfactory epithelium, however, inactivation, but not induction, occurs. Classical inducing agents were reported to have no effect upon olfactory cytochrome P-450, and in the present study neither phenobarbitone

  14. Cytochrome P450 2C9-natural antiarthritic interactions: Evaluation of inhibition magnitude and prediction from in vitro data.

    PubMed

    Tan, Boon Hooi; Ahemad, Nafees; Pan, Yan; Palanisamy, Uma Devi; Othman, Iekhsan; Yiap, Beow Chin; Ong, Chin Eng

    2018-04-01

    Many dietary supplements are promoted to patients with osteoarthritis (OA) including the three naturally derived compounds, glucosamine, chondroitin and diacerein. Despite their wide spread use, research on interaction of these antiarthritic compounds with human hepatic cytochrome P450 (CYP) enzymes is limited. This study aimed to examine the modulatory effects of these compounds on CYP2C9, a major CYP isoform, using in vitro biochemical assay and in silico models. Utilizing valsartan hydroxylase assay as probe, all forms of glucosamine and chondroitin exhibited IC 50 values beyond 1000 μM, indicating very weak potential in inhibiting CYP2C9. In silico docking postulated no interaction with CYP2C9 for chondroitin and weak bonding for glucosamine. On the other hand, diacerein exhibited mixed-type inhibition with IC 50 value of 32.23 μM and K i value of 30.80 μM, indicating moderately weak inhibition. Diacerein's main metabolite, rhein, demonstrated the same mode of inhibition as diacerein but stronger potency, with IC 50 of 6.08 μM and K i of 1.16 μM. The docking of both compounds acquired lower CDOCKER interaction energy values, with interactions dominated by hydrogen and hydrophobic bondings. The ranking with respect to inhibition potency for the investigated compounds was generally the same in both in vitro enzyme assay and in silico modeling with order of potency being diacerein/rhein > various glucosamine/chondroitin forms. In vitro-in vivo extrapolation of inhibition kinetics (using 1 + [I]/K i ratio) demonstrated negligible potential of diacerein to cause interaction in vivo, whereas rhein was predicted to cause in vivo interaction, suggesting potential interaction risk with the CYP2C9 drug substrates. Copyright © 2018 John Wiley & Sons, Ltd.

  15. Simultaneous Assessment of Clearance, Metabolism, Induction, and Drug-Drug Interaction Potential Using a Long-Term In Vitro Liver Model for a Novel Hepatitis B Virus Inhibitor.

    PubMed

    Kratochwil, Nicole A; Triyatni, Miriam; Mueller, Martina B; Klammers, Florian; Leonard, Brian; Turley, Dan; Schmaler, Josephine; Ekiciler, Aynur; Molitor, Birgit; Walter, Isabelle; Gonsard, Pierre-Alexis; Tournillac, Charles A; Durrwell, Alexandre; Marschmann, Michaela; Jones, Russell; Ullah, Mohammed; Boess, Franziska; Ottaviani, Giorgio; Jin, Yuyan; Parrott, Neil J; Fowler, Stephen

    2018-05-01

    Long-term in vitro liver models are now widely explored for human hepatic metabolic clearance prediction, enzyme phenotyping, cross-species metabolism, comparison of low clearance drugs, and induction studies. Here, we present studies using a long-term liver model, which show how metabolism and active transport, drug-drug interactions, and enzyme induction in healthy and diseased states, such as hepatitis B virus (HBV) infection, may be assessed in a single test system to enable effective data integration for physiologically based pharmacokinetic (PBPK) modeling. The approach is exemplified in the case of (3S)-4-[[(4R)-4-(2-Chloro-4-fluorophenyl)-5-methoxycarbonyl-2-thiazol-2-yl-1,4-dihydropyrimidin-6-yl]methyl]morpholine-3-carboxylic acid RO6889678, a novel inhibitor of HBV with a complex absorption, distribution, metabolism, and excretion (ADME) profile. RO6889678 showed an intracellular enrichment of 78-fold in hepatocytes, with an apparent intrinsic clearance of 5.2 µ l/min per mg protein and uptake and biliary clearances of 2.6 and 1.6 µ l/min per mg protein, respectively. When apparent intrinsic clearance was incorporated into a PBPK model, the simulated oral human profiles were in good agreement with observed data at low doses but were underestimated at high doses due to unexpected overproportional increases in exposure with dose. In addition, the induction potential of RO6889678 on cytochrome P450 (P450) enzymes and transporters at steady state was assessed and cotreatment with ritonavir revealed a complex drug-drug interaction with concurrent P450 inhibition and moderate UDP-glucuronosyltransferase induction. Furthermore, we report on the first evaluation of in vitro pharmacokinetics studies using HBV-infected HepatoPac cocultures. Thus, long-term liver models have great potential as translational research tools exploring pharmacokinetics of novel drugs in vitro in health and disease. Copyright © 2018 The Author(s).

  16. The Interaction of Ethanol Ingestion and Social Interaction with an Intoxicated Peer on the Odor-Mediated Response to the Drug in Adolescent Rats.

    PubMed

    Eade, Amber M; Youngentob, Lisa M; Youngentob, Steven L

    2016-04-01

    Using a social transmission of food preference paradigm in rats, we previously demonstrated that ethanol (EtOH) exposure during adolescence, as either an observer (interaction with an intoxicated conspecific) or demonstrator (intragastric infusion with EtOH), altered the reflexive odor-mediated responses to the drug. The 2 modes of exposure were equivalent in the magnitude of their effects. Human adolescents, however, are likely to experience the drug in a social setting as both an EtOH observer and demonstrator. That is, both interacting with an intoxicated peer and experiencing EtOH's postingestive consequences in conjunction with hematogenic olfaction. Therefore, we tested whether combined adolescent exposure as both an observer and demonstrator differed from either form of individual experience. Beginning on postnatal day (P) 29, naïve rats received EtOH or water exposures in a social interaction paradigm as either an observer, a demonstrator, or combined experience (where each animal in the interaction was, itself, an observer and demonstrator). Exposures occurred 4 times, once every 48 hours. On P37, the reflexive behavioral response to EtOH odor was tested, using whole-body plethysmography. The odor-mediated responses of adolescent EtOH observers, demonstrators, and combined exposure animals all significantly differed from controls. Compared to controls, however, the magnitude of the behavioral effect was greatest in the combined exposure animals. Moreover, combined exposure as both an EtOH observer and demonstrator significantly differed from either form of individual EtOH experience. EtOH's component chemosensory qualities are known to be central contributors to its acceptance and increases in the acceptability of EtOH's odor, resulting from a social transmission experience, are predictive of enhanced EtOH avidity in adolescence. Our findings demonstrate that combined exposure as an observer and demonstrator, within a socially relevant framework, may

  17. Analysis of Cytochrome P450 CYP119 Ligand-dependent Conformational Dynamics by Two-dimensional NMR and X-ray Crystallography*

    PubMed Central

    Basudhar, Debashree; Madrona, Yarrow; Kandel, Sylvie; Lampe, Jed N.; Nishida, Clinton R.; de Montellano, Paul R. Ortiz

    2015-01-01

    Defining the conformational states of cytochrome P450 active sites is critical for the design of agents that minimize drug-drug interactions, the development of isoform-specific P450 inhibitors, and the engineering of novel oxidative catalysts. We used two-dimensional 1H,15N HSQC chemical shift perturbation mapping of 15N-labeled Phe residues and x-ray crystallography to examine the ligand-dependent conformational dynamics of CYP119. Active site Phe residues were most affected by the binding of azole inhibitors and fatty acid substrates, in agreement with active site localization of the conformational changes. This was supported by crystallography, which revealed movement of the F-G loop with various azoles. Nevertheless, the NMR chemical shift perturbations caused by azoles and substrates were distinguishable. The absence of significant chemical shift perturbations with several azoles revealed binding of ligands to an open conformation similar to that of the ligand-free state. In contrast, 4-phenylimidazole caused pronounced NMR changes involving Phe-87, Phe-144, and Phe-153 that support the closed conformation found in the crystal structure. The same closed conformation is observed by NMR and crystallography with a para-fluoro substituent on the 4-phenylimidazole, but a para-chloro or bromo substituent engendered a second closed conformation. An open conformation is thus favored in solution with many azole ligands, but para-substituted phenylimidazoles give rise to two closed conformations that depend on the size of the para-substituent. The results suggest that ligands selectively stabilize discrete cytochrome P450 conformational states. PMID:25670859

  18. Analysis of Cytochrome P450 CYP119 Ligand-dependent Conformational Dynamics by Two-dimensional NMR and X-ray Crystallography

    DOE PAGES

    Basudhar, Debashree; Madrona, Yarrow; Kandel, Sylvie; ...

    2015-02-10

    Defining the conformational states of cytochrome P450 active sites is critical for the design of agents that minimize drug-drug interactions, the development of isoform-specific P450 inhibitors, and the engineering of novel oxidative catalysts. In this paper, we used two-dimensional 1H,15N HSQC chemical shift perturbation mapping of 15N-labeled Phe residues and x-ray crystallography to examine the ligand-dependent conformational dynamics of CYP119. Active site Phe residues were most affected by the binding of azole inhibitors and fatty acid substrates, in agreement with active site localization of the conformational changes. This was supported by crystallography, which revealed movement of the F-G loop withmore » various azoles. Nevertheless, the NMR chemical shift perturbations caused by azoles and substrates were distinguishable. The absence of significant chemical shift perturbations with several azoles revealed binding of ligands to an open conformation similar to that of the ligand-free state. In contrast, 4-phenylimidazole caused pronounced NMR changes involving Phe-87, Phe-144, and Phe-153 that support the closed conformation found in the crystal structure. The same closed conformation is observed by NMR and crystallography with a para-fluoro substituent on the 4-phenylimidazole, but a para-chloro or bromo substituent engendered a second closed conformation. An open conformation is thus favored in solution with many azole ligands, but para-substituted phenylimidazoles give rise to two closed conformations that depend on the size of the para-substituent. Finally, the results suggest that ligands selectively stabilize discrete cytochrome P450 conformational states.« less

  19. Analysis of Cytochrome P450 CYP119 Ligand-dependent Conformational Dynamics by Two-dimensional NMR and X-ray Crystallography

    SciTech Connect

    Basudhar, Debashree; Madrona, Yarrow; Kandel, Sylvie

    Defining the conformational states of cytochrome P450 active sites is critical for the design of agents that minimize drug-drug interactions, the development of isoform-specific P450 inhibitors, and the engineering of novel oxidative catalysts. In this paper, we used two-dimensional 1H,15N HSQC chemical shift perturbation mapping of 15N-labeled Phe residues and x-ray crystallography to examine the ligand-dependent conformational dynamics of CYP119. Active site Phe residues were most affected by the binding of azole inhibitors and fatty acid substrates, in agreement with active site localization of the conformational changes. This was supported by crystallography, which revealed movement of the F-G loop withmore » various azoles. Nevertheless, the NMR chemical shift perturbations caused by azoles and substrates were distinguishable. The absence of significant chemical shift perturbations with several azoles revealed binding of ligands to an open conformation similar to that of the ligand-free state. In contrast, 4-phenylimidazole caused pronounced NMR changes involving Phe-87, Phe-144, and Phe-153 that support the closed conformation found in the crystal structure. The same closed conformation is observed by NMR and crystallography with a para-fluoro substituent on the 4-phenylimidazole, but a para-chloro or bromo substituent engendered a second closed conformation. An open conformation is thus favored in solution with many azole ligands, but para-substituted phenylimidazoles give rise to two closed conformations that depend on the size of the para-substituent. Finally, the results suggest that ligands selectively stabilize discrete cytochrome P450 conformational states.« less

  20. Deletion of P399{sub E}401 in NADPH cytochrome P450 oxidoreductase results in partial mixed oxidase deficiency

    SciTech Connect

    Flueck, Christa E., E-mail: christa.flueck@dkf.unibe.ch; Mallet, Delphine; Hofer, Gaby

    2011-09-09

    Highlights: {yields} Mutations in human POR cause congenital adrenal hyperplasia. {yields} We are reporting a novel 3 amino acid deletion mutation in POR P399{sub E}401del. {yields} POR mutation P399{sub E}401del decreased P450 activities by 60-85%. {yields} Impairment of steroid metabolism may be caused by multiple hits. {yields} Severity of aromatase inhibition is related to degree of in utero virilization. -- Abstract: P450 oxidoreductase (POR) is the electron donor for all microsomal P450s including steroidogenic enzymes CYP17A1, CYP19A1 and CYP21A2. We found a novel POR mutation P399{sub E}401del in two unrelated Turkish patients with 46,XX disorder of sexual development. Recombinant PORmore » proteins were produced in yeast and tested for their ability to support steroid metabolizing P450 activities. In comparison to wild-type POR, the P399{sub E}401del protein was found to decrease catalytic efficiency of 21-hydroxylation of progesterone by 68%, 17{alpha}-hydroxylation of progesterone by 76%, 17,20-lyase action on 17OH-pregnenolone by 69%, aromatization of androstenedione by 85% and cytochrome c reduction activity by 80%. Protein structure analysis of the three amino acid deletion P399{sub E}401 revealed reduced stability and flexibility of the mutant. In conclusion, P399{sub E}401del is a novel mutation in POR that provides valuable genotype-phenotype and structure-function correlation for mutations in a different region of POR compared to previous studies. Characterization of P399{sub E}401del provides further insight into specificity of different P450s for interaction with POR as well as nature of metabolic disruptions caused by more pronounced effect on specific P450s like CYP17A1 and aromatase.« less

  1. RS-predictor: a new tool for predicting sites of cytochrome P450-mediated metabolism applied to CYP 3A4.

    PubMed

    Zaretzki, Jed; Bergeron, Charles; Rydberg, Patrik; Huang, Tao-wei; Bennett, Kristin P; Breneman, Curt M

    2011-07-25

    This article describes RegioSelectivity-Predictor (RS-Predictor), a new in silico method for generating predictive models of P450-mediated metabolism for drug-like compounds. Within this method, potential sites of metabolism (SOMs) are represented as "metabolophores": A concept that describes the hierarchical combination of topological and quantum chemical descriptors needed to represent the reactivity of potential metabolic reaction sites. RS-Predictor modeling involves the use of metabolophore descriptors together with multiple-instance ranking (MIRank) to generate an optimized descriptor weight vector that encodes regioselectivity trends across all cases in a training set. The resulting pathway-independent (O-dealkylation vs N-oxidation vs Csp(3) hydroxylation, etc.), isozyme-specific regioselectivity model may be used to predict potential metabolic liabilities. In the present work, cross-validated RS-Predictor models were generated for a set of 394 substrates of CYP 3A4 as a proof-of-principle for the method. Rank aggregation was then employed to merge independently generated predictions for each substrate into a single consensus prediction. The resulting consensus RS-Predictor models were shown to reliably identify at least one observed site of metabolism in the top two rank-positions on 78% of the substrates. Comparisons between RS-Predictor and previously described regioselectivity prediction methods reveal new insights into how in silico metabolite prediction methods should be compared.

  2. Expression, function and regulation of mouse cytochrome P450 enzymes: comparison with human P450 enzymes.

    PubMed

    Hrycay, E G; Bandiera, S M

    2009-12-01

    The present review focuses on the expression, function and regulation of mouse cytochrome P450 (Cyp) enzymes. Information compiled for mouse Cyp enzymes is compared with data collected for human CYP enzymes. To date, approximately 40 pairs of orthologous mouse-human CYP genes have been identified that encode enzymes performing similar metabolic functions. Recent knowledge concerning the tissue expression of mouse Cyp enzymes from families 1 to 51 is summarized. The catalytic activities of microsomal, mitochondrial and recombinant mouse Cyp enzymes are discussed and their involvement in the metabolism of exogenous and endogenous compounds is highlighted. The role of nuclear receptors, such as the aryl hydrocarbon receptor, constitutive androstane receptor and pregnane X receptor, in regulating the expression of mouse Cyp enzymes is examined. Targeted disruption of selected Cyp genes has generated numerous Cyp null mouse lines used to decipher the role of Cyp enzymes in metabolic, toxicological and biological processes. In conclusion, the laboratory mouse is an indispensable model for exploring human CYP-mediated activities.

  3. Effects of Electro-Acupuncture on Ovarian P450arom, P450c17α and mRNA Expression Induced by Letrozole in PCOS Rats

    PubMed Central

    Wu, Huangan; Zhao, Jimeng; Cui, Yunhua; Liu, Huirong; Wu, Lingxiang; Shi, Yin; Zhu, Bing

    2013-01-01

    Hyperandrogenism is a core factor in the series of reproductive and endocrine metabolic disorders involved in polycystic ovary syndrome (PCOS). Abnormalities in enzymatic activity and the expression of ovarian granular cell layer P450arom and theca cell P450c17α can lead to an atypical environment of local ovarian hormones, including excessive androgen levels. Rat models prepared with letrozole exhibit similar endocrine and histological changes to those that occur in human PCOS. We used such a model to study the role of electro-acupuncture (EA) in regulating ovarian P450arom and P450c17α enzymatic activity and mRNA expression in PCOS rats. Female Sprague Dawley (SD) rats aged 42 days were randomly divided into 3 groups (control, PCOS, and PCOS EA) consisting of 10 rats each. The PCOS and PCOS EA groups were administered a gavage of 1.0 mg/kg−1 of letrozole solution once daily for 21 consecutive days. Beginning in the ninth week, the PCOS EA group was administered low-frequency EA treatment daily for 14 consecutive days. After the treatment, we obtained the following results. The estrous cycles were restored in 8 of the 10 rats in the PCOS EA group, and their ovarian morphologies and ultrastructures normalized. The peripheral blood measurements (with ELISA) showed significantly decreased androgens (i.e., androstenedione and testosterone) with significantly increased estrogens (i.e., estrone, estradiol) and increased P450arom with decreased P450C17α. Immunohistochemistry and Western blotting methods showed enhanced expression of ovarian granular cell layer P450arom as well as decreased expression of theca cell layer P450C17α. Fluorescence quantitative PCR methods showed enhanced expression of ovarian granular cell layer P450arom mRNA as well as decreased expression of theca cell layer P450C17α mRNA. These results may help explain the effects of electro-acupuncture in changing the local ovarian hyperandrogenic environment and improving reproductive and

  4. Effects of electro-acupuncture on ovarian P450arom, P450c17α and mRNA expression induced by letrozole in PCOS rats.

    PubMed

    Sun, Jie; Jin, Chunlan; Wu, Huangan; Zhao, Jimeng; Cui, Yunhua; Liu, Huirong; Wu, Lingxiang; Shi, Yin; Zhu, Bing

    2013-01-01

    Hyperandrogenism is a core factor in the series of reproductive and endocrine metabolic disorders involved in polycystic ovary syndrome (PCOS). Abnormalities in enzymatic activity and the expression of ovarian granular cell layer P450arom and theca cell P450c17α can lead to an atypical environment of local ovarian hormones, including excessive androgen levels. Rat models prepared with letrozole exhibit similar endocrine and histological changes to those that occur in human PCOS. We used such a model to study the role of electro-acupuncture (EA) in regulating ovarian P450arom and P450c17α enzymatic activity and mRNA expression in PCOS rats. Female Sprague Dawley (SD) rats aged 42 days were randomly divided into 3 groups (control, PCOS, and PCOS EA) consisting of 10 rats each. The PCOS and PCOS EA groups were administered a gavage of 1.0 mg/kg(-1) of letrozole solution once daily for 21 consecutive days. Beginning in the ninth week, the PCOS EA group was administered low-frequency EA treatment daily for 14 consecutive days. After the treatment, we obtained the following results. The estrous cycles were restored in 8 of the 10 rats in the PCOS EA group, and their ovarian morphologies and ultrastructures normalized. The peripheral blood measurements (with ELISA) showed significantly decreased androgens (i.e., androstenedione and testosterone) with significantly increased estrogens (i.e., estrone, estradiol) and increased P450arom with decreased P450C17α. Immunohistochemistry and Western blotting methods showed enhanced expression of ovarian granular cell layer P450arom as well as decreased expression of theca cell layer P450C17α. Fluorescence quantitative PCR methods showed enhanced expression of ovarian granular cell layer P450arom mRNA as well as decreased expression of theca cell layer P450C17α mRNA. These results may help explain the effects of electro-acupuncture in changing the local ovarian hyperandrogenic environment and improving reproductive and

  5. Recruitment and Regulation of the Non-ribosomal Peptide Synthetase Modifying Cytochrome P450 Involved in Nikkomycin Biosynthesis.

    PubMed

    Wise, Courtney E; Makris, Thomas M

    2017-05-19

    The β-hydroxylation of l-histidine is the first step in the biosynthesis of the imidazolone base of the antifungal drug nikkomycin. The cytochrome P450 (NikQ) hydroxylates the amino acid while it is appended via a phosphopantetheine linker to the non-ribosomal peptide synthetase (NRPS) NikP1. The latter enzyme is comprised of an MbtH and single adenylation and thiolation domains, a minimal composition that allows for detailed binding and kinetics studies using an intact and homogeneous NRPS substrate. Electron paramagnetic resonance studies confirm that a stable complex is formed with NikQ and NikP1 when the amino acid is tethered. Size exclusion chromatography is used to further refine the principal components that are required for this interaction. NikQ binds NikP1 in the fully charged state, but binding also occurs when NikP1 is lacking both the phosphopantetheine arm and appended amino acid. This demonstrates that the interaction is mainly guided by presentation of the thiolation domain interface, rather than the attached amino acid. Electrochemistry and transient kinetics have been used to probe the influence of l-His-NikP1 binding on catalysis by NikQ. Unlike many P450s, the binding of substrate fails to induce significant changes on the redox potential and autoxidation properties of NikQ and slows down the binding of dioxygen to the ferrous enzyme to initiate catalysis. Collectively, these studies demonstrate a complex interplay between the NRPS maturation process and the recruitment and regulation of an auxiliary tailoring enzyme required for natural product biosynthesis.

  6. Cytochrome P450 inactivation by serum from humans with a viral infection and serum from rabbits with a turpentine-induced inflammation: the role of cytokines.

    PubMed

    Bleau, A M; Levitchi, M C; Maurice, H; du Souich, P

    2000-08-01

    Serum from humans with an acute upper respiratory viral infection and from rabbits with turpentine-induced inflammation reduce the catalytic activity of hepatic cytochrome P450 (P450). The aim of this study was to identify the serum mediators responsible for the decrease in P450 activity. Rabbit and human sera were fractionated by size exclusion chromatography and the fractions tested for their ability to reduce the activity and amount of P450 after 4 h of incubation with hepatocytes from turpentine-treated rabbits (H(INF)). Rabbit and human sera decreased P450 activity by around 40% without any change in the amount of CYP1A1 and 1A2 apoproteins. In rabbit serum, the fraction containing proteins of M(r) 23-15 kDa decreased P450 content by 41%, but did not alter the amount of the apoproteins. Anti-IL-6 antibody added to the M(r) 23-15 kDa fraction restored P450 content to 97% of control values, while anti-IL-1beta, TNF-alpha and IFN-gamma antibodies had no effect. Supporting the role of IL-6, incubation of H(INF) in the presence of IL-6 for 4 h reduced P450 content by 40%. In human serum, the fraction containing proteins of M(r) >95 kDa lowered P450 content by 43% without modifying the amounts of CYP1A1/2. Neutralization experiments showed that IFN-gamma, IL-6, and IL-1beta contributed to the decrease in P450 content. In conclusion, the present results demonstrate that IL-6, and IFN-gamma, IL-6 and IL-1beta are the serum mediators released in vivo by a turpentine-induced inflammatory reaction in the rabbit and an upper respiratory viral infection in humans, respectively, inactivating hepatic P450.

  7. Cytochrome P450 inactivation by serum from humans with a viral infection and serum from rabbits with a turpentine-induced inflammation: the role of cytokines

    PubMed Central

    Bleau, Anne-Marie; Levitchi, Mihaela C; Maurice, Hélène; du Souich, Patrick

    2000-01-01

    Serum from humans with an acute upper respiratory viral infection and from rabbits with turpentine-induced inflammation reduce the catalytic activity of hepatic cytochrome P450 (P450). The aim of this study was to identify the serum mediators responsible for the decrease in P450 activity.Rabbit and human sera were fractionated by size exclusion chromatography and the fractions tested for their ability to reduce the activity and amount of P450 after 4 h of incubation with hepatocytes from turpentine-treated rabbits (HINF). Rabbit and human sera decreased P450 activity by around 40% without any change in the amount of CYP1A1 and 1A2 apoproteins.In rabbit serum, the fraction containing proteins of Mr 23–15 kDa decreased P450 content by 41%, but did not alter the amount of the apoproteins. Anti-IL-6 antibody added to the Mr 23–15 kDa fraction restored P450 content to 97% of control values, while anti-IL-1β, TNF-α and IFN-γ antibodies had no effect. Supporting the role of IL-6, incubation of HINF in the presence of IL-6 for 4 h reduced P450 content by 40%.In human serum, the fraction containing proteins of Mr >95 kDa lowered P450 content by 43% without modifying the amounts of CYP1A1/2. Neutralization experiments showed that IFN-γ, IL-6, and IL-1β contributed to the decrease in P450 content.In conclusion, the present results demonstrate that IL-6, and IFN-γ, IL-6 and IL-1β are the serum mediators released in vivo by a turpentine-induced inflammatory reaction in the rabbit and an upper respiratory viral infection in humans, respectively, inactivating hepatic P450. PMID:10952665

  8. Regulation of Organic Anion Transporting Polypeptides (OATP) 1B1- and OATP1B3-Mediated Transport: An Updated Review in the Context of OATP-Mediated Drug-Drug Interactions.

    PubMed

    Alam, Khondoker; Crowe, Alexandra; Wang, Xueying; Zhang, Pengyue; Ding, Kai; Li, Lang; Yue, Wei

    2018-03-14

    Organic anion transporting polypeptides (OATP) 1B1 and OATP1B3 are important hepatic transporters that mediate the uptake of many clinically important drugs, including statins from the blood into the liver. Reduced transport function of OATP1B1 and OATP1B3 can lead to clinically relevant drug-drug interactions (DDIs). Considering the importance of OATP1B1 and OATP1B3 in hepatic drug disposition, substantial efforts have been given on evaluating OATP1B1/1B3-mediated DDIs in order to avoid unwanted adverse effects of drugs that are OATP substrates due to their altered pharmacokinetics. Growing evidences suggest that the transport function of OATP1B1 and OATP1B3 can be regulated at various levels such as genetic variation, transcriptional and post-translational regulation. The present review summarizes the up to date information on the regulation of OATP1B1 and OATP1B3 transport function at different levels with a focus on potential impact on OATP-mediated DDIs.

  9. Regulation of Organic Anion Transporting Polypeptides (OATP) 1B1- and OATP1B3-Mediated Transport: An Updated Review in the Context of OATP-Mediated Drug-Drug Interactions

    PubMed Central

    Alam, Khondoker; Crowe, Alexandra; Wang, Xueying; Zhang, Pengyue; Ding, Kai; Li, Lang; Yue, Wei

    2018-01-01

    Organic anion transporting polypeptides (OATP) 1B1 and OATP1B3 are important hepatic transporters that mediate the uptake of many clinically important drugs, including statins from the blood into the liver. Reduced transport function of OATP1B1 and OATP1B3 can lead to clinically relevant drug-drug interactions (DDIs). Considering the importance of OATP1B1 and OATP1B3 in hepatic drug disposition, substantial efforts have been given on evaluating OATP1B1/1B3-mediated DDIs in order to avoid unwanted adverse effects of drugs that are OATP substrates due to their altered pharmacokinetics. Growing evidences suggest that the transport function of OATP1B1 and OATP1B3 can be regulated at various levels such as genetic variation, transcriptional and post-translational regulation. The present review summarizes the up to date information on the regulation of OATP1B1 and OATP1B3 transport function at different levels with a focus on potential impact on OATP-mediated DDIs. PMID:29538325

  10. Peroxisome Proliferator-Activated Receptor α Activation Suppresses Cytochrome P450 Induction Potential in Mice Treated with Gemfibrozil.

    PubMed

    Shi, Cunzhong; Min, Luo; Yang, Julin; Dai, Manyun; Song, Danjun; Hua, Huiying; Xu, Gangming; Gonzalez, Frank J; Liu, Aiming

    2017-09-01

    Gemfibrozil, a peroxisome proliferator-activated receptor α (PPARα) agonist, is widely used for hypertriglyceridaemia and mixed hyperlipidaemia. Drug-drug interaction of gemfibrozil and other PPARα agonists has been reported. However, the role of PPARα in cytochrome P450 (CYP) induction by fibrates is not well known. In this study, wild-type mice were first fed gemfibrozil-containing diets (0.375%, 0.75% and 1.5%) for 14 days to establish a dose-response relationship for CYP induction. Then, wild-type mice and Pparα-null mice were treated with a 0.75% gemfibrozil-containing diet for 7 days. CYP3a, CYP2b and CYP2c were induced in a dose-dependent manner by gemfibrozil. In Pparα-null mice, their mRNA level, protein level and activity were induced more than those in wild-type mice. So, gemfibrozil induced CYP, and this action was inhibited by activated PPARα. These data suggested that the induction potential of CYPs was suppressed by activated PPARα, showing a potential role of this receptor in drug-drug interactions and metabolic diseases treated with fibrates. © 2017 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society).

  11. Electrochemical Detection of Anti-Breast-Cancer Agents in Human Serum by Cytochrome P450-Coated Carbon Nanotubes

    PubMed Central

    Baj-Rossi, Camilla; De Micheli, Giovanni; Carrara, Sandro

    2012-01-01

    We report on the electrochemical detection of anti-cancer drugs in human serum with sensitivity values in the range of 8–925 nA/μM. Multi-walled carbon nanotubes were functionalized with three different cytochrome P450 isoforms (CYP1A2, CYP2B6, and CYP3A4). A model used to effectively describe the cytochrome P450 deposition onto carbon nanotubes was confirmed by Monte Carlo simulations. Voltammetric measurements were performed in phosphate buffer saline (PBS) as well as in human serum, giving well-defined current responses upon addition of increasing concentrations of anti-cancer drugs. The results assert the capability to measure concentration of drugs in the pharmacological ranges in human serum. Another important result is the possibility to detect pairs of drugs present in the same sample, which is highly required in case of therapies with high side-effects risk and in anti-cancer pharmacological treatments based on mixtures of different drugs. Our technology holds potentials for inexpensive multi-panel drug-monitoring in personalized therapy. PMID:22778656

  12. Chemotherapeutic Drug Based Metal-Organic Particles for Microvesicle-Mediated Deep Penetration and Programmable pH/NIR/Hypoxia Activated Cancer Photochemotherapy.

    PubMed

    Zhang, Da; Wu, Ming; Cai, Zhixiong; Liao, Naishun; Ke, Kun; Liu, Hongzhi; Li, Ming; Liu, Gang; Yang, Huanghao; Liu, Xiaolong; Liu, Jingfeng

    2018-02-01

    A novel metal-organic particle (MOP) based nanodrug formed by mild self-assembly of chemotherapeutic drugs, including banoxantrone and doxorubicin, through Cu(II)-mediated coordination effects, is reported. In this nanodrug, Cu(II) acts as a bridge to join AQ4N and DOX, and then, self-assembly of [-AQ4N-Cu(II)-(DOX) 2 -Cu(II)-] n complexes forms nanosized MOPs (referred to as ADMOPs) through multiple interactions including host-metal-guest coordination, hydrophobic interactions, π-stacking, and van der Waals force. The ADMOPs reported here have several important features over conventional drugs, including tumor microenvironment pH-sensitive drug release that can be tracked by "turning on" the fluorescence of AQ4N or DOX through proton competition with Cu(II) to break the coordination bonds and much deeper penetration into solid tumors via microvesicle-mediated intercellular transfer. Most strikingly, the ADMOPs can serve as stimuli-responsive nanocarriers to efficiently load the photosensitizer phthalocyanine due to their inherent highly porous characteristics. Thus, the ADMOPs significantly enhance the chemotherapeutic efficacy by "on-demand" photodynamic therapy, which further induces a hypoxic environment that enhances the reduction of AQ4N to systematically increase the therapeutic efficiency. Taken together, the designed ADMOPs composed of chemotherapeutic drugs may serve as a potential programmable controlled synergistic agent for cancer therapy.

  13. Key Mutations Alter the Cytochrome P450 BM3 Conformational Landscape and Remove Inherent Substrate Bias*

    PubMed Central

    Butler, Christopher F.; Peet, Caroline; Mason, Amy E.; Voice, Michael W.; Leys, David; Munro, Andrew W.

    2013-01-01

    Cytochrome P450 monooxygenases (P450s) have enormous potential in the production of oxychemicals, due to their unparalleled regio- and stereoselectivity. The Bacillus megaterium P450 BM3 enzyme is a key model system, with several mutants (many distant from the active site) reported to alter substrate selectivity. It has the highest reported monooxygenase activity of the P450 enzymes, and this catalytic efficiency has inspired protein engineering to enable its exploitation for biotechnologically relevant oxidations with structurally diverse substrates. However, a structural rationale is lacking to explain how these mutations have such effects in the absence of direct change to the active site architecture. Here, we provide the first crystal structures of BM3 mutants in complex with a human drug substrate, the proton pump inhibitor omeprazole. Supported by solution data, these structures reveal how mutation alters the conformational landscape and decreases the free energy barrier for transition to the substrate-bound state. Our data point to the importance of such “gatekeeper” mutations in enabling major changes in substrate recognition. We further demonstrate that these mutants catalyze the same 5-hydroxylation reaction as performed by human CYP2C19, the major human omeprazole-metabolizing P450 enzyme. PMID:23828198

  14. Transposable elements are enriched within or in close proximity to xenobiotic-metabolizing cytochrome P450 genes

    PubMed Central

    Chen, Song; Li, Xianchun

    2007-01-01

    Background Transposons, i.e. transposable elements (TEs), are the major internal spontaneous mutation agents for the variability of eukaryotic genomes. To address the general issue of whether transposons mediate genomic changes in environment-adaptation genes, we scanned two alleles per each of the six xenobiotic-metabolizing Helicoverpa zea cytochrome P450 loci, including CYP6B8, CYP6B27, CYP321A1, CYP321A2, CYP9A12v3 and CYP9A14, for the presence of transposon insertions by genome walking and sequence analysis. We also scanned thirteen Drosophila melanogaster P450s genes for TE insertions by in silico mapping and literature search. Results Twelve novel transposons, including LINEs (long interspersed nuclear elements), SINEs (short interspersed nuclear elements), MITEs (miniature inverted-repeat transposable elements), one full-length transib-like transposon, and one full-length Tcl-like DNA transpson, are identified from the alleles of the six H. zea P450 genes. The twelve transposons are inserted into the 5'flanking region, 3'flanking region, exon, or intron of the six environment-adaptation P450 genes. In D. melanogaster, seven out of the eight Drosophila P450s (CYP4E2, CYP6A2, CYP6A8, CYP6A9, CYP6G1, CYP6W1, CYP12A4, CYP12D1) implicated in insecticide resistance are associated with a variety of transposons. By contrast, all the five Drosophila P450s (CYP302A1, CYP306A1, CYP307A1, CYP314A1 and CYP315A1) involved in ecdysone biosynthesis and developmental regulation are free of TE insertions. Conclusion These results indicate that TEs are selectively retained within or in close proximity to xenobiotic-metabolizing P450 genes. PMID:17381843

  15. Structural comparison of cytochromes P450 2A6, 2A13, and 2E1 with pilocarpine

    SciTech Connect

    DeVore, Natasha M.; Meneely, Kathleen M.; Bart, Aaron G.

    2013-11-20

    Human xenobiotic-metabolizing cytochrome P450 (CYP) enzymes can each bind and monooxygenate a diverse set of substrates, including drugs, often producing a variety of metabolites. Additionally, a single ligand can interact with multiple CYP enzymes, but often the protein structural similarities and differences that mediate such overlapping selectivity are not well understood. Even though the CYP superfamily has a highly canonical global protein fold, there are large variations in the active site size, topology, and conformational flexibility. We have determined how a related set of three human CYP enzymes bind and interact with a common inhibitor, the muscarinic receptor agonist drugmore » pilocarpine. Pilocarpine binds and inhibits the hepatic CYP2A6 and respiratory CYP2A13 enzymes much more efficiently than the hepatic CYP2E1 enzyme. To elucidate key residues involved in pilocarpine binding, crystal structures of CYP2A6 (2.4 {angstrom}), CYP2A13 (3.0 {angstrom}), CYP2E1 (2.35 {angstrom}), and the CYP2A6 mutant enzyme, CYP2A6 I208S/I300F/G301A/S369G (2.1 {angstrom}) have been determined with pilocarpine in the active site. In all four structures, pilocarpine coordinates to the heme iron, but comparisons reveal how individual residues lining the active sites of these three distinct human enzymes interact differently with the inhibitor pilocarpine.« less

  16. Effect of p-amino-diphenyl ethers on hepatic microsomal cytochrome P450.

    PubMed

    Jiang, Huidi; Xuan, Guida

    2003-09-01

    The present paper aims to investigate whether p-amino-2',4'-dichlorodiphenyl ether and p-amino-4'-methyldiphenyl ether are inhibitors as well as inducers of P450. Mice were given daily intraperitoneal (ip) injections of p-amino-2',4'-dichlorodiphenyl ether (0.25 mmol/kg) or p-amino-4'-methyldiphenyl ether (0.25 mmol/kg) for 4 days and tested at 24 h and 48 h after the last dose injection. The results showed the mice pentobarbital sleeping time was shorter and the P450 content of hepatic microsome increased significantly in the group pretreated with p-amino-4'-methyldiphenyl ether when compared with the control group, while in mice pretreated with p-amino-2',4'-dichlorodiphenyl ether the hepatic microsome P450 content increased but the pentobarbital sleeping time was extended in clear contrast to the control group. The sleeping time of the phenobarbital group (80 mg/kg daily ip injection for 4 days) was shortened at 24 h after the last injection with increased P450 content of hepatic microsome, but it showed no difference at 48 h. The zoxazolamine-paralysis times of mice treated with p-amino-2',4'-dichlorodiphenyl ether were longer than those of the control mice, while the same dose of zoxazolamine did not lead to paralysis in mice pretreated with BNF. p-Amino-2',4'-dichlorodiphenyl ether and p-amino-4'-methyldiphenyl ether inhibited the activity of 7-ethoxyresorufin O-deethylase from rat hepatic microsome induced by BNF in vitro by 70.0% and 50.1% respectively. These results suggest that p-amino-2',4'-dichlorodiphenyl ether and p-amino-4'-methyldiphenyl ether are inhibitors as well as inducers of P450.

  17. Comparative Analysis of P450 Signature Motifs EXXR and CXG in the Large and Diverse Kingdom of Fungi: Identification of Evolutionarily Conserved Amino Acid Patterns Characteristic of P450 Family

    PubMed Central

    Syed, Khajamohiddin; Mashele, Samson Sitheni

    2014-01-01

    Cytochrome P450 monooxygenases (P450s) are heme-thiolate proteins distributed across the biological kingdoms. P450s are catalytically versatile and play key roles in organisms primary and secondary metabolism. Identification of P450s across the biological kingdoms depends largely on the identification of two P450 signature motifs, EXXR and CXG, in the protein sequence. Once a putative protein has been identified as P450, it will be assigned to a family and subfamily based on the criteria that P450s within a family share more than 40% homology and members of subfamilies share more than 55% homology. However, to date, no evidence has been presented that can distinguish members of a P450 family. Here, for the first time we report the identification of EXXR- and CXG-motifs-based amino acid patterns that are characteristic of the P450 family. Analysis of P450 signature motifs in the under-explored fungal P450s from four different phyla, ascomycota, basidiomycota, zygomycota and chytridiomycota, indicated that the EXXR motif is highly variable and the CXG motif is somewhat variable. The amino acids threonine and leucine are preferred as second and third amino acids in the EXXR motif and proline and glycine are preferred as second and third amino acids in the CXG motif in fungal P450s. Analysis of 67 P450 families from biological kingdoms such as plants, animals, bacteria and fungi showed conservation of a set of amino acid patterns characteristic of a particular P450 family in EXXR and CXG motifs. This suggests that during the divergence of P450 families from a common ancestor these amino acids patterns evolve and are retained in each P450 family as a signature of that family. The role of amino acid patterns characteristic of a P450 family in the structural and/or functional aspects of members of the P450 family is a topic for future research. PMID:24743800

  18. Co-Prescription Trends in a Large Cohort of Subjects Predict Substantial Drug-Drug Interactions

    PubMed Central

    Sutherland, Jeffrey J.; Daly, Thomas M.; Liu, Xiong; Goldstein, Keith; Johnston, Joseph A.; Ryan, Timothy P.

    2015-01-01

    Pharmaceutical prescribing and drug-drug interaction data underlie recommendations on drug combinations that should be avoided or closely monitored by prescribers. Because the number of patients taking multiple medications is increasing, a comprehensive view of prescribing patterns in patients is important to better assess real world pharmaceutical response and evaluate the potential for multi-drug interactions. We obtained self-reported prescription data from NHANES surveys between 1999 and 2010, and confirm the previously reported finding of increasing drug use in the elderly. We studied co-prescription drug trends by focusing on the 2009-2010 survey, which contains prescription data on 690 drugs used by 10,537 subjects. We found that medication profiles were unique for individuals aged 65 years or more, with ≥98 unique drug regimens encountered per 100 subjects taking 3 or more medications. When drugs were viewed by therapeutic class, it was found that the most commonly prescribed drugs were not the most commonly co-prescribed drugs for any of the 16 drug classes investigated. We cross-referenced these medication lists with drug interaction data from Drugs.com to evaluate the potential for drug interactions. The number of drug alerts rose proportionally with the number of co-prescribed medications, rising from 3.3 alerts for individuals prescribed 5 medications to 11.7 alerts for individuals prescribed 10 medications. We found 22% of elderly subjects taking both a substrate and inhibitor of a given cytochrome P450 enzyme, and 4% taking multiple inhibitors of the same enzyme simultaneously. By examining drug pairs prescribed in 0.1% of the population or more, we found low agreement between co-prescription rate and co-discussion in the literature. These data show that prescribing trends in treatment could drive a large extent of individual variability in drug response, and that current pairwise approaches to assessing drug-drug interactions may be inadequate for

  19. Pharmacokinetic and pharmacodynamic drug interactions with ethanol (alcohol).

    PubMed

    Chan, Lingtak-Neander; Anderson, Gail D

    2014-12-01

    Ethanol (alcohol) is one of the most widely used legal drugs in the world. Ethanol is metabolized by alcohol dehydrogenase (ADH) and the cytochrome P450 (CYP) 2E1 drug-metabolizing enzyme that is also responsible for the biotransformation of xenobiotics and fatty acids. Drugs that inhibit ADH or CYP2E1 are the most likely theoretical compounds that would lead to a clinically significant pharmacokinetic interaction with ethanol, which include only a limited number of drugs. Acute ethanol primarily alters the pharmacokinetics of other drugs by changing the rate and extent of absorption, with more limited effects on clearance. Both acute and chronic ethanol use can cause transient changes to many physiologic responses in different organ systems such as hypotension and impairment of motor and cognitive functions, resulting in both pharmacokinetic and pharmacodynamic interactions. Evaluating drug interactions with long-term use of ethanol is uniquely challenging. Specifically, it is difficult to distinguish between the effects of long-term ethanol use on liver pathology and chronic malnutrition. Ethanol-induced liver disease results in decreased activity of hepatic metabolic enzymes and changes in protein binding. Clinical studies that include patients with chronic alcohol use may be evaluating the effects of mild cirrhosis on liver metabolism, and not just ethanol itself. The definition of chronic alcohol use is very inconsistent, which greatly affects the quality of the data and clinical application of the results. Our study of the literature has shown that a significantly higher volume of clinical studies have focused on the pharmacokinetic interactions of ethanol and other drugs. The data on pharmacodynamic interactions are more limited and future research addressing pharmacodynamic interactions with ethanol, especially regarding the non-central nervous system effects, is much needed.

  20. A cellular stress response (CSR) that interacts with NADPH-P450 reductase (NPR) is a new regulator of hypoxic response.

    PubMed

    Oguro, Ami; Koyama, Chika; Xu, Jing; Imaoka, Susumu

    2014-02-28

    NADPH-P450 reductase (NPR) was previously found to contribute to the hypoxic response of cells, but the mechanism was not clarified. In this study, we identified a cellular stress response (CSR) as a new factor interacting with NPR by a yeast two-hybrid system. Overexpression of CSR enhanced the induction of erythropoietin and hypoxia response element (HRE) activity under hypoxia in human hepatocarcinoma cell lines (Hep3B), while knockdown of CSR suppressed them. This new finding regarding the interaction of NPR with CSR provides insight into the function of NPR in hypoxic response. Copyright © 2014 Elsevier Inc. All rights reserved.

  1. Promising Tools in Prostate Cancer Research: Selective Non-Steroidal Cytochrome P450 17A1 Inhibitors

    PubMed Central

    Bonomo, Silvia; Hansen, Cecilie H.; Petrunak, Elyse M.; Scott, Emily E.; Styrishave, Bjarne; Jørgensen, Flemming Steen; Olsen, Lars

    2016-01-01

    Cytochrome P450 17A1 (CYP17A1) is an important target in the treatment of prostate cancer because it produces androgens required for tumour growth. The FDA has approved only one CYP17A1 inhibitor, abiraterone, which contains a steroidal scaffold similar to the endogenous CYP17A1 substrates. Abiraterone is structurally similar to the substrates of other cytochrome P450 enzymes involved in steroidogenesis, and interference can pose a liability in terms of side effects. Using non-steroidal scaffolds is expected to enable the design of compounds that interact more selectively with CYP17A1. Therefore, we combined a structure-based virtual screening approach with density functional theory (DFT) calculations to suggest non-steroidal compounds selective for CYP17A1. In vitro assays demonstrated that two such compounds selectively inhibited CYP17A1 17α-hydroxylase and 17,20-lyase activities with IC50 values in the nanomolar range, without affinity for the major drug-metabolizing CYP2D6 and CYP3A4 enzymes and CYP21A2, with the latter result confirmed in human H295R cells. PMID:27406023

  2. Cytochrome P450 in the central nervous system as a therapeutic target in neurodegenerative diseases.

    PubMed

    Navarro-Mabarak, Cynthia; Camacho-Carranza, Rafael; Espinosa-Aguirre, Jesús Javier

    2018-05-01

    Cytochromes P450 (CYPs) constitute a family of enzymes that can be found in the endoplasmic reticulum (ER), mitochondria or the cell surface of the cells. CYPs are characterized by carrying out the oxidation of organic compounds and they are mainly recognized as mediators of the biotransformation of xenobiotics to polar hydrophilic metabolites that can be eliminated from the organism. However, these enzymes play a key role in many other physiological processes, being involved in diverse indispensable metabolic pathways since they metabolize many endogenous substrates. Various CYP isoforms are expressed in the brain, and it is believed that this could be in part due to the particular function of brain CYPs. In the brain, CYPs are involved in the cholesterol turnover, the biosynthesis of dopamine, serotonin, morphine, hormones, and protective lipid mediators (epoxyeicosatrienoic acids), in addition to their already recognized role in xenobiotics detoxification and psychotropic drug metabolism. Increasing evidence suggests that this group of enzymes is fundamental for the normal functioning and maintenance of brain homeostasis. This review is focused on highlighting the importance of CYP-mediated endogenous metabolism in the central nervous system (CNS) and its relationship with recent findings regarding CYP involvement in neurodegenerative diseases. Some therapeutic approaches focused on CYP regulation are also discussed.

  3. Potential for drug interactions mediated by polymorphic flavin-containing monooxygenase 3 in human livers.

    PubMed

    Shimizu, Makiko; Shiraishi, Arisa; Sato, Ayumi; Nagashima, Satomi; Yamazaki, Hiroshi

    2015-02-01

    Human flavin-containing monooxygenase 3 (FMO3) in the liver catalyzes a variety of oxygenations of nitrogen- and sulfur-containing medicines and xenobiotic substances. Because of growing interest in drug interactions mediated by polymorphic FMO3, benzydamine N-oxygenation by human FMO3 was investigated as a model reaction. Among the 41 compounds tested, trimethylamine, methimazole, itopride, and tozasertib (50 μM) suppressed benzydamine N-oxygenation at a substrate concentration of 50 μM by approximately 50% after co-incubation. Suppression of N-oxygenation of benzydamine, trimethylamine, itopride, and tozasertib and S-oxygenation of methimazole and sulindac sulfide after co-incubation with the other five of these six substrates was compared using FMO3 proteins recombinantly expressed in bacterial membranes. Apparent competitive inhibition by methimazole (0-50 μM) of sulindac sulfide S-oxygenation was observed with FMO3 proteins. Sulindac sulfide S-oxygenation activity of Arg205Cys variant FMO3 protein was likely to be suppressed more by methimazole than wild-type or Val257Met variant FMO3 protein was. These results suggest that genetic polymorphism in the human FMO3 gene may lead to changes of drug interactions for N- or S-oxygenations of xenobiotics and endogenous substances and that a probe battery system of benzydamine N-oxygenation and sulindac sulfide S-oxygenation activities is recommended to clarify the drug interactions mediated by FMO3. Copyright © 2014 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.

  4. Thyroid hormone stimulation of NADPH P450 reductase expression in liver and extrahepatic tissues. Regulation by multiple mechanisms.

    PubMed

    Ram, P A; Waxman, D J

    1992-02-15

    The role of thyroid hormone in regulating the expression of the flavoprotein NADPH cytochrome P450 reductase was studied in adult rats. Depletion of circulating thyroid hormone by hypophysectomy, or more selectively, by treatment with the anti-thyroid drug methimazole led to a 75-85% depletion of hepatic microsomal P450 reductase activity and protein in both male and female rats. Thyroxine substantially restored P450 reductase activity at a dose that rendered the thyroid-depleted rats euthyroid. Microsomal P450 reductase activity in several extrahepatic tissues was also dependent on thyroid hormone, but to a lesser extent than in liver (30-50% decrease in kidney, adrenal, lung, and heart but not in testis from hypothyroid rats). Hepatic P450 reductase mRNA levels were also decreased in the hypothyroid state, indicating that the loss of P450 reductase activity is not a consequence of the associated decreased availability of the FMN and FAD cofactors of P450 reductase. Parallel analysis of S14 mRNA, which has been studied extensively as a model thyroid-regulated liver gene product, indicated that P450 reductase and S14 mRNA respond similarly to these changes in thyroid state. In contrast, while the expression of S14 and several other thyroid hormone-dependent hepatic mRNAs is stimulated by feeding a high carbohydrate, fat-free diet, hepatic P450 reductase expression was not increased by this lipogenic diet. Injection of hypothyroid rats with T3 at a supraphysiologic, receptor-saturating dose stimulated a major induction of hepatic P450 reductase mRNA that was detectable 4 h after the T3 injection, and peaked at approximately 650% of euthyroid levels by 12 h. However, this same treatment stimulated a biphasic increase in P450 reductase protein and activity that required 3 days to reach normal euthyroid levels. T3 treatment of euthyroid rats also stimulated a major induction of P450 reductase mRNA that was maximal (12-fold increase) by 12 h, but in this case no major

  5. A Novel Semi-biosynthetic Route for Artemisinin Production Using Engineered Substrate-Promiscuous P450BM3

    SciTech Connect

    Dietrich, Jeffrey; Yoshikuni, Yasuo; Fisher, Karl

    2009-11-30

    Production of fine heterologus pathways in microbial hosts is frequently hindered by insufficient knowledge of the native metabolic pathway and its cognate enzymes; often the pathway is unresolved and enzymes lack detailed characterization. An alternative paradigm to using native pathways is de novo pathway design using well-characterized, substrate-promiscuous enzymes. We demonstrate this concept using P450BM3 from Bacillus megaterium. Using a computer model, we illustrate how key P450BM3 activ site mutations enable binding of non-native substrate amorphadiene, incorporating these mutations into P450BM3 enabled the selective oxidation of amorphadiene arteminsinic-11s,12-epoxide, at titers of 250 mg L"1 in E. coli. We also demonstratemore » high-yeilding, selective transformations to dihydroartemisinic acid, the immediate precursor to the high value anti-malarial drug artemisinin.« less

  6. The role of renal proximal tubule P450 enzymes in chloroform-induced nephrotoxicity: Utility of renal specific P450 reductase knockout mouse models

    SciTech Connect

    Liu, Senyan; Wadsworth Center, New York State Department of Health, and School of Public Health, State University of New York, Albany, NY 12201; Yao, Yunyi

    The kidney is a primary target for numerous toxic compounds. Cytochrome P450 enzymes (P450) are responsible for the metabolic activation of various chemical compounds, and in the kidney are predominantly expressed in proximal tubules. The aim of this study was to test the hypothesis that renal proximal tubular P450s are critical for nephrotoxicity caused by chemicals such as chloroform. We developed two new mouse models, one having proximal tubule-specific deletion of the cytochrome P450 reductase (Cpr) gene (the enzyme required for all microsomal P450 activities), designated proximal tubule-Cpr-null (PTCN), and the other having proximal tubule-specific rescue of CPR activity withmore » the global suppression of CPR activity in all extra-proximal tubular tissues, designated extra-proximal tubule-Cpr-low (XPT-CL). The PTCN, XPT-CL, Cpr-low (CL), and wild-type (WT) mice were treated with a single oral dose of chloroform at 200 mg/kg. Blood, liver and kidney samples were obtained at 24 h after the treatment. Renal toxicity was assessed by measuring BUN and creatinine levels, and by pathological examination. The blood and tissue levels of chloroform were determined. The severity of toxicity was less in PTCN and CL mice, compared with that of WT and XPT-CL mice. There were no significant differences in chloroform levels in the blood, liver, or kidney, between PTCN and WT mice, or between XPT-CL and CL mice. These findings indicate that local P450-dependent activities play an important role in the nephrotoxicity induced by chloroform. Our results also demonstrate the usefulness of these novel mouse models for studies of chemical-induced kidney toxicity. - Highlights: • New mouse models were developed with varying P450 activities in the proximal tubule. • These mouse models were treated with chloroform, a nephrotoxicant. • Studies showed the importance of local P450s in chloroform-induced nephrotoxicity.« less

  7. Analysis of cytochrome P450 CYP119 ligand-dependent conformational dynamics by two-dimensional NMR and X-ray crystallography.

    PubMed

    Basudhar, Debashree; Madrona, Yarrow; Kandel, Sylvie; Lampe, Jed N; Nishida, Clinton R; de Montellano, Paul R Ortiz

    2015-04-17

    Defining the conformational states of cytochrome P450 active sites is critical for the design of agents that minimize drug-drug interactions, the development of isoform-specific P450 inhibitors, and the engineering of novel oxidative catalysts. We used two-dimensional (1)H,(15)N HSQC chemical shift perturbation mapping of (15)N-labeled Phe residues and x-ray crystallography to examine the ligand-dependent conformational dynamics of CYP119. Active site Phe residues were most affected by the binding of azole inhibitors and fatty acid substrates, in agreement with active site localization of the conformational changes. This was supported by crystallography, which revealed movement of the F-G loop with various azoles. Nevertheless, the NMR chemical shift perturbations caused by azoles and substrates were distinguishable. The absence of significant chemical shift perturbations with several azoles revealed binding of ligands to an open conformation similar to that of the ligand-free state. In contrast, 4-phenylimidazole caused pronounced NMR changes involving Phe-87, Phe-144, and Phe-153 that support the closed conformation found in the crystal structure. The same closed conformation is observed by NMR and crystallography with a para-fluoro substituent on the 4-phenylimidazole, but a para-chloro or bromo substituent engendered a second closed conformation. An open conformation is thus favored in solution with many azole ligands, but para-substituted phenylimidazoles give rise to two closed conformations that depend on the size of the para-substituent. The results suggest that ligands selectively stabilize discrete cytochrome P450 conformational states. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. Thermodynamics of camphor migration in cytochrome P450cam by atomistic simulations.

    PubMed

    Rydzewski, J; Nowak, W

    2017-08-10

    Understanding the mechanisms of ligand binding to enzymes is of paramount importance for the design of new drugs. Here, we report on the use of a novel biased molecular dynamics (MD) methodology to study the mechanism of camphor binding to cytochrome P450cam. Microsecond-long MD simulations allowed us to observe reaction coordinates characterizing ligand diffusion from the active site of cytochrome P450cam to solvent via three egress routes. These atomistic simulations were used to estimate thermodynamic quantities along the reaction coordinates and indicate diverse binding configurations. The results suggest that the diffusion of camphor along the pathway near the substrate recognition site (SRS) is thermodynamically preferred. In addition, we show that the diffusion near the SRS is triggered by a transition from a heterogeneous collection of closed ligand-bound conformers to the basin comprising the open conformations of cytochrome P450cam. The conformational change accompanying this switch is characterized by the retraction of the F and G helices and the disorder of the B' helix. These results are corroborated by experimental studies and provide detailed insight into ligand binding and conformational behavior of the cytochrome family. The presented methodology is general and can be applied to other ligand-protein systems.

  9. Modeling of Anopheles minimus Mosquito NADPH-Cytochrome P450 Oxidoreductase (CYPOR) and Mutagenesis Analysis

    PubMed Central

    Sarapusit, Songklod; Lertkiatmongkol, Panida; Duangkaew, Panida; Rongnoparut, Pornpimol

    2013-01-01

    Malaria is one of the most dangerous mosquito-borne diseases in many tropical countries, including Thailand. Studies in a deltamethrin resistant strain of Anopheles minimus mosquito, suggest cytochrome P450 enzymes contribute to the detoxification of pyrethroid insecticides. Purified A. minimus CYPOR enzyme (AnCYPOR), which is the redox partner of cytochrome P450s, loses flavin-adenosine di-nucleotide (FAD) and FLAVIN mono-nucleotide (FMN) cofactors that affect its enzyme activity. Replacement of leucine residues at positions 86 and 219 with phenylalanines in FMN binding domain increases FMN binding, enzyme stability, and cytochrome c reduction activity. Membrane-Bound L86F/L219F-AnCYPOR increases A. minimus P450-mediated pyrethroid metabolism in vitro. In this study, we constructed a comparative model structure of AnCYPOR using a rat CYPOR structure as a template. Overall model structure is similar to rat CYPOR, with some prominent differences. Based on primary sequence and structural analysis of rat and A. minimus CYPOR, C427R, W678A, and W678H mutations were generated together with L86F/L219F resulting in three soluble Δ55 triple mutants. The C427R triple AnCYPOR mutant retained a higher amount of FAD binding and increased cytochrome c reduction activity compared to wild-type and L86F/L219F-Δ55AnCYPOR double mutant. However W678A and W678H mutations did not increase FAD and NAD(P)H bindings. The L86F/L219F double and C427R triple membrane-bound AnCYPOR mutants supported benzyloxyresorufin O-deakylation (BROD) mediated by mosquito CYP6AA3 with a two-to three-fold increase in efficiency over wild-type AnCYPOR. The use of rat CYPOR in place of AnCYPOR most efficiently supported CYP6AA3-mediated BROD compared to all AnCYPORs. PMID:23325047

  10. Cytochrome P450 diversity in the tree of life.

    PubMed

    Nelson, David R

    2018-01-01

    Sequencing in all areas of the tree of life has produced >300,000 cytochrome P450 (CYP) sequences that have been mined and collected. Nomenclature has been assigned to >41,000 CYP sequences and the majority of the remainder has been sorted by BLAST searches into clans, families and subfamilies in preparation for naming. The P450 sequence space is being systematically explored and filled in. Well-studied groups like vertebrates are covered in greater depth while new insights are being added into uncharted territories like horseshoe crab (Limulus polyphemus), tardigrades (Hypsibius dujardini), velvet worm (Euperipatoides_rowelli), and basal land plants like hornworts, liverworts and mosses. CYPs from the fungi, one of the most diverse groups, are being explored and organized as nearly 800 fungal species are now sequenced. The CYP clan structure in fungi is emerging with 805 CYP families sorting into 32 CYP clans. >3000 bacterial sequences are named, mostly from terrestrial or freshwater sources. Of 18,379 bacterial sequences downloaded from the CYPED database, all are >43% identical to named CYPs. Therefore, they fit in the 602 named P450 prokaryotic families. Diversity in this group is becoming saturated, however 25% of 3305 seawater bacterial P450s did not match known P450 families, indicating marine bacterial CYPs are not as well sampled as land/freshwater based bacterial CYPs. Future sequencing plans of the Genome 10K project, i5k and GIGA (Global Invertebrate Genomics Alliance) are expected to produce more than one million cytochrome P450 sequences by 2020. This article is part of a Special Issue entitled: Cytochrome P450 biodiversity and biotechnology, edited by Erika Plettner, Gianfranco Gilardi, Luet Wong, Vlada Urlacher, Jared Goldstone. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. Metronidazole reduces the expression of cytochrome P450 enzymes in HepaRG cells and cryopreserved human hepatocytes.

    PubMed

    Kudo, Toshiyuki; Endo, Yumiko; Taguchi, Rina; Yatsu, Masami; Ito, Kiyomi

    2015-05-01

    1. Blood levels of S-warfarin have been reported to be increased by concomitant administration of metronidazole (MTZ), an antiprotozoal imidazole derivative. 2. To elucidate the mechanism of this interaction and to identify other possible drug-drug interactions, we conducted an in vitro study with the human hepatoma HepaRG cells and cryopreserved human hepatocytes on the ability of MTZ to reduce the expression of cytochrome P450 (CYP) as well as nuclear receptors that regulate the expression of these enzymes. 3. HepaRG cells and cryopreserved human hepatocytes were treated with MTZ (20 to 500 µM) and were then analyzed by real-time RT-PCR to determine mRNA levels of drug-metabolizing enzymes and nuclear receptors. 4. In both cells, the expressions of CYP2C8, CYP2C9, CYP3A4 and constitutive androstane receptor (CAR) were decreased by MTZ treatment. Particularly, in HepaRG cells, their mRNA levels were decreased by MTZ treatment in a concentration-dependent manner. 5. Our findings suggest that the interaction between MTZ and S-warfarin may be due to the MTZ-induced down-regulation of CYP2C9, the primary enzyme responsible for S-warfarin hydroxylation, and CAR, which regulates CYP2C9 expression. We also found that MTZ use may alter the disposition of drugs metabolized by the CYP isozymes investigated.

  12. Overexpression of cytochrome P450 CYP6BG1 may contribute to chlorantraniliprole resistance in Plutella xylostella (L.).

    PubMed

    Li, Xiuxia; Li, Ran; Zhu, Bin; Gao, Xiwu; Liang, Pei

    2018-06-01

    The diamondback moth Plutella xylostella (L.) is the most widely distributed pest of cruciferous crops and has developed resistance to most commonly used insecticides, including chlorantraniliprole. Resistance to chlorantraniliprole is likely caused by mutations of the target, the ryanodine receptor, and/or mediated by an increase in detoxification enzyme activities. Although target-site resistance is documented in detail, resistance mediated by increased metabolism has rarely been reported. The activity of cytochrome P450 was significantly higher in two resistant P. xylostella populations than in a susceptible one. Among ten detected cytochrome P450 genes, CYP6BG1 was significantly overexpressed (over 80-fold) in a field-resistant population compared with expression in a susceptible one. Knockdown of CYP6BG1 by RNA interference dramatically reduced the 7-ethoxycoumarin-O-deethylase (7-ECOD) activity of P450 by 45.5% and increased the toxicity of chlorantraniliprole toward P. xylostella by 26.8% at 48 h postinjection of double-stranded RNA. By contrast, overexpression of CYP6BG1 in a transgenic Drosophila melanogaster line significantly decreased the toxicity of the insecticide to the transgenic flies. Overexpression of CYP6BG1 may contribute to chlorantraniliprole resistance in P. xylostella. Our findings will provide new insights into the mechanisms of resistance to diamide insecticides in other insect pests. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

  13. Molecular characterization and expression analysis of a suite of cytochrome P450 enzymes implicated in insect hydrocarbon degradation in the entomopathogenic fungus Beauveria bassiana.

    PubMed

    Pedrini, Nicolás; Zhang, Shizhu; Juárez, M Patricia; Keyhani, Nemat O

    2010-08-01

    The insect epicuticle or waxy layer comprises a heterogeneous mixture of lipids that include abundant levels of long-chain alkanes, alkenes, wax esters and fatty acids. This structure represents the first barrier against microbial attack and for broad-host-range insect pathogens, such as Beauveria bassiana, it is the initial interface mediating the host-pathogen interaction, since these organisms do not require any specialized mode of entry and infect target hosts via the cuticle. B. bassiana is able to grow on straight chain alkanes up to n-C(33) as a sole source of carbon and energy. The cDNA and genomic sequences, including putative regulatory elements, for eight cytochrome P450 enzymes, postulated to be involved in alkane and insect epicuticle degradation, were isolated and characterized. Expression studies using a range of alkanes as well as an insect-derived epicuticular extract from the blood-sucking bug Triatomas infestans revealed a differential expression pattern for the P450 genes examined, and suggest that B. bassiana contains a series of hydrocarbon-assimilating enzymes with overlapping specificity in order to target the surface lipids of insect hosts. Phylogenetic analysis of the translated ORFs of the sequences revealed that the enzyme which displayed the highest levels of induction on both alkanes and the insect epicuticular extract represents the founding member of a new cytochrome P450 family, with three of the other sequences assigned as the first members of new P450 subfamilies. The remaining four proteins clustered with known P450 families whose members include alkane monooxygenases.

  14. Impact of Fusarium mycotoxins on hepatic and intestinal mRNA expression of cytochrome P450 enzymes and drug transporters, and on the pharmacokinetics of oral enrofloxacin in broiler chickens.

    PubMed

    Antonissen, Gunther; Devreese, Mathias; De Baere, Siegrid; Martel, An; Van Immerseel, Filip; Croubels, Siska

    2017-03-01

    Cytochrome P450 (CYP450) drug biotransformation enzymes and multidrug resistance (MDR) proteins may influence drug disposition processes. The first part of the study aimed to evaluate the effect of mycotoxins deoxynivalenol (DON) and/or fumonisins (FBs), at contamination levels approaching European Union guidance levels, on intestinal and hepatic CYP450 enzymes and MDR proteins gene expression in broiler chickens. mRNA expression of genes encoding CYP450 enzymes (CYP3A37, CYP1A4 and CYP1A5) and drug transporters (MDR1/ABCB1 and MRP2/ABCC2) was determined using qRT-PCR. A significant up-regulation of CYP1A4 (P = 0.037) and MDR1 (P = 0.036) was observed in the jejunum of chickens fed a diet contaminated with FBs. The second part of this study aimed to investigate the impact of feeding a FBs contaminated diet on the oral absorption of enrofloxacin (10 mg/kg BW), a MDR1 substrate. A significant (P = 0.045), however small, decreased area under the plasma concentration-time curve (AUC 0-48  h, mean ± SD) was observed for enrofloxacin in chickens fed the FBs contaminated diet compared to the control group, 16.28 ± 1.82 h μg/mL versus 18.27 ± 1.79 h μg/mL. These findings suggest that concurrent administration of drugs with FBs contaminated feed might alter the pharmacokinetic characteristics of CYP1A4 substrate drugs and MDR1 substrates, such as enrofloxacin. Copyright © 2017 Elsevier Ltd. All rights reserved.

  15. Identification of human cytochrome P450 and flavin-containing monooxygenase enzymes involved in the metabolism of lorcaserin, a novel selective human 5-hydroxytryptamine 2C agonist.

    PubMed

    Usmani, Khawja A; Chen, Weichao G; Sadeque, Abu J M

    2012-04-01

    Lorcaserin, a selective serotonin 5-hydroxytryptamine 2C receptor agonist, is being developed for weight management. The oxidative metabolism of lorcaserin, mediated by recombinant human cytochrome P450 (P450) and flavin-containing monooxygenase (FMO) enzymes, was examined in vitro to identify the enzymes involved in the generation of its primary oxidative metabolites, N-hydroxylorcaserin, 7-hydroxylorcaserin, 5-hydroxylorcaserin, and 1-hydroxylorcaserin. Human CYP1A2, CYP2A6, CYP2B6, CYP2C19, CYP2D6, CYP3A4, and FMO1 are major enzymes involved in N-hydroxylorcaserin; CYP2D6 and CYP3A4 are enzymes involved in 7-hydroxylorcaserin; CYP1A1, CYP1A2, CYP2D6, and CYP3A4 are enzymes involved in 5-hydroxylorcaserin; and CYP3A4 is an enzyme involved in 1-hydroxylorcaserin formation. In 16 individual human liver microsomal preparations (HLM), formation of N-hydroxylorcaserin was correlated with CYP2B6, 7-hydroxylorcaserin was correlated with CYP2D6, 5-hydroxylorcaserin was correlated with CYP1A2 and CYP3A4, and 1-hydroxylorcaserin was correlated with CYP3A4 activity at 10.0 μM lorcaserin. No correlation was observed for N-hydroxylorcaserin with any P450 marker substrate activity at 1.0 μM lorcaserin. N-Hydroxylorcaserin formation was not inhibited by CYP1A2, CYP2A6, CYP2B6, CYP2C19, CYP2D6, and CYP3A4 inhibitors at the highest concentration tested. Furafylline, quinidine, and ketoconazole, selective inhibitors of CYP1A2, CYP2D6, and CYP3A4, respectively, inhibited 5-hydroxylorcaserin (IC(50) = 1.914 μM), 7-hydroxylorcaserin (IC(50) = 0.213 μM), and 1-hydroxylorcaserin formation (IC(50) = 0.281 μM), respectively. N-Hydroxylorcaserin showed low and high K(m) components in HLM and 7-hydroxylorcaserin showed lower K(m) than 5-hydroxylorcaserin and 1-hydroxylorcaserin in HLM. The highest intrinsic clearance was observed for N-hydroxylorcaserin, followed by 7-hydroxylorcaserin, 5-hydroxylorcaserin, and 1-hydroxylorcaserin in HLM. Multiple human P450 and FMO enzymes catalyze

  16. Classification of highly unbalanced CYP450 data of drugs using cost sensitive machine learning techniques.

    PubMed

    Eitrich, T; Kless, A; Druska, C; Meyer, W; Grotendorst, J

    2007-01-01

    In this paper, we study the classifications of unbalanced data sets of drugs. As an example we chose a data set of 2D6 inhibitors of cytochrome P450. The human cytochrome P450 2D6 isoform plays a key role in the metabolism of many drugs in the preclinical drug discovery process. We have collected a data set from annotated public data and calculated physicochemical properties with chemoinformatics methods. On top of this data, we have built classifiers based on machine learning methods. Data sets with different class distributions lead to the effect that conventional machine learning methods are biased toward the larger class. To overcome this problem and to obtain sensitive but also accurate classifiers we combine machine learning and feature selection methods with techniques addressing the problem of unbalanced classification, such as oversampling and threshold moving. We have used our own implementation of a support vector machine algorithm as well as the maximum entropy method. Our feature selection is based on the unsupervised McCabe method. The classification results from our test set are compared structurally with compounds from the training set. We show that the applied algorithms enable the effective high throughput in silico classification of potential drug candidates.

  17. Antiplatelet drug interactions with proton pump inhibitors

    PubMed Central

    Scott, Stuart A; Obeng, Aniwaa Owusu; Hulot, Jean-Sébastien

    2014-01-01

    Introduction Non-aspirin antiplatelet agents (e.g., clopidogrel, prasugrel, ticagrelor) are commonly prescribed for the prevention of recurrent cardiovascular events among patients with acute coronary syndromes (ACS) and/or those undergoing percutaneous coronary intervention (PCI). In addition, combination therapy with proton pump inhibitors (PPIs) is often recommended to attenuate gastrointestinal bleeding risk, particularly during dual antiplatelet therapy (DAPT) with clopidogrel and aspirin. Importantly, a pharmacological interaction between clopidogrel and some PPIs has been proposed based on mutual CYP450-dependent metabolism, but available evidence is inconsistent. Areas covered This article provides an overview of the currently approved antiplatelet agents and PPIs, including their metabolic pathways. Additionally, the CYP450 isoenzyme at the center of the drug interaction, CYP2C19, is described in detail, and the available evidence on both the potential pharmacological interaction and influence on clinical outcomes are summarized and evaluated. Expert opinion Although concomitant DAPT and PPI use reduces clopidogrel active metabolite levels and ex vivo-measured platelet inhibition, the influence of the drug interaction on clinical outcomes has been conflicting and largely reported from non-randomized observational studies. Despite this inconsistency, a clinically important interaction cannot be definitively excluded, particularly among patient subgroups with higher overall cardiovascular risk and potentially among CYP2C19 loss-of-function allele carriers. PMID:24205916

  18. Mediator MED23 regulates basal transcription in vivo via an interaction with P-TEFb.

    PubMed

    Wang, Wei; Yao, Xiao; Huang, Yan; Hu, Xiangming; Liu, Runzhong; Hou, Dongming; Chen, Ruichuan; Wang, Gang

    2013-01-01

    The Mediator is a multi-subunit complex that transduces regulatory information from transcription regulators to the RNA polymerase II apparatus. Growing evidence suggests that Mediator plays roles in multiple stages of eukaryotic transcription, including elongation. However, the detailed mechanism by which Mediator regulates elongation remains elusive. In this study, we demonstrate that Mediator MED23 subunit controls a basal level of transcription by recruiting elongation factor P-TEFb, via an interaction with its CDK9 subunit. The mRNA level of Egr1, a MED23-controlled model gene, is reduced 4-5 fold in Med23 (-/-) ES cells under an unstimulated condition, but Med23-deficiency does not alter the occupancies of RNAP II, GTFs, Mediator complex, or activator ELK1 at the Egr1 promoter. Instead, Med23 depletion results in a significant decrease in P-TEFb and RNAP II (Ser2P) binding at the coding region, but no changes for several other elongation regulators, such as DSIF and NELF. ChIP-seq revealed that Med23-deficiency partially reduced the P-TEFb occupancy at a set of MED23-regulated gene promoters. Further, we demonstrate that MED23 interacts with CDK9 in vivo and in vitro. Collectively, these results provide the mechanistic insight into how Mediator promotes RNAP II into transcription elongation.

  19. p62 regulates CD40-mediated NFκB activation in macrophages through interaction with TRAF6

    SciTech Connect

    Seibold, Kristina; Ehrenschwender, Martin, E-mail: martin.ehrenschwender@ukr.de

    CD40 is a member of the tumor necrosis factor (TNF) receptor family. Activation-induced recruitment of adapter proteins, so-called TNF-receptor-associated factors (TRAFs) to the cytoplasmic tail of CD40 triggers signaling cascades important in the immune system, but has also been associated with excessive inflammation in diseases such as atherosclerosis and rheumatoid arthritis. Especially, pro-inflammatory nuclear factor κB (NFκB) signaling emanating from CD40-associated TRAF6 appears to be a key pathogenic driving force. Consequently, targeting the CD40-TRAF6 interaction is emerging as a promising therapeutic strategy, but the underlying molecular machinery of this signaling axis is to date poorly understood. Here, we identified themore » multifunctional adaptor protein p62 as a critical regulator in CD40-mediated NFκB signaling via TRAF6. CD40 activation triggered formation of a TRAF6-p62 complex. Disturbing this interaction tremendously reduced CD40-mediated NFκB signaling in macrophages, while TRAF6-independent signaling pathways remained unaffected. This highlights p62 as a potential target in hyper-inflammatory, CD40-associated pathologies. - Highlights: • CD40 activation triggers interaction of the adapter protein TRAF6 with p62. • TRAF6-p62 interaction regulates CD40-mediated NFκB signaling in macrophages. • Defective TRAF6-p62 interaction reduces CD40-mediated NFκB activation in macrophages.« less

  20. Clinically significant drug-drug interactions involving opioid analgesics used for pain treatment in patients with cancer: a systematic review.

    PubMed

    Kotlinska-Lemieszek, Aleksandra; Klepstad, Pål; Haugen, Dagny Faksvåg

    2015-01-01

    Opioids are the most frequently used drugs to treat pain in cancer patients. In some patients, however, opioids can cause adverse effects and drug-drug interactions. No advice concerning the combination of opioids and other drugs is given in the current European guidelines. To identify studies that report clinically significant drug-drug interactions involving opioids used for pain treatment in adult cancer patients. Systematic review with searches in Embase, MEDLINE, and Cochrane Central Register of Controlled Trials from the start of the databases (Embase from 1980) through January 2014. In addition, reference lists of relevant full-text papers were hand-searched. Of 901 retrieved papers, 112 were considered as potentially eligible. After full-text reading, 17 were included in the final analysis, together with 15 papers identified through hand-searching of reference lists. All of the 32 included publications were case reports or case series. Clinical manifestations of drug-drug interactions involving opioids were grouped as follows: 1) sedation and respiratory depression, 2) other central nervous system symptoms, 3) impairment of pain control and/or opioid withdrawal, and 4) other symptoms. The most common mechanisms eliciting drug-drug interactions were alteration of opioid metabolism by inhibiting the activity of cytochrome P450 3A4 and pharmacodynamic interactions due to the combined effect on opioid, dopaminergic, cholinergic, and serotonergic activity in the central nervous system. Evidence for drug-drug interactions associated with opioids used for pain treatment in cancer patients is very limited. Still, the cases identified in this systematic review give some important suggestions for clinical practice. Physicians prescribing opioids should recognize the risk of drug-drug interactions and if possible avoid polypharmacy.

  1. Albendazole sulfonation by rat liver cytochrome P-450c.

    PubMed

    Souhaili-El Amri, H; Mothe, O; Totis, M; Masson, C; Batt, A M; Delatour, P; Siest, G

    1988-08-01

    The metabolism of albendazole (ABZ) was studied in perfused livers from control and ABZ-treated rats (10.6 mg/kg, per os, each day for 10 days). In the perfusion fluid, the concentration of ABZ-sulfoxide (SO-ABZ) remained unchanged in treated, as compared to control animals, whereas ABZ-sulfone (SO2-ABZ) was increased in treated animals. In bile, only SO-ABZ was present. The transformation kinetics of SO-ABZ to SO2-ABZ in microsomes from rats treated with ABZ, 3-methylcholanthrene, Aroclor and isosafrole were biphasic. This suggests that enzyme activity was a consequence of two enzyme systems, one characterized by low affinity and high capacity, the other by high affinity and low capacity, the latter could be induced by 3-methylcholanthrene, ABZ, Aroclor and isosafrole. Cytochrome P-450c was induced potently in vivo by ABZ as proven by increased monooxygenase (7-ethoxyresorufin and 7-ethoxycoumarin-O-deethylase) activities and by Elisa test (a 5-fold increase in hemoprotein concentration was observed). Purified and reconstituted cytochrome P-450c from 3-methylcholanthrene or ABZ-treated rat liver were able to produce SO2-ABZ (2.01 and 1.70 nmol/mg/15 min, respectively, whereas cytochrome P-450b produced 10 times less SO2-ABZ). Immunological assays, as well as activity measurements showed a relationship between cytochrome P-450c-3-methylcholanthrene and cytochrome P-450c-ABZ. We conclude that induction of cytochrome P-450c by ABZ is the probable explanation for the enhanced formation of SO2-ABZ in vivo.

  2. Comparison of inhibitory effects of the proton pump-inhibiting drugs omeprazole, esomeprazole, lansoprazole, pantoprazole, and rabeprazole on human cytochrome P450 activities.

    PubMed

    Li, Xue-Qing; Andersson, Tommy B; Ahlström, Marie; Weidolf, Lars

    2004-08-01

    The human clearance of proton pump inhibitors (PPIs) of the substituted benzimidazole class is conducted primarily by the hepatic cytochrome P450 (P450) system. To compare the potency and specificity of the currently used PPIs (i.e., omeprazole, esomeprazole, lansoprazole, pantoprazole, and rabeprazole) as inhibitors of four cytochrome P450 enzymes (CYP2C9, 2C19, 2D6, and 3A4), we performed in vitro studies using human liver microsomal preparations and recombinant CYP2C19. Sample analysis was done using selected reaction monitoring liquid chromatography/tandem mass spectometry. With several systems for CYP2C19 activity (two marker reactions, S-mephenytoin 4'-hydroxylation and R-omeprazole 5-hydroxylation, tested in either human liver microsomes or recombinant CYP2C19), the five PPIs showed competitive inhibition of CYP2C19 activity with K(i) of 0.4 to 1.5 microM for lansoprazole, 2 to 6 microM for omeprazole, approximately 8 microM for esomeprazole, 14 to 69 microM for pantoprazole, and 17 to 21 microM for rabeprazole. Pantoprazole was a competitive inhibitor of both CYP2C9-catalyzed diclofenac 4'-hydroxylation and CYP3A4-catalyzed midazolam 1'-hydroxylation (K(i) of 6 and 22 microM, respectively), which were at least 2 times more potent than the other PPIs. All PPIs were poor inhibitors of CYP2D6-mediated bufuralol 1'-hydroxylation with IC(50) > 200 microM. The inhibitory potency of a nonenzymatically formed product of rabeprazole, rabeprazole thioether, was also investigated and showed potent, competitive inhibition with K(i) values of 6 microM for CYP2C9, 2 to 8 microM for CYP2C19, 12 microM for CYP2D6, and 15 microM for CYP3A4. The inhibitory potency of R-omeprazole on the four studied P450 enzymes was also studied and showed higher inhibitory potency than its S-isomer on CYP2C9 and 2C19 activities. Our data suggest that, although the inhibitory profiles of the five studied PPIs were similar, lansoprazole and pantoprazole are the most potent in vitro inhibitors

  3. Differential Consequences of Tramadol in Overdosing: Dilemma of a Polymorphic Cytochrome P450 2D6-Mediated Substrate.

    PubMed

    Srinivas, Nuggehally R

    2015-09-01

    Tramadol is a centrally acting opioid analgesic that is prone to polymorphic metabolism via cytochrome P450 (CYP) 2D6. The generation of the active metabolite, O-desmethyltramadol, which occurs through the CYP 2D6 pathway, significantly contributes to the drug's activity. However, dosage adjustments of tramadol are typically not practiced in the clinic when treating patients who are homozygous extensive metabolizers, heterozygous extensive metabolizers, or poor metabolizers. In the event of a tramadol overdose, the consequences may be influenced importantly by the genotype or phenotype status of the subject. Depending on the individual subject's CYP 2D6 status, one may see excessive miotic-related toxicity driven by the excessive availability of O-desmethyltramadol or one may manifest mydriatic-related toxicity driven by the excessive availability of tramadol. This report provides pharmacokinetic perspectives in situations of tramadol overdosing.

  4. PROPICONAZOLE-INDUCED CYTOCHROME P450 GENE EXPRESSION AND ENZYMATIC ACTIVITIES IN RAT AND MOUSE LIVER

    EPA Science Inventory

    Conazoles are N-substituted azole antifungal agents used as both pesticides and drugs. Some of these compounds are hepatocarcinogenic in mice and some can induce thyroid tumors in rats. Many of these compounds are able to induce and/or inhibit mammalian hepatic cytochrome P450s t...

  5. Different structure of the complexes of two cytochrome P-450 isozymes with acetanilide by 1H-NMR relaxation and spectrophotometry.

    PubMed

    Woldman YaYu; Weiner, L M; Lyakhovich, V V

    1993-05-28

    The functional and spectral characteristics of the interaction of acetanilide with phenobarbital- and methylcholanthrene- induced rat liver microsomes, as well as with corresponding major isozymes (cytochromes P-450b and P-450c) have been compared. The magnitude of the reverse 1st type binding spectra proved to be negatively correlated with the acetanilide oxidation on isozymes under study. The data on paramagnetic relaxation of acetanilide protons in the presence of P-450 have shown the structure of the enzyme-substrate complex to be different for two isozymes, acetanilide molecule being closer to Fe ion in the active site in the case of P-450c, which is active towards acetanilide oxidation. For the P-450c-acetanilide complex the group oxidized (phenyl) is the closest to Fe ion.

  6. Hop (Humulus lupulus L.) Extract and 6-Prenylnaringenin Induce P450 1A1 Catalyzed Estrogen 2-Hydroxylation

    PubMed Central

    2016-01-01

    Humulus lupulus L. (hops) is a popular botanical dietary supplement used by women as a sleep aid and for postmenopausal symptom relief. In addition to its efficacy for menopausal symptoms, hops can also modulate the chemical estrogen carcinogenesis pathway and potentially protect women from breast cancer. In the present study, an enriched hop extract and the key bioactive compounds [6-prenylnarigenin (6-PN), 8-prenylnarigenin (8-PN), isoxanthohumol (IX), and xanthohumol (XH)] were tested for their effects on estrogen metabolism in breast cells (MCF-10A and MCF-7). The methoxyestrones (2-/4-MeOE1) were analyzed as biomarkers for the nontoxic P450 1A1 catalyzed 2-hydroxylation and the genotoxic P450 1B1 catalyzed 4-hydroxylation pathways, respectively. The results indicated that the hop extract and 6-PN preferentially induced the 2-hydroxylation pathway in both cell lines. 8-PN only showed slight up-regulation of metabolism in MCF-7 cells, whereas IX and XH did not have significant effects in either cell line. To further explore the influence of hops and its bioactive marker compounds on P450 1A1/1B1, mRNA expression and ethoxyresorufin O-dealkylase (EROD) activity were measured. The results correlated with the metabolism data and showed that hop extract and 6-PN preferentially enhanced P450 1A1 mRNA expression and increased P450 1A1/1B1 activity. The aryl hydrocarbon receptor (AhR) activation by the isolated compounds was tested using xenobiotic response element (XRE) luciferase construct transfected cells. 6-PN was found to be an AhR agonist that significantly induced XRE activation and inhibited 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induced XRE activity. 6-PN mediated induction of EROD activity was also inhibited by the AhR antagonist CH223191. These data show that the hop extract and 6-PN preferentially enhance the nontoxic estrogen 2-hydroxylation pathway through AhR mediated up-regulation of P450 1A1, which further emphasizes the importance of

  7. Molecular Insights into the Effects of Media-Drug and Carrier-Drug Interactions on pH-Responsive Drug Carriers.

    PubMed

    Katiyar, Ratna S; Jha, Prateek K

    2018-05-10

    We have performed two sets of all atom molecular dynamics (MD) simulations of poly(acrylic acid) (PAA) oligomers, considered as a model pH-responsive drug carrier. In the first set, multiple oligomers of PAA are simulated in model gastric and intestinal fluids, where the degree of deprotonation of PAA oligomers is varied with the medium pH. Since the gastric fluid has a pH substantially lower than that of intestinal fluid, PAA is relatively lesser ionized in gastric fluid and forms aggregates. In the second set, we simulated multiple oligomers of PAA with multiple molecules of a cationic anticancer drug, doxorubicin (DOX), for a range of pH values representative of various physiological conditions. The diffusion coefficient of DOX decreases with an increase in pH due to an increase in the ionic complexation of PAA with DOX, despite a decrease in PAA aggregation. Our findings are in agreement with recent experimental reports on pH-triggered targeting of tumor cells by the PAA-DOX system. Results of these two sets of studies establish that both carrier aggregation and carrier-drug interactions are competing influences that together determine the drug release from pH-responsive polymers.

  8. Environmentally persistent free radical-containing particulate matter competitively inhibits metabolism by cytochrome P450 1A2

    SciTech Connect

    Reed, James R., E-mail: rreed@lsuhsc.edu; Cruz, Albert Leo N. dela, E-mail: adelac2@tigers.lsu.edu; Lomnicki, Slawo M., E-mail: slomni1@lsu.edu

    Combustion processes generate different types of particulate matter (PM) that can have deleterious effects on the pulmonary and cardiovascular systems. Environmentally persistent free radicals (EPFRs) represent a type of particulate matter that is generated after combustion of environmental wastes in the presence of redox-active metals and aromatic hydrocarbons. Cytochromes P450 (P450/CYP) are membrane-bound enzymes that are essential for the phase I metabolism of most lipophilic xenobiotics. The EPFR formed by chemisorption of 2-monochlorophenol to silica containing 5% copper oxide (MCP230) has been shown to generally inhibit the activities of different forms of P450s without affecting those of cytochrome P450 reductasemore » and heme oxygenase-1. The mechanism of inhibition of rat liver microsomal CYP2D2 and purified rabbit CYP2B4 by MCP230 has been shown previously to be noncompetitive with respect to substrate. In this study, MCP230 was shown to competitively inhibit metabolism of 7-benzyl-4-trifluoromethylcoumarin and 7-ethoxyresorufin by the purified, reconstituted rabbit CYP1A2. MCP230 is at least 5- and 50-fold more potent as an inhibitor of CYP1A2 than silica containing 5% copper oxide and silica, respectively. Thus, even though PM generally inhibit multiple forms of P450, PM interacts differently with the forms of P450 resulting in different mechanisms of inhibition. P450s function as oligomeric complexes within the membrane. We also determined the mechanism by which PM inhibited metabolism by the mixed CYP1A2–CYP2B4 complex and found that the mechanism was purely competitive suggesting that the CYP2B4 is dramatically inhibited when bound to CYP1A2. - Highlights: • Combustion of organic pollutants generates long-lived particulate radicals (EPFRs). • Particulate matter (PM) competitively inhibited CYP1A2 activity. • EPFRs were much more potent CYP1A2 inhibitors than other types of PM. • PM interacts differently with different forms of P450.

  9. Effects of soy containing diet and isoflavones on cytochrome P450 enzyme expression and activity.

    PubMed

    Ronis, Martin J J

    2016-08-01

    Cytochromes P450 (CYPs) play an important role in metabolism and clearance of most clinically utilized drugs and other xenobiotics. They are important in metabolism of endogenous compounds including fatty acids, sterols, steroids and lipid-soluble vitamins. Dietary factors such as phytochemicals are capable of affecting CYP expression and activity, which may be important in diet-drug interactions and in the development of fatty liver disease, cardiovascular disease and cancer. One important diet-CYP interaction is with diets containing plant proteins, particularly soy protein. Soy diets are traditionally consumed in Asian countries and are linked to lower incidence of several cancers and of cardiovascular disease in Asian populations. Soy is also an important protein source in vegetarian and vegan diets and the sole protein source in soy infant formulas. Recent studies suggest that consumption of soy can inhibit induction of CY1 enzymes by polycyclic aromatic hydrocarbons (PAHs) which may contribute to cancer prevention. In addition, there are data to suggest that soy components promiscuously activate several nuclear receptors including PXR, PPAR and LXR resulting in increased expression of CYP3As, CYP4As and CYPs involved in metabolism of cholesterol to bile acids. Such soy-CYP interactions may alter drug pharmacokinetics and therapeutic efficacy and are associated with improved lipid homeostasis and reduced risk of cardiovascular disease. The current review summarizes results from in vitro; in vivo and clinical studies of soy-CYP interactions and examines the evidence linking the effects of soy diets on CYP expression to isoflavone phytoestrogens, particularly, genistein and daidzein that are associated with soy protein.

  10. Upgrading cytochrome P450 activity in HepG2 cells co-transfected with adenoviral vectors for drug hepatotoxicity assessment.

    PubMed

    Tolosa, Laia; Donato, M Teresa; Pérez-Cataldo, Gabriela; Castell, José Vicente; Gómez-Lechón, M José

    2012-12-01

    In a number of adverse drug reactions leading to hepatotoxicity, drug metabolism is thought to be involved by the generation of reactive metabolites from non-toxic drugs. The use of hepatoma cell lines, such as HepG2 cell line, for the evaluation of drug-induced hepatotoxicity is hampered by their low cytochrome P450 expression which makes impossible the study of the toxicity produced by bioactivable compounds. Genetically manipulated cells constitute promising tools for hepatotoxicity applications. HepG2 cells were simultaneously transfected with recombinant adenoviruses encoding CYP1A2, CYP2C9 and CYP3A4 to confer them drug-metabolic competence. Upgraded cells (Adv-HepG2) were highly able to metabolize the toxin studied in contrast to the reduced metabolic capacity of HepG2 cells. Aflatoxin B1-induced hepatotoxicity was studied as a proof of concept in metabolically competent and non-competent HepG2 cells by using high content screening technology. Significant differences in mitochondrial membrane potential, intracellular calcium concentration, nuclear morphology and cell viability after treatment with aflatoxin B1 were observed in Adv-HepG2 when compared to HepG2 cells. Rotenone (non bioactivable) and citrate (non hepatotoxic) were analysed as negative controls. This cell model showed to be a suitable hepatic model to test hepatotoxicity of bioactivable drugs and constitutes a valuable alternative for hepatotoxicity testing. Copyright © 2011 Elsevier Ltd. All rights reserved.

  11. MDR1 and BCRP Transporter-Mediated Drug-Drug Interaction between Rilpivirine and Abacavir and Effect on Intestinal Absorption

    PubMed Central

    Reznicek, Josef; Ceckova, Martina; Ptackova, Zuzana; Martinec, Ondrej; Tupova, Lenka; Cerveny, Lukas

    2017-01-01

    ABSTRACT Rilpivirine (TMC278) is a highly potent nonnucleoside reverse transcriptase inhibitor (NNRTI) representing an effective component of combination antiretroviral therapy (cART) in the treatment of HIV-positive patients. Many antiretroviral drugs commonly used in cART are substrates of ATP-binding cassette (ABC) and/or solute carrier (SLC) drug transporters and, therefore, are prone to pharmacokinetic drug-drug interactions (DDIs). The aim of our study was to evaluate rilpivirine interactions with abacavir and lamivudine on selected ABC and SLC transporters in vitro and assess its importance for pharmacokinetics in vivo. Using accumulation assays in MDCK cells overexpressing selected ABC or SLC drug transporters, we revealed rilpivirine as a potent inhibitor of MDR1 and BCRP, but not MRP2, OCT1, OCT2, or MATE1. Subsequent transport experiments across monolayers of MDCKII-MDR1, MDCKII-BCRP, and Caco-2 cells demonstrated that rilpivirine inhibits MDR1- and BCRP-mediated efflux of abacavir and increases its transmembrane transport. In vivo experiments in male Wistar rats confirmed inhibition of MDR1/BCRP in the small intestine, leading to a significant increase in oral bioavailability of abacavir. In conclusion, rilpivirine inhibits MDR1 and BCRP transporters and may affect pharmacokinetic behavior of concomitantly administered substrates of these transporters, such as abacavir. PMID:28696229

  12. Photosystem I from plants as a bacterial cytochrome P450 surrogate electron donor: terminal hydroxylation of branched hydrocarbon chains.

    PubMed

    Jensen, Kenneth; Johnston, Jonathan B; de Montellano, Paul R Ortiz; Møller, Birger Lindberg

    2012-02-01

    The ability of cytochrome P450 enzymes to catalyze highly regio- and stereospecific hydroxylations makes them attractive alternatives to approaches based on chemical synthesis but they require expensive cofactors, e.g. NAD(P)H, which limits their commercial potential. Ferredoxin (Fdx) is a multifunctional electron carrier that in plants accepts electrons from photosystem I (PSI) and facilitates photoreduction of NADP(+) to NADPH mediated by ferredoxin-NAD(P)H oxidoreductase (FdR). In bacteria, the electron flow is reversed and Fdx accepts electrons from NADPH via FdR and serves as the direct electron donor to bacterial P450s. By combining the two systems, we demonstrate that irradiation of PSI can drive the activity of a bacterial P450, CYP124 from Mycobacterium tuberculosis. The substitution of the costly cofactor NADPH with sunlight illustrates the potential of the light-driven hydroxylation system for biotechnology applications.

  13. Quantitation of Human Cytochrome P450 2D6 Protein with Immunoblot and Mass Spectrometry Analysis

    PubMed Central

    Yu, Ai-Ming; Qu, Jun; Felmlee, Melanie A.; Cao, Jin; Jiang, Xi-Ling

    2009-01-01

    Accurate quantification of cytochrome P450 (P450) protein contents is essential for reliable assessment of drug safety, including the prediction of in vivo clearance from in vitro metabolism data, which may be hampered by the use of uncharacterized standards and existence of unknown allelic isozymes. Therefore, this study aimed to delineate the variability in absolute quantification of polymorphic CYP2D6 drug-metabolizing enzyme and compare immunoblot and nano liquid chromatography coupled to mass spectrometry (nano-LC/MS) methods in identification and relative quantification of CYP2D6.1 and CYP2D6.2 allelic isozymes. Holoprotein content of in-house purified CYP2D6 isozymes was determined according to carbon monoxide difference spectrum, and total protein was quantified with bicinchoninic acid protein assay. Holoprotein/total CYP2D6 protein ratio was markedly higher for purified CYP2D6.1 (71.0%) than that calculated for CYP2D6.1 Supersomes (35.5%), resulting in distinct linear calibration range (0.05–0.50 versus 0.025–0.25 pmol) that was determined by densitometric analysis of immunoblot bands. Likewise, purified CYP2D6.2 and CYP2D6.10 and the CYP2D6.10 Supersomes all showed different holoprotein/total CYP2D6 protein ratios and distinct immunoblot linear calibration ranges. In contrast to immunoblot, nano-LC/MS readily distinguished CYP2D6.2 (R296C and S486T) from CYP2D6.1 by isoform-specific proteolytic peptides that contain the altered amino acid residues. In addition, relative quantitation of the two allelic isozymes was successfully achieved with label-free protein quantification, consistent with the nominated ratio. Because immunoblot and nano-LC/MS analyses measure total P450 protein (holoprotein and apoprotein) in a sample, complete understanding of holoprotein and apoprotein contents in P450 standards is desired toward reliable quantification. Our data also suggest that nano-LC/MS not only facilitates P450 quantitation but also provides genotypic

  14. Pharmacokinetic and pharmacodynamic drug interactions of carbamazepine and glibenclamide in healthy albino Wistar rats

    PubMed Central

    Prashanth, S.; Kumar, A. Anil; Madhu, B.; Rama, N.; Sagar, J. Vidya

    2011-01-01

    Aims: To find out the pharmacokinetic and pharmacodynamic drug interaction of carbamazepine, a protype drug used to treat painful diabetic neuropathy with glibenclamide in healthy albino Wistar rats following single and multiple dosage treatment. Materials and Methods: Therapeutic doses (TD) of glibenclamide and TD of carbamazepine were administered to the animals. The blood glucose levels were estimated by GOD/POD method and the plasma glibenclamide concentrations were estimated by a sensitive RP HPLC method to calculate pharmacokinetic parameters. Results: In single dose study the percentage reduction of blood glucose levels and glibenclamide concentrations of rats treated with both carbamazepine and glibenclamide were significantly increased when compared with glibenclamide alone treated rats and the mechanism behind this interaction may be due to inhibition of P-glycoprotein mediated transport of glibenclamide by carbamazepine, but in multiple dose study the percentage reduction of blood glucose levels and glibenclamide concentrations were reduced and it may be due to inhibition of P-glycoprotein mediated transport and induction of CYP2C9, the enzyme through which glibenclamide is metabolised. Conclusions: In the present study there is a pharmacokinetic and pharmacodynamic interaction between carbamazepine and glibenclamide was observed. The possible interaction involves both P-gp and CYP enzymes. To investigate this type of interactions pre-clinically are helpful to avoid drug-drug interactions in clinical situation. PMID:21701639

  15. Studying the Inhibitory Effect of Quercetin and Thymoquinone on Human Cytochrome P450 Enzyme Activities.

    PubMed

    Elbarbry, Fawzy; Ung, Aimy; Abdelkawy, Khaled

    2018-01-01

    Quercetin (QR) and thymoquinone (TQ) are herbal remedies that are currently extensively used by the general population to prevent and treat various chronic conditions. Therefore, investigating the potential of pharmacokinetic interactions caused by the concomitant use of these herbal remedies and conventional medicine is warranted to ensure patient safety. This study was conducted to determine the inhibitory effect of QR and TQ, two commonly used remedies, on the activities of selected cytochrome P450 (CYP) enzymes that play an important role in drug metabolism and/or toxicology. The in vitro studies were conducted using fluorescence-based high throughput assays using human c-DNA baculovirus expressed CYP enzymes. For measuring CYP2E1 activity, a validated High-performance liquid chromatography (HPLC) assay was utilized to measure the formation of 6-hydroxychlorzoxazone. The obtained half-maximum inhibitory concentration values with known positive control inhibitors of this study were comparable to the published values indicating accurate experimental techniques. Although QR did not show any significant effect on CYP1A2 and CYP2E1, it exhibited a strong inhibitory effect against CYP2D6 and a moderate effect against CYP2C19 and CYP3A4. On the other hand, TQ demonstrated a strong and a moderate inhibitory effect against CYP3A4 and CYP2C19, respectively. The findings of this study may indicate that consumption of QR or TQ, in the form of food or dietary supplements, with drugs that are metabolized by CYP2C19, CYP2D6, or CYP3A4 may cause significant herb-drug interactions. Neither QR nor TQ has any significant inhibitory effect on the activity of CYP1A2 or CYP2E1 enzymesBoth QR and TQ have a moderate to strong inhibitory effect on CYP3A4 activityQR has a moderate inhibitory effect on CYP2C19 and a strong inhibitory effect on CYP2D6Both QR and TQ are moderate inhibitors of the CYP2C9 activity. Abbreviations used: ABT: Aminobenztriazole, BZF: 7,8 Benzoflavone, CYP

  16. Two-way pharmacokinetic interaction studies between saxagliptin and cytochrome P450 substrates or inhibitors: simvastatin, diltiazem extended-release, and ketoconazole

    PubMed Central

    Patel, Chirag G; Li, Li; Girgis, Suzette; Kornhauser, David M; Frevert, Ernest U; Boulton, David W

    2011-01-01

    Background Many medicines, including several cholesterol-lowering agents (eg, lovastatin, simvastatin), antihypertensives (eg, diltiazem, nifedipine, verapamil), and antifungals (eg, ketoconazole) are metabolized by and/or inhibit the cytochrome P450 (CYP) 3A4 metabolic pathway. These types of medicines are commonly coprescribed to treat comorbidities in patients with type 2 diabetes mellitus (T2DM) and the potential for drug-drug interactions of these medicines with new medicines for T2DM must be carefully evaluated. Objective To investigate the effects of CYP3A4 substrates or inhibitors, simvastatin (substrate), diltiazem (moderate inhibitor), and ketoconazole (strong inhibitor) on the pharmacokinetics and safety of saxagliptin, a CYP3A4/5 substrate; and the effects of saxagliptin on these agents in three separate studies. Methods Healthy subjects were administered saxagliptin 10 mg or 100 mg. Simvastatin, diltiazem extended-release, and ketoconazole doses of 40 mg once daily, 360 mg once daily, and 200 mg twice daily, respectively, were used to determine two-way pharmacokinetic interactions. Results Coadministration of simvastatin, diltiazem extended-release, or ketoconazole increased mean area under the concentration-time curve values (AUC) of saxagliptin by 12%, 109%, and 145%, respectively, versus saxagliptin alone. Mean exposure (AUC) of the CYP3A4-generated active metabolite of saxagliptin, 5-hydroxy saxagliptin, decreased with coadministration of simvastatin, diltiazem, and ketoconazole by 2%, 34%, and 88%, respectively. All adverse events were considered mild or moderate in all three studies; there were no serious adverse events or deaths. Conclusion Saxagliptin, when coadministered with simvastatin, diltiazem extended-release, or ketoconazole, was safe and generally well tolerated in healthy subjects. Clinically meaningful interactions of saxagliptin with simvastatin and diltiazem extended-release are not expected. The dose of saxagliptin does not need

  17. Oral and inhaled corticosteroids: Differences in P-glycoprotein (ABCB1) mediated efflux

    SciTech Connect

    Crowe, Andrew, E-mail: a.p.crowe@curtin.edu.au; Tan, Ai May

    There is concern that P-glycoprotein mediated efflux contributes to steroid resistance. Therefore, this study examined bidirectional corticosteroid transport and induction capabilities for P-glycoprotein (P-gp) to understand which of the systemic and inhaled corticosteroids interacted with P-gp to the greatest extent. Hydrocortisone, prednisolone, prednisone, methylprednisolone, and dexamethasone represented systemically active drugs, while fluticasone propionate, beclomethasone dipropionate, ciclesonide and budesonide represented inhaled corticosteroids. Aldosterone and fludrocortisone represented mineralocorticoids. All drugs were detected using individually optimised HPLC protocols. Transport studies were conducted through Caco-2 monolayers. Hydrocortisone and aldosterone had efflux ratios below 1.5, while prednisone showed a P-gp mediated efflux ratio of onlymore » 1.8 compared to its active drug, prednisolone, with an efflux ratio of 4.5. Dexamethasone and beclomethasone had efflux ratios of 2.1 and 3.3 respectively, while this increased to 5.1 for methylprednisolone. Fluticasone showed an efflux ratio of 2.3. Protein expression studies suggested that all of the inhaled corticosteroids were able to induce P-gp expression, from 1.6 to 2 times control levels. Most of the systemic corticosteroids had higher passive permeability (> 20 × 10{sup −6} cm/s) compared to the inhaled corticosteroids (> 5 × 10{sup −6} cm/s), except for budesonide, with permeability similar to the systemic corticosteroids. Inhaled corticosteroids are not transported by P-gp to the same extent as systemic corticosteroids. However, they are able to induce P-gp production. Thus, inhaled corticosteroids may have greater interactions with other P-gp substrates, but P-gp itself is less likely to influence resistance to the drugs. -- Highlights: ► Inhaled corticosteroids are only weak substrates for P-gp, including budesonide. ► Inhaled corticosteroid potent P-gp inducers especially

  18. Inhibition of drug metabolizing cytochrome P450s by the aromatase inhibitor drug letrozole and its major oxidative metabolite 4,4′-methanol-bisbenzonitrile in vitro

    PubMed Central

    Jeong, Seongwook; Woo, Margaret M.; Flockhart, David A.

    2009-01-01

    Purpose To determine the inhibitory potency of letrozole and its main human metabolite, 4,4′-methanol-bisbenzonitrilee, on the activities of eight cytochrome P450 (CYP) enzymes. Methods Letrozole and its metabolite were incubated with human liver microsomes (HLMs) (or expressed CYP isoforms) and NADPH in the absence (control) and presence of the test inhibitor. Results Letrozole was a potent competitive inhibitor of CYP2A6 (Ki 4.6 ± 0.05 μM and 5.0 ± 2.4 μM in HLMs and CYP2A6, respectively) and a weak inhibitor of CYP2C19 (Ki 42.2 μM in HLMs and 33.3 μM in CYP2C19), while its metabolite showed moderate inhibition of CYP2C19 and CYP2B6. Letrozole or its metabolite had negligible effect on other CYPs. Conclusions Based on the in vitro Ki values, letrozole is predicted to be a weak inhibitor of CYP2A6 in vivo. Letrozole and its major human metabolite show inhibitory activity towards other CYPs, but clinically relevant drug interactions seem less likely as the Ki values are above the therapeutic plasma concentrations of letrozole. PMID:19198839

  19. Cytochrome P450IA mRNA expression in feral Hudson River tomcod

    SciTech Connect

    Kreamer, G.L.; Squibb, K.; Gioeli, D.

    1991-06-01

    The authors sought to determine if levels of cytochrome P450IA gene expression are environmentally induced in feral populations of Hudson River tomcod, a cancer prone fish, and whether laboratory exposure of tomcod to artificially spiked and naturally contaminated Hudson sediments can elicit a significant response. Using Northern blot analysis, they found levels of P450IA mRNA in tomcod collected from two Hudson River sites higher than those in tomcod from a river in Maine. Depuration of environmentally induced Hudson tomcod P450IA mRNA was rapid, with an initial detectable decline in P450 gene expression by 8 hr and basal levels reached bymore » 5 days. Intraperitoneal injection of {beta}-napthoflavone in depurated Hudson tomcod resulted in a 15-fold induction of P450 gene expression within 26 hr. Exposure of depurated Hudson tomcod to natural sediment spiked with two PAHs resulted in a 7-fold induction of P450 gene expression. Exposure of depurated tomcod to sediment from a contaminated Hudson site also resulted in a 7- to 15-fold induction of P450IA mRNA expression. Northern blot analysis revealed a second polymorphic cytochrome P450IA mRNA band in some tomcod which was also detected by Southern blot analysis. Induction of cytochrome P450IA mRNA in Atlantic tomcod may provide a sensitive biomarker of environmentally relevant concentrations of some pollutants in the Hudson and other northeastern tidal rivers.« less

  20. Homology modelling of Drosophila cytochrome P450 enzymes associated with insecticide resistance.

    PubMed

    Jones, Robert T; Bakker, Saskia E; Stone, Deborah; Shuttleworth, Sally N; Boundy, Sam; McCart, Caroline; Daborn, Phillip J; ffrench-Constant, Richard H; van den Elsen, Jean M H

    2010-10-01

    Overexpression of the cytochrome P450 gene Cyp6g1 confers resistance against DDT and a broad range of other insecticides in Drosophila melanogaster Meig. In the absence of crystal structures of CYP6G1 or complexes with its substrates, structural studies rely on homology modelling and ligand docking to understand P450-substrate interactions. Homology models are presented for CYP6G1, a P450 associated with resistance to DDT and neonicotinoids, and two other enzymes associated with insecticide resistance in D. melanogaster, CYP12D1 and CYP6A2. The models are based on a template of the X-ray structure of the phylogenetically related human CYP3A4, which is known for its broad substrate specificity. The model of CYP6G1 has a much smaller active site cavity than the template. The cavity is also 'V'-shaped and is lined with hydrophobic residues, showing high shape and chemical complementarity with the molecular characteristics of DDT. Comparison of the DDT-CYP6G1 complex and a non-resistant CYP6A2 homology model implies that tight-fit recognition of this insecticide is important in CYP6G1. The active site can accommodate differently shaped substrates ranging from imidacloprid to malathion but not the pyrethroids permethrin and cyfluthrin. The CYP6G1, CYP12D1 and CYP6A2 homology models can provide a structural insight into insecticide resistance in flies overexpressing P450 enzymes with broad substrate specificities.

  1. Food-drug interactions precipitated by fruit juices other than grapefruit juice: An update review.

    PubMed

    Chen, Meng; Zhou, Shu-Yi; Fabriaga, Erlinda; Zhang, Pian-Hong; Zhou, Quan

    2018-04-01

    This review addressed drug interactions precipitated by fruit juices other than grapefruit juice based on randomized controlled trials (RCTs). Literature was identified by searching PubMed, Cochrane Library, Scopus and Web of Science till December 30 2017. Among 46 finally included RCTs, six RCTs simply addressed pharmacodynamic interactions and 33 RCTs studied pharmacokinetic interactions, whereas seven RCTs investigated both pharmacokinetic and pharmacodynamic interactions. Twenty-two juice-drug combinations showed potential clinical relevance. The beneficial combinations included orange juice-ferrous fumarate, lemon juice- 99m Tc-tetrofosmin, pomegranate juice-intravenous iron during hemodialysis, cranberry juice-triple therapy medications for H. pylori, blueberry juice-etanercept, lime juice-antimalarials, and wheat grass juice-chemotherapy. The potential adverse interactions included decreased drug bioavailability (apple juice-fexofenadine, atenolol, aliskiren; orange juice-aliskiren, atenolol, celiprolol, montelukast, fluoroquinolones, alendronate; pomelo juice-sildenafil; grape juice-cyclosporine), increased bioavailability (Seville orange juice-felodipine, pomelo juice-cyclosporine, orange-aluminum containing antacids). Unlike furanocoumarin-rich grapefruit juice which could primarily precipitate drug interactions by strong inhibition of cytochrome P450 3A4 isoenzyme and P-glycoprotein and thus cause deadly outcomes due to co-ingestion with some medications, other fruit juices did not precipitate severely detrimental food-drug interaction despite of sporadic case reports. The extent of a juice-drug interaction may be associated with volume of drinking juice, fruit varieties, type of fruit, time between juice drinking and drug intake, genetic polymorphism in the enzymes or transporters and anthropometric variables. Pharmacists and health professionals should properly screen for and educate patients about potential adverse juice-drug interactions and help

  2. Evidence for complexation of P-450 IIC6 by an orphenadrine metabolite.

    PubMed

    Reidy, G F; Murray, M

    1990-01-30

    Removal of the orphenadrine metabolite from its complex with rat liver P-450 IIB1 is associated with a discrepancy in the reactivation of IIB1 activity. Two possible explanations are that either (1) NADPH-P-450-reductase is inaccessible to the restored IIB1, or (2) complexation of other P-450s may occur. Exogenous P-450 reductase increased all pathways of steroid hydroxylation (1.9 to 3.6-fold) but did not enhance reactivation of IIB1-dependent steroid 16 beta-hydroxylation. Instead, P-450 IIC6-dependent progesterone 21-hydroxylase activity was increased after dissociation to 122% of control. IIC6 activity was also inhibited in vitro in microsomes from phenobarbital-induced rats (ki = 151 microM). Thus, orphenadrine appears to complex P-450 IIC6 as well as IIB1 in rat liver.

  3. Cytochrome P450-2D6 Screening Among Elderly Using Antidepressants (CYSCE)

    ClinicalTrials.gov

    2017-08-15

    Depression; Depressive Disorder; Poor Metabolizer Due to Cytochrome P450 CYP2D6 Variant; Intermediate Metabolizer Due to Cytochrome P450 CYP2D6 Variant; Ultrarapid Metabolizer Due to Cytochrome P450 CYP2D6 Variant

  4. INTERINDIVIDUAL VARIANCE OF CYTOCHROME P450 FORMS IN HUMAN HEPATIC MICROSOMES: CORRELATION OF INDIVIDUAL FORMS WITH XENOBIOTIC METABOLISM AND IMPLICATIONS IN RISK ASSESSMENT

    EPA Science Inventory

    Differences in biotransformation activities may alter the bioavailability or efficacy of drugs, provide protection from certain xenobiotic and environmental agents, or increase toxicity of others. Cytochrome P450 (CYP450) enzymes are responsible for the majority of oxidation reac...

  5. The crystal structure of P450-TT heme-domain provides the first structural insights into the versatile class VII P450s.

    PubMed

    Tavanti, Michele; Porter, Joanne L; Levy, Colin W; Gómez Castellanos, J Rubén; Flitsch, Sabine L; Turner, Nicholas J

    2018-07-02

    The first crystal structure of a class VII P450, CYP116B46 from Tepidiphilus thermophilus, has been solved at 1.9 Å resolution. The structure reveals overall conservation of the P450-fold and a water conduit around the I-helix. Active site residues have been identified and sequence comparisons have been made with other class VII enzymes. A structure similarity search demonstrated that the P450-TT structure is similar to enzymes capable of oxy-functionalization of fatty acids, terpenes, macrolides, steroids and statins. The insight gained from solving this structure will provide a guideline for future engineering and modelling studies on this catalytically promiscuous class of enzymes. Copyright © 2018 Elsevier Inc. All rights reserved.

  6. Polycyclic Aromatic Hydrocarbons (PAHs) Mediate Transcriptional Activation of the ATP Binding Cassette Transporter ABCB6 Gene via the Aryl Hydrocarbon Receptor (AhR)*

    PubMed Central

    Chavan, Hemantkumar; Krishnamurthy, Partha

    2012-01-01

    Liver is endowed with a mechanism to induce hepatic cytochromes P450 (CYP450s) in response to therapeutic drugs and environmental contaminants, leading to increased detoxification and elimination of the xenobiotics. Each CYP450 is composed of an apoprotein moiety and a heme prosthetic group, which is required for CYP450 activity. Thus, under conditions of CYP450 induction, there is a coordinate increase in heme biosynthesis to compensate for the increased expression of CYP450s. ABCB6, a mitochondrial ATP binding cassette transporter, which regulates coproporphyrinogen transport from the cytoplasm into the mitochondria to complete heme biosynthesis, represents a previously unrecognized rate-limiting step in heme biosynthesis. However, it is not known if exposure to drugs and environmental contaminants induces ABCB6 expression, to assure an adequate and apparently coordinated supply of heme for the generation of functional cytochrome holoprotein. In the present study, we demonstrate that polycyclic aromatic hydrocarbons (PAHs), the widely distributed environmental toxicants shown to induce porphyrin accumulation causing hepatic porphyria, up-regulate ABCB6 expression in both mice and humans. Using siRNA technology and Abcb6 knock-out mice, we demonstrate that PAH-mediated increase in hepatic porphyrins is compromised in the absence of ABCB6. Moreover, in vivo studies in aryl hydrocarbon receptor (AhR) knock-out mice demonstrate that PAH induction of ABCB6 is mediated by AhR. Promoter ac