Exploring the Role of CYP3A4 Mediated Drug Metabolism in the Pharmacological Modulation of Nitric Oxide Production (original) (raw)
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European Journal of Drug Metabolism and Pharmacokinetics, 2016
Background and objectives The role of metabolite(s) to elicit potential clinical drug-drug interaction (DDI) via cytochrome P450 enzymes (CYP) is gaining momentum. In this context, the role of N-oxides for in vitro CYP inhibition has not been evaluated. The objectives of this study were: (a) to examine in vitro CYP inhibition of Noxides of clozapine, levofloxacin, roflumilast, voriconazole and zopiclone in a tiered approach and (b) evaluate in vitro fate of aforementioned N-oxides examined in recombinant CYPs, human microsomes and hepatocytes. Methods CYP enzymes evaluated in the work included: CYP1A2, 2B6, 2C9, 2C19, 2D6 and 3A4 using standard procedures for incubation with appropriate probe substrates. The initial cutoff for CYP inhibition was C50% using 2 and 10 lM concentrations of various N-oxide metabolites (Tier 1). IC 50 values were constructed for the CYP pathway(s) that showed C50% inhibition (Tier 2). In addition, co-incubation of N-oxides with parent was performed to evaluate potentiation of CYP inhibition (Tier 3). Results N-oxides of clozapine (CYP2B6/2C19) and voriconazole (CYP2C9/3A4) showed CYP inhibition C50%. Clozapine-N-oxide inhibited CYP2B6 and CYP2C19 pathways with IC 50 of 8.3 and 8.7 lM, respectively. Voriconazole-N-oxide inhibited CYP2B6 and CYP2C19 pathways with IC 50 of 10.5 and 11.2 lM, respectively. Co-incubation of clozapine-N-oxide with clozapine potentiated CYP2B6/2C19 pathways; however, incubation of voriconazole-N-oxide with voriconazole did not appear to potentiate the CYP pathways because parent caused an inhibition of almost 80%. None of the N-oxides appeared to further undergo biotransformation as judged by the in vitro metabolic fate experiments (stage 2). Conclusions Clinical DDI potential of specific CYP enzymes needs to be considered arising due to circulatory concentrations of certain N-oxides depending on the dose size and/or frequency of dosing of the respective parent drugs. Key Points Recently, the role of metabolite(s) to elicit potential clinical drug-drug interaction (DDI) has become a topic of interest In this context, the potential role of N-oxides of clozapine, levofloxacin, roflumilast, voriconazole and zopiclone to inhibit in vitro cytochrome P450 (CYP) enzymes and their metabolic fate were investigated with appropriate tiered experimental designs Based on the generated IC 50 values for in vitro CYP inhibition of various isozymes, involvement of either clozapine-N-oxide or voriconazole-N-oxide in clinical DDI may need further probing
Characterization of Inducible Nitric-oxide Synthase by Cytochrome P-450 Substrates and Inhibitors
Journal of Biological Chemistry, 1997
Nitric-oxide synthases (NOS, EC 1.14.13.39) are hemecontaining enzymes that catalyze the formation of nitric oxide from L-Arg. General cytochrome P-450 inhibitors and cytochrome P-450 isoform-selective substrates and inhibitors were used to characterize the activity of recombinant human inducible NOS (iNOS). Classical cytochrome P-450 ligands such as the mechanism-based inactivator 1-aminobenzotriazole did not inhibit iNOS. Of a panel of 30 human cytochrome P-450 isoform-selective substrates and inhibitors, only chlorzoxazone, a cytochrome P-450 2E1 (CYP2E1) substrate, showed any significant inhibition of iNOS activity. Chlorzoxazone was not a substrate for iNOS but was a potent competitive inhibitor with respect to L-Arg with K i ؍ 3.3 ؎ 0.7 M. The binding of chlorzoxazone to iNOS and human and rat liver microsomal cytochrome P-450 induced a high spin, type I spectra, which was reversed by imidazole. Although the binding of chlorzoxazone to iNOS and its inhibition of iNOS activity suggest some similarity between iNOS and CYP2E1 activity, other CYP2E1 substrates and inhibitors including zoxazolamine were not inhibitors of iNOS. Overall, iNOS activity is distinctly different from the major cytochrome P-450 enzymes in human liver microsomes.
Evaluation of Time-Dependent Cytochrome P450 Inhibition Using Cultured Human Hepatocytes
Drug Metabolism and Disposition, 2006
Primary human hepatocytes in culture are commonly used to evaluate cytochrome P450 (P450) induction via an enzyme activity endpoint. However, other processes can confound data interpretation. To this end, the impact of time-dependent P450 inhibition in this system was evaluated. Using a substrate-cassette approach, P450 activities were determined after incubation with the prototypic inhibitors tienilic acid (CYP2C9), erythromycin, troleandomycin, and fluoxetine (CYP3A4). Kinetic analysis of enzyme inactivation in hepatocytes was used to describe the effect of these time-dependent inhibitors and derive the inhibition parameters k inact and K I , which generally were in good agreement with the values derived using recombinant P450s and human liver microsomes (HLMs). Tienilic acid selectively inhibited CYP2C9-dependent diclofenac 4-hydroxylation activity, and erythromycin, troleandomycin, and fluoxetine inhibited CYP3A4-dependent midazolam 1hydroxylation in a time-and concentration-dependent manner.
Cytochromes P450 and experimental models of drug metabolism
Journal of Cellular and Molecular Medicine, 2002
For the development of new drugs, evaluation of drug-drug interactions with already known compounds, as well as for better understanding of metabolism pathways of various toxicants and pollutants, we studied the drug metabolism mediated by cytochromes P450. The experimental approach is based on animal drug-metabolising systems. From the ethical as well as rational reasons, the selection of an appropriate system is crucial. Here, it is necessary to decide on the basis of expected CYP system involved. For CYP1A-mediated pathways, all the commonly used experimental models are appropriate except probably the dog. On the contrary, the dog seems to be suitable for modelling of processes depending on the CYP2D. With CYP2C, which is possibly the most large and complicated subfamily, the systems based on monkey (Maccacus rhesus) may be a good representative. The CYP3A seems to be well modelled by pig or minipig CYP3A29. Detailed studies on activities with individual isolated CYP forms are needed to understand in full all aspects of inter-species differences and variations.
Acta Pharmacologica et Toxicologica, 1983
The rate of conversion of p-nitrophenetole to p-nitrophenol by rat liver microsomes was studied. Inhibition of the reaction by CO and by SKF 525A and the absolute dependence on NADPH and oxygen indicate that cytochrome P-450 catalyzes the reaction. The apparent K, for oxygen was 0.07 pM. Furthermore, cytochrome bs seemed to be involved in the formation of p-nitrophenol. The effect on p-nitrophenol formation of drugs known to be involved in drug interaction in clinical practice was studied. There was a competitive inhibition by phenytoin (inhibitor constant, K,, 30 pM), disulfiram (K,, 2pM) and chloramphenicol (K,, 20 pM), whereas a mixed-type inhibition by isoniazid was observed (
Chromatographic assays of drug oxidation by human cytochrome P450 3A4
Nature Protocols, 2009
Cytochrome P450 enzymes (P450s) are heme-thiolate mono-oxygenases involved in the oxidation of many endogenous and exogenous substrates. Herein, we describe two protocols for measuring the activity of a key enzyme of drug metabolism, P450 3A4. In this protocol, the substrate is incubated with human liver microsomes, the reaction is quenched, and the substrates and products are extracted and subjected to liquid chromatography (LC) separation and detection. Oxidation of the calcium-channel blocker nifedipine is measured using UV-Vis spectroscopy in-line with high performance liquid chromatography (HPLC). 6b-Hydroxytestosterone formation from testosterone is measured by HPLC coupled to mass spectrometry (MS). Both of these procedures are rapid, requiring 2 h or less, and can be used to confirm and measure P450 3A4 activity and can also be used as a guide for developing other assays for measuring P450 catalysis. The separation strategy described here is more rapid than many available methods, except when ultra-performance liquid chromatography (UPLC) is used.
Identification of human liver cytochrome P450 isoforms mediating omeprazole metabolism
British Journal of Clinical Pharmacology, 1993
1. The in vitro metabolism of omeprazole was studied in human liver microsomes in order to define the secondary metabolic pathways and identify the cytochrome P450 (CYP) isoforms responsible for the formation of the secondary metabolites of omeprazole. 2. The major secondary omeprazole metabolite was the hydroxysulphone, which was formed during incubation with both hydroxyomeprazole and omeprazole sulphone. A second metabolite, tentatively identified as pyridine-N-oxide omeprazole sulphone, was also formed during incubation with omeprazole sulphone. The formation kinetics of these two metabolites from omeprazole sulphone were biphasic suggesting the involvement of multiple CYP isoforms in each case. In contrast, the formation kinetics of hydroxysulphone from hydroxyomeprazole were linear. 3. Inhibition studies, performed with omeprazole sulphone as substrate at concentrations at which the high affinity activities predominated, with a series of isoform selective inhibitors as well as with an anti-CYP2C3 antibody suggested a dominant role of S-mephenytoin hydroxylase in the formation of hydroxysulphone from omeprazole sulphone. By contrast, CYP3A activities were predominant in the formation of hydroxysulphone from hydroxyomeprazole as well as in the formation of pyridine-N-oxide omeprazole sulphone from omeprazole sulphone.
Evidence of Significant Contribution from CYP3A5 to Hepatic Drug Metabolism
Drug Metabolism and Disposition, 2004
CYP3A4 and CYP3A5 exhibit significant overlap in substrate specificity but can differ in product regioselectivity and formation activity. To further explore this issue, we compared the kinetics of product formation for eight different substrates, using heterologously expressed CYP3A4 and CYP3A5 and phenotyped human liver microsomes. Both enzymes displayed allosteric behavior toward six of the substrates. When it occurred, the "maximal" intrinsic clearance was used for quantitative comparisons. Based on this parameter, CYP3A5 was more active than CYP3A4 in catalyzing total midazolam hydroxylation (3-fold) and lidocaine demethylation (1.4-fold). CYP3A5 exhibited comparable metabolic activity as CYP3A4 (90-110%) toward dextromethorphan N-demethylation and carbamazepine epoxidation. CYP3A5-catalyzed erythromycin N-demethylation, total flunitrazepam hydroxylation, testosterone 6-hydroxylation, and terfenadine alcohol formation occurred with an intrinsic clearance that was less than 65% that of CYP3A4. Using two sets of human liver microsomes with equivalent CYP3A4-specific content but markedly different CYP3A5 content (group 1, predominantly CYP3A4; group 2, CYP3A4 ؉ CYP3A5), we assessed the contribution of CYP3A5 to product formation rates determined at low substrate concentrations (<K m). Mean product formation rates for group 2 microsomes were 1.4-to 2.2-fold higher than those of group 1 (p < 0.05 for 5 of 8 substrates). After adjusting for CYP3A4 activity (itraconazole hydroxylation), mean product formation rates for group 2 microsomes were still significantly higher than those of group 1 (p < 0.05 for 3 substrates). We suggest that, under conditions when CYP3A5 content represents a significant fraction of the total hepatic CYP3A pool, the contribution of CYP3A5 to the clearance of some drugs may be an important source of interindividual variability.
Personalized Medicine Universe, 2019
The cytochrome P450 (CYP) 3A family of enzymes metabolize the majority of clinically used drugs. CYP3A4 and CYP3A5 are the two major CYP3A isoforms, but exhinbit different substrate specificity. The aim of this study was to establish a simple screening method to determine the relative contributions of CYP3A4 and CYP3A5 to drug metabolism in vitro. Methods: A screening method was developed based on competitive inhibition using luciferin-PPXE (L-PPXE), a luminogenic CYP3A substrate. CYP3cide, tacrolimus, and midazolam were selected as standard compounds metabolized by CYP3A4 or CYP3A5. Nine clinically-used drugs were evaluated for their abilities to inhibit luminescence resulting from L-PPXE metabolism. Appropriate reaction conditions for the screening method were determined using recombinant CYP3A4 and CYP3A5. Results: A significant decrease in luminescence resulting from L-PPXE metabolism by CYP3A4 and CYP3A5 was observed only for drugs reported to be metabolized by CYP3As. The substrate specificities of CYP3A4 or CYP3A5 for the proposed CYP3A substrates using our screening method were consistent with those of previous reports or available drug information from pharmaceutical companies. The reaction volume for this method was 50 μL, and the time required for the entire procedure was 70 min. To Personalized Medicine Universe, Short communication 4 Furthermore, this screening can be performed using a single tube with minimal training. Conclusions: Through the establishment of our screening method in the present study, we are sure it is useful to determine the relative contributions of CYP3A4 and CYP3A5 to drug metabolism in vitro.