An improved substrate cocktail for assessing direct inhibition and time-dependent inhibition of multiple cytochrome P450s.
Ontology highlight
ABSTRACT:
SUBMITTER: Chen ZH
PROVIDER: S-EPMC4857547 | biostudies-other | 2016 May
REPOSITORIES: biostudies-other
Similar Datasets
Project description: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.
| S-EPMC5938745 | biostudies-literature
Project description:Genetic polymorphisms in CYP2C8 can influence the metabolism of important therapeutic agents and cause interindividual variation in drug response and toxicity. The significance of the variant CYP2C8*3 has been controversial with reports of higher in vivo but lower in vitro activity compared to CYP2C8*1. In this study, the contribution of the redox partners cytochrome P450 reductase (CPR) and cytochrome b5 to the substrate dependent activity of CYP2C8.3 (R139K, K399R) was investigated in human liver microsomes (HLMs) and Escherichia coli expressed recombinant CYP2C8 proteins using amodiaquine, paclitaxel, rosiglitazone and cerivastatin as probe substrates. For recombinant CYP2C8.3, clearance values were two- to five-fold higher compared to CYP2C8.1. CYP2C8.3's higher k(cat) seems to be dominated by a higher, but substrate specific affinity, towards cytochrome b5 and CPR (K(D) and K(m,red)) which resulted in increased reaction coupling. A stronger binding affinity of ligands to CYP2C8.3, based on a two site binding model, in conjunction with a five fold increase in amplitude of heme spin change during binding of ligands and redox partners could potentially contribute to a higher k(cat). In HLMs, carriers of the CYP2C8*1/*3 genotype were as active as CYP2C8*1/*1 towards the CYP2C8 specific reaction amodiaquine N-deethylation. Large excess of cytochrome b5 compared to CYP2C8 in recombinant systems and HLMs inhibited metabolic clearance, diminishing the difference in k(cat) between the two enzymes, and may provide an explanation for the discrepancy to in vivo data. In silico studies illustrate the genetic differences between wild type and variant on the molecular level.
| S-EPMC3159548 | biostudies-literature
Project description:SIRT1 is the most extensively studied human sirtuin with a broad spectrum of endogenous targets. It has been implicated in the regulation of a myriad of cellular events, such as gene transcription, mitochondria biogenesis, insulin secretion as well as glucose and lipid metabolism. From a mechanistic perspective, nicotinamide (NAM), a byproduct of a sirtuin-catalyzed reaction, reverses a reaction intermediate to regenerate NAD+ through "base exchange", leading to the inhibition of the forward deacetylation. NAM has been suggested as a universal sirtuin negative regulator. Sirtuins have evolved different strategies in response to NAM regulation. Here, we report the detailed kinetic analysis of SIRT1-catalyzed reactions using endogenous substrate-based synthetic peptides. A novel substrate-dependent sensitivity of SIRT1 to NAM inhibition was observed. Additionally, SIRT1 demonstrated pH-dependent deacetylation with normal solvent isotope effects (SIEs), consistent with proton transfer in the rate-limiting step. Base exchange, in contrast, was insensitive to pH changes with no apparent SIEs, indicative of lack of proton transfer in the rate-limiting step. Consequently, NAM inhibition was attenuated at a high pH in proteated buffers. Our study provides new evidence for "activation by de-repression" as an effective sirtuin activation strategy.
| S-EPMC7922766 | biostudies-literature
Project description:Activity of human cytochrome P450 enzymes (CYPs) shows high inter-and intra-individual variability, which is determined by genetic and non-genetic factors. Using a combination of CYP-specific probe drugs, phenotyping cocktails allow simultaneous assessment of the activity of different CYP isoforms. The objective of this study was to characterize the phenotyping metrics of the Basel cocktail in healthy male subjects with induced and inhibited CYP activity.In a randomized crossover study, the probe drugs for simultaneous phenotyping of CYP1A2 (caffeine), CYP2B6 (efavirenz), CYP2C9 (losartan), 2C19 (omeprazole), CYP2D6 (metoprolol), and CYP3A4 (midazolam) were administered to 16 subjects without pretreatment (baseline), after pretreatment with a combination of CYP inhibitors (ciprofloxacin, ketoconazole, and paroxetine), and after CYP induction with rifampicin. All subjects were genotyped. Pharmacokinetic profiles of the probe drugs and their main metabolites and metabolic ratios 2, 4, 6, and 8 h after probe drug application were determined in plasma and compared with the corresponding area under the plasma concentration-time curve (AUC) ratios.The Basel phenotyping cocktail was well tolerated by all subjects independent of pretreatment. Good correlations of metabolic ratios with AUC ratios of the corresponding probe drugs and their metabolites for all three conditions (baseline, CYP inhibition, and CYP induction) were found at 2 h after probe drug administration for CYP3A4, at 4 h for CYP1A2 and CYP2C19, and at 6 h for CYP2B6 and CYP2D6. While CYP inhibition significantly changed AUC ratios and metabolic ratios at these time points for all six CYP isoforms, CYP induction did not significantly change AUC ratios for CYP2C9. For CYP3A4, total 1'-hydroxymidazolam concentrations after pretreatment of samples with ?-glucuronidase were needed to obtain adequate reflection of CYP induction by the metabolic ratio.Inhibition of CYP activity can be detected with the Basel phenotyping cocktail for all six tested CYP isoforms at the proposed time points. The AUC ratio of losartan:losartan carboxylic acid in plasma does not seem suitable to detect induction of CYP2C9. The observed metabolic ratios for inhibited and induced CYP activity need to be confirmed for extensive metabolizers, and typical ratios for subjects with genetically altered CYP activity will need to be established in subsequent studies. ClinicalTrials.gov-ID: NCT01386593.
| S-EPMC4712254 | biostudies-literature
Project description:This study was designed to evaluate the effects of cenobamate, an antiseizure medication for focal seizures, on the pharmacokinetics of cytochrome P450 probes (bupropion, CYP2B6; midazolam, CYP3A4/5; warfarin, CYP2C9; and omeprazole, CYP2C19) in healthy subjects. Probes were administered alone on days 1 (bupropion) and 7 (midazolam/warfarin/omeprazole), and with cenobamate 100 mg/day on day 69 (midazolam) and cenobamate 200 mg/day on days 99 (bupropion) and 105 (midazolam/warfarin/omeprazole). No significant interaction was concluded if 90% confidence intervals (CIs) for geometric mean ratios (GMRs) for area under the curve (AUC) and maximum concentration of CYP substrates and/or their metabolites were within the no-effect interval (0.80-1.25). When co-administered with cenobamate 100 mg/day, AUC from time of administration up to the time of the last quantifiable concentration (AUC0-last ) GMR (90% CIs) for midazolam was 0.734 (0.647-0.832). When co-administered with cenobamate 200 mg/day, AUC0-last GMRs (90% CI) for midazolam, bupropion, S-warfarin, and omeprazole were 0.277 (0.238-0.323), 0.615 (0.522-0.724), 1.14 (1.10-1.18), and 2.07 (1.44-2.98), respectively. Co-administration of cenobamate with midazolam and bupropion probes led to values that were outside and below the no effect boundary, indicating that cenobamate induces the CYP3A4/5 and CYP2B6 enzymes. Co-administration of cenobamate led to omeprazole values which were outside and above the no-effect boundary, but with high variability, suggesting that cenobamate may moderately inhibit CYP2C19 activity. No effect on CYP2C9 was observed with the cenobamate and warfarin combination. Co-administration of cenobamate with these probes drugs was well-tolerated. In this study, 200 mg/day cenobamate moderately induced CYP3A4/5 (dose-dependently; 100 mg/day was a weak inducer), was a weak inducer of CYP2B6, moderately inhibited CYP2C19, and had a negligible effect on CYP2C9.
| S-EPMC9010261 | biostudies-literature
Project description:BackgroundMembers of the cytochrome P450 1A family metabolize many procarcinogens such as polycyclicaromatic hydrocarbons and heterocyclic amines. Inactivation of these enzymes is a prerequisite for cancer prevention and treatment in certain cases. Mechanism-based inhibition (time and co-factor dependent) is an effective method for the inactivation of these enzymes. Our recent study on emodin analogs revealed an anthraquinone with ortho-methylarylamine moiety that exhibited timedependent inhibition of P450 enzymes 1A1 and 1A2.MethodsTo determine whether the amino group or the methyl group or both were responsible for the time-dependent inhibition of these enzymes, a set of eleven compounds containing the orthomethylarylamine moiety were identified through a database search, and studied for the inhibition of the P450 enzymes 1A1, 1A2, 2A6 and 2B1. Our earlier studies on carbazole derivatives provided us with highly selective P450 1A2 inhibitors. Glycine scanning studies were performed on the docked proteinligand complexes of compounds 1-20 in order to understand the contribution of different protein residues towards the ligand binding.ResultsFour compounds were found to cause selective time-dependent inhibition of P450 1A1 with KI values ranging from 0.24 to 8.25 mM. These compounds exhibited only direct inhibition of P450 1A2. Molecular modeling studies of these molecules indicated that the shapes of the molecules, their binding modes, and the methyl substituent in close proximity (4.5-5.7 Å) to the heme-Fe all contributed to their selective time-dependent inhibition activity on P450 1A1. Glycine scanning studies for P450 1A1 indicated that ligand interaction with Phe123 was the strongest binding contributor and similar studies for P450 1A2 indicated that ligand interactions with the phenylalanine residues 226 and 260 were the largest binding contributors.ConclusionFour compounds have been identified that exhibit selective time-dependent inhibition of P450 1A1. Modeling studies have indicated that the proximity of the aromatic methyl group to the heme-Fe could be the main contributor for time-dependent inhibition. Future studies will focus on the confirmation of the involvement of the aromatic methyl group in enzyme inactivation.
| S-EPMC6697106 | biostudies-literature
Project description:There is growing evidence that some members of cytochrome P450 enzymes contribute to regulation of normal prenatal development. CYP epoxygenases (CYP2C and CYP2J subfamilies) convert arachidonic acid into four regioisomeric epoxyeicosatrienoic acids (EETs), biologically active molecules involved in mitogenesis and cell signaling. Almost nothing is known about localization of their expression in tissues during human prenatal development. The spatio-temporal expression pattern of CYP2C8, CYP2C9, CYP2C19 and CYP2J2 in human embryonic/fetal intestines, liver, and kidney was investigated by immunohistochemical method. CYP epoxygenases are expressed already in early stages of development in these embryonic/fetal tissues (as early as 7th week of IUD in the intestines, 5th week of IUD in the liver, and 6th week of IUD in the kidney). In kidney, CYP epoxygenases are expressed in the metanephrogenic blastema (but not in the uninduced mesenchyme) and in the tubular system. In the intestines, diverse CYP epoxygenases distribution along crypt-villus axis could suggest role in cell differentiation. Moreover, we detected higher CYP2J2 level in these organs than in adult tissue samples.
| S-EPMC4049895 | biostudies-literature
Project description:The multidrug and toxin extruders 1- and 2-K (MATE1 and MATE2-K) are expressed in the luminal membrane of renal proximal tubule cells and provide the active step in the secretion of molecules that carry a net positive charge at physiologic pH, so-called organic cations. The present study tested whether structurally distinct MATE substrates can display different quantitative profiles of inhibition when interacting with structurally distinct ligands. The tested ligands were three structurally similar cationic ionic liquids (ILs, salts in the liquid state: N-butylpyridinium, NBuPy; 1-methyl-3-butylimidazolium, Bmim; and N-butyl-N-methylpyrrolidinium, BmPy). Uptake was measured using Chinese hamster ovary cells that stably expressed MATE1 or MATE2-K. By trans-stimulation, all three ILs were transported by both MATE transporters. The three ILs also inhibited uptake of three structurally distinct MATE substrates: 1-methyl-4-phenylpyridinium (MPP), triethylmethylammonium (TEMA), and N,N,N-trimethyl-2-[methyl(7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino]ethanaminium (NBD-MTMA). MATE1 displayed a higher affinity for the pyridinium-based NBuPy (IC50 values, 2-4 µM) than for either the pyrrolidinium- (BmPy; 20-70 µM) or imidazolium-based ILs (Bmim; 15-60 µM). Inhibition of MPP, TEMA, and NBD-MTMA transport by NBuPy was competitive, with comparable Ki values against all substrates. Bmim also competitively blocked the three substrates but with Ki values that differed significantly (20 µM against MPP and 30 µM against NBD-MTMA versus 60 µM against TEMA). Together, these data indicate that renal secretion of ILs by the human kidney involves MATE transporters and suggest that the mechanism of transport inhibition is ligand-dependent, supporting the hypothesis that the binding of substrates to MATE transporters involves interaction with a binding surface with multiple binding sites.
| S-EPMC3876781 | biostudies-literature
Project description:Mechanical cues like the rigidity of the substrate are main determinants for the decision-making of adherent cells. Here we use a mechano-chemical model to predict the cellular response to varying substrate stiffnesses. The model equations combine the mechanics of contractile actin filament bundles with a model for the Rho-signaling pathway triggered by forces at cell-matrix contacts. A bifurcation analysis of cellular contractility as a function of substrate stiffness reveals a bistable response, thus defining a lower threshold of stiffness, below which cells are not able to build up contractile forces, and an upper threshold of stiffness, above which cells are always in a strongly contracted state. Using the full dynamical model, we predict that rate-dependent hysteresis will occur in the cellular traction forces when cells are exposed to substrates of time-dependent stiffness.
| S-EPMC2895361 | biostudies-literature
Project description:To optimally manage an environment with predators, prey must correctly distinguish between cues that are risky and cues that are safe. Even a specific cue that is safe in one area or at a certain time may be dangerous in other situations, and vice versa. Latent inhibition is a cognitive mechanism by which animals fail to learn that a stimulus as risky because they have already learned it as non-threatening via previous encounters with the stimulus in the absence of negative consequences. Here, we demonstrate that latent inhibition of predator-recognition learning in wood frog tadpoles, Lithobates sylvaticus, depends on the timing of their learning opportunities. For 6 days, tadpoles were exposed daily to an initially novel stimulus (salamander odour), either in the morning (11.00-13.00 h) or evening (16.00-18.00 h). The following day, we conditioned tadpoles to recognize the salamander odour as a predator by pairing it with injured tadpole cues, either at the same time as their previous experience or at the opposite time. When tested the following day, latent inhibition occurred under each scenario where the timing of conditioning matched the timing of the pre-exposure. However, tadpoles tested in the morning showed learned fright responses when conditioned in the morning if their pre-exposure had occurred in the evening, whereas individuals tested in the evening showed learned fright responses when conditioned in the evening if their pre-exposure had occurred in the morning. This is the first report of time-dependent latent inhibition of predator-recognition learning, which is likely an important mechanism for correctly managing predation risk and safety.
| S-EPMC6548720 | biostudies-literature