- Development and validation of probe drug cocktails for the characterization of CYP450-mediated metabolism by human heart microsomes. [Journal Article]
- XXenobiotica 2018 Feb 16; :1-46
- The objective of our study was to develop and validate a cocktail approach to allow the simultaneous characterization of various CYP450-mediated oxydations by human heart microsomes for nine probe dr...
The objective of our study was to develop and validate a cocktail approach to allow the simultaneous characterization of various CYP450-mediated oxydations by human heart microsomes for nine probe drug substrates namely. ethoxyresorufin. bupropion. repaglinide. tolbutamide. bufuralol. chlorzoxazone. ebastine. midazolam and dodecanoic acid. The first validation step was conducted using recombinant human CYP450 isoenzymes by comparing activity measured for each probe drug as a function of 1) buffer used. 2) selectivity towards specific isoenzymes. and 3) drug interactions between probes. Activity was all measured by validated LC-MSMS methods. Two cocktails were then constituted with 7 of the 9 drugs and subjected to kinetic validation. Finally. all probe drugs were incubated with human heart microsomes prepared from ventricular tissues obtained from twelve patients undergoing cardiac transplantation. Validated cocktail #1 including bupropion. chlorzoxazone. ebastine and midazolam was used to characterize CYP2B6. 2E1. 2J2 and 3A5-mediated metabolism in human hearts. Cocktail #2 which includes bufuralol. ethoxyresorufin and repaglinide failed the validation step. Substrates in cocktail #2 as well as tolbutamide and dodecanoic acid had to be incubated separately because of their physico-chemical characteristics (solubility. ionization) or drug interactions. Activity in HHM was the highest towards ebastine. chlorzoxazone and tolbutamide.
- Generation and Characterization of a CYP2C11-null Rat Model by using the CRISPR/Cas9 method. [Journal Article]
- DMDrug Metab Dispos 2018 Feb 14
- CYP2C11 is involved in the metabolism of many drugs in rats. To assess the roles of CYP2C11 in physiology and drug metabolism, a CYP2C11-null rat model was generated using the clustered regularly int...
CYP2C11 is involved in the metabolism of many drugs in rats. To assess the roles of CYP2C11 in physiology and drug metabolism, a CYP2C11-null rat model was generated using the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 method. A 2-bp insertion was added to exon 6 of CYP2C11 in Sprague-Dawley rats. CYP2C11 was not detected by Western blotting in liver microsomes of CYP2C11-null rats. No off-target effects were found at eleven predicted sites of the knockout model. The CYP2C11-null rats were viable and had no obvious abnormalities, with the exception of reduced fertility. Puberty in CYP2C11-null rats appeared to be delayed by ~20 days, and the average litter size fell by 43%. Tolbutamide was used as a probe in this drug metabolism study. In the liver microsomes of CYP2C11-null rats, Vmax and CLint decreased by ~21% and ~44%, respectively, compared to those of wild-type rats. The Km increased by 138% compared to that of wild-types. However, our pharmacokinetics study showed no major differences in any parameters between the two strains, in both males and females. In conclusion, a CYP2C11-null rat model was successfully generated and is a valuable tool to study the in vivo function of CYP2C11.
- Influences of Anlotinib on Cytochrome P450 Enzymes in Rats Using a Cocktail Method. [Journal Article]
- BRBiomed Res Int 2017; 2017:3619723
- The present study aimed to investigate the effect of anlotinib (AL3818) on pharmacokinetics of cytochrome P450 (CYP) enzymes (CYP1A2, CYP2C6, CYP2D1, CYP2D2, and CYP3A1/2) by using five cocktail prob...
The present study aimed to investigate the effect of anlotinib (AL3818) on pharmacokinetics of cytochrome P450 (CYP) enzymes (CYP1A2, CYP2C6, CYP2D1, CYP2D2, and CYP3A1/2) by using five cocktail probe drugs in vivo. After pretreatment for 7 days with anlotinib (treatment group) or saline (control group) by oral administration, probe drugs phenacetin, tolbutamide, omeprazole, metoprolol, and midazolam were administered to rats by oral administration. Blood samples were obtained at a series of time-points and the concentrations of five probe drugs in plasma were determined by a UHPLC-MS/MS method. The results showed that treatment with anlotinib had no significant effect on rat CYP1A2, CYP2D2, and CYP2C6. However, anlotinib had a significant inductive effect on CYP2D1 and CYP3A1/2. Therefore, caution is needed during the concomitant use of anlotinib with other drugs metabolized by CYP2D1 and CYP3A1/2 because of potential drug-anlotinib interactions.
- Simultaneous Assessment in vitro of Transporter and Metabolic Processes in Hepatic Drug Clearance: Use of a media loss approach. [Journal Article]
- DMDrug Metab Dispos 2018 Feb 08
- Hepatocyte drug depletion-time assays are well established for the determination of metabolic clearance in vitro. The present study focuses on the refinement and evaluation of a "media loss" assay, a...
Hepatocyte drug depletion-time assays are well established for the determination of metabolic clearance in vitro. The present study focuses on the refinement and evaluation of a "media loss" assay, an adaptation of the conventional depletion assay involving centrifugation of hepatocytes prior to sampling, allowing estimation of uptake in addition to metabolism. Using experimental procedures consistent with a high throughput, a selection of 12 compounds with a range of uptake and metabolism characteristics (atorvastatin, cerivastatin, clarithromycin, erythromycin, indinavir, pitavastatin, repaglinide, rosuvastatin, saquinavir and valsartan, together with two control compounds - midazolam and tolbutamide) were investigated in the presence and absence of the cytochrome P450 inhibitor 1-aminobenzotriazole and organic anion transporter protein inhibitor rifamycin SV. Data generated simultaneously for a given drug provided, through the use of a mechanistic cell model, clearance terms characterising metabolism, active and passive uptake, together with intracellular binding and partitioning parameters. Results were largely consistent with the particular drug characteristics, with active uptake, passive diffusion and metabolic clearances ranging between 0.4 - 777, 3 - 383 and 2 - 236 μL/min/mg protein, respectively. The same experiments provided total and unbound drug cellular partition coefficients ranging between 3.8 - 254 and 2.3 - 8.3, respectively and intracellular unbound fractions between 0.014 - 0.263. Following in vitro to in vivo extrapolation, the lowest prediction bias was noted using uptake clearance, compared to metabolic clearance or apparent clearance from the media loss assay alone. This approach allows rapid and comprehensive characterisation of hepatocyte drug disposition valuable for prediction of hepatic processes in vivo.
- Evaluating the impact of type 2 diabetes mellitus on CYP450 metabolic activities: protocol for a case-control pharmacokinetic study. [Journal Article]
- BOBMJ Open 2018 02 08; 8(2):e020922
- Diabetes affects more than 9% of the adult population worldwide. Patients with type 2 diabetes mellitus (T2DM) show variable responses to some drugs which may be due, in part, to variability in the f...
Diabetes affects more than 9% of the adult population worldwide. Patients with type 2 diabetes mellitus (T2DM) show variable responses to some drugs which may be due, in part, to variability in the functional activity of drug-metabolising enzymes including cytochromes P450 (CYP450s). CYP450 is a superfamily of enzymes responsible for xenobiotic metabolism. Knowledge must be gained on the impact of T2DM and related inflammatory processes on drug metabolism and its consequences on drug response. The aim of this study is to characterise the activity of CYP1A2, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4/5 in T2DM versus non-T2DM subjects following the administration of a cocktail of probe drug substrates.
- Crystal Nucleation of Tolbutamide in Solution: Relationship to Solvent, Solute Conformation, and Solution Structure. [Journal Article]
- CChemistry 2018 Feb 12
- The influence of the solvent in nucleation of Tolbutamide has been explored by measuring nucleation induction times, estimating solvent-solute interaction enthalpies using molecular modelling and cal...
The influence of the solvent in nucleation of Tolbutamide has been explored by measuring nucleation induction times, estimating solvent-solute interaction enthalpies using molecular modelling and calorimetric data, probing interactions and clustering with spectroscopy, and modelling solvent-dependence of molecular conformation in solution. The nucleation driving force required to reach the same induction time is strongly solvent-dependent, increasing in the order: acetonitrile < ethyl acetate < n-propanol < toluene. The combined DFT and MD modelling results show that in acetonitrile, ethyl acetate and n-propanol the nucleation difficulty is a function of the strength of solvent-solute interaction. A clear exception from this rule is the most difficult nucleation in toluene despite the weakest solvent-solute interactions. However molecular dynamics modelling predicts that tolbutamide assumes an intramolecularly H-bonded conformation in toluene and this presents an additional barrier to nucleation. This explains why nucleation in toluene is the most difficult and why the relatively higher propensity for aggregation of tolbutamide molecules in toluene solution, as observed with FTIR spectroscopy, does not translate into easier nucleation. Thus, our combined experimental and molecular modelling study suggests that the solvent can influence on the nucleation not only via differences in the desolvation but also through the influence on molecular conformation.
- Modulation of Excitability of Stellate Neurons in the Ventral Cochlear Nucleus of Mice by ATP-Sensitive Potassium Channels. [Journal Article]
- JMJ Membr Biol 2018 Jan 29
- Major voltage-activated ionic channels of stellate cells in the ventral part of cochlear nucleus (CN) were largely characterized previously. However, it is not known if these cells are equipped with ...
Major voltage-activated ionic channels of stellate cells in the ventral part of cochlear nucleus (CN) were largely characterized previously. However, it is not known if these cells are equipped with other ion channels apart from the voltage-sensitive ones. In the current study, it was aimed to study subunit composition and function of ATP-sensitive potassium channels (KATP) in stellate cells of the ventral cochlear nucleus. Subunits of KATP channels, Kir6.1, Kir6.2, SUR1, and SUR2, were expressed at the mRNA level and at the protein level in the mouse VCN tissue. The specific and clearly visible bands for all subunits but that for Kir6.1 were seen in Western blot. Using immunohistochemical staining technique, stellate cells were strongly labeled with SUR1 and Kir6.2 antibodies and moderately labeled with SUR2 antibody, whereas the labeling signals for Kir6.1 were too weak. In patch clamp recordings, KATP agonists including cromakalim (50 µM), diazoxide (0.2 mM), 3-Amino-1,2,4-triazole (ATZ) (1 mM), 2,2-Dithiobis (5-nitro pyridine) (DTNP) (330 µM), 6-Chloro-3-isopropylamino- 4H-thieno[3,2-e]-1,2,4-thiadiazine 1,1-dioxide (NNC 55-0118) (1 µM), 6-chloro-3-(methylcyclopropyl)amino-4H-thieno[3,2-e]-1,2,4-thiadiazine 1,1-dioxide (NN414) (1 µM), and H2O2 (0.88 mM) induced marked responses in stellate cells, characterized by membrane hyperpolarization which were blocked by KATP antagonists. Blockers of KATP channels, glibenclamide (0.2 mM), tolbutamide (0.1 mM) as well as 5-hydroxydecanoic acid (1 mM), and catalase (500 IU/ml) caused depolarization of stellate cells, increasing spontaneous action potential firing. In conclusion, KATP channels seemed to be composed dominantly of Kir 6.2 subunit and SUR1 and SUR2 and activation or inhibition of KATP channels regulates firing properties of stellate cells by means of influencing resting membrane potential and input resistance.
- Predicted contributions of cytochrome P450s to drug metabolism in human liver microsomes using relative activity factor were dependent on probes. [Journal Article]
- XXenobiotica 2018 Feb 08; :1-8
- Contributions of cytochrome P450 (CYP450) isoforms to drug metabolism are often predicted using relative activity factor (RAF) method, assuming RAF values were independent of probe. We aimed to repor...
Contributions of cytochrome P450 (CYP450) isoforms to drug metabolism are often predicted using relative activity factor (RAF) method, assuming RAF values were independent of probe. We aimed to report probe-dependent characteristic of RAF values using CYP3A4 or CYP2C9 probes. Metabolism of four CYP3A4 probes (testosterone, midazolam, verapamil and atorvastatin) and three CYP2C9 probes (tolbutamide, diclofenac and S-warfarin) in human liver microsomes (HLM) and cDNA-expressed recombinant CYP450 (Rec-CYP450) systems were characterized and RAFCLvalue was estimated as ratio of probe intrinsic clearance in HLM to that in Rec-CYP450. CYP450i contributions to metabolic reaction of a probe were predicted using other probes and compared with data from specific inhibitions. Contributions of CYP3A4 and CYP2C9 to metabolism of deoxypodophyllotoxin and nateglinide were also predicted. RAF values were dependent on probes, leading to probe-dependently predicted contributions. Predicted contributions of CYP3A4 to formations of 6β-hydroxytestosterone, 1'-hydroxymidazolam, norverapamil, ortho-hydroxyatorvastatin and para-hydroxyatorvastatin using other probes were 47.46-219.46%, 21.62-98.87%, 186.49-462.44%, 21.87-101.15% and 53.62-247.97%, respectively. Predicted contributions of CYP3A4 and CYP2C9 to nateglinide metabolism were 8.18-37.84% and 36.08-94.04%, separately. In conclusion, CYP450i contribution to drug metabolism in HLM estimated using RAF approach were probe-dependent. Therefore, contribution of each isoform must be confirmed by multiple probes.
- Comprehensive PBPK Model of Rifampicin for Quantitative Prediction of Complex Drug-Drug Interactions: CYP3A/2C9 Induction and OATP Inhibition Effects. [Journal Article]
- CPCPT Pharmacometrics Syst Pharmacol 2018 Jan 25
- This study aimed to construct a physiologically based pharmacokinetic (PBPK) model of rifampicin that can accurately and quantitatively predict complex drug-drug interactions (DDIs) involving its sat...
This study aimed to construct a physiologically based pharmacokinetic (PBPK) model of rifampicin that can accurately and quantitatively predict complex drug-drug interactions (DDIs) involving its saturable hepatic uptake and auto-induction. Using in silico and in vitro parameters, and reported clinical pharmacokinetic data, rifampicin PBPK model was built and relevant parameters for saturable hepatic uptake and UDP-glucuronosyltransferase (UGT) auto-induction were optimized by fitting. The parameters for cytochrome P450 (CYP) 3A and CYP2C9 induction by rifampicin were similarly optimized using clinical DDI data with midazolam and tolbutamide as probe substrates, respectively. For validation, our current PBPK model was applied to simulate complex DDIs with glibenclamide (a substrate of CYP3A/2C9 and hepatic organic anion transporting polypeptides (OATPs)). Simulated results were in quite good accordance with the observed data. Altogether, our constructed PBPK model of rifampicin demonstrates the robustness and utility in quantitatively predicting CYP3A/2C9 induction-mediated and/or OATP inhibition-mediated DDIs with victim drugs.
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- Storage stability study of porcine hepatic and intestinal cytochrome P450 isoenzymes by use of a newly developed and fully validated highly sensitive HPLC-MS/MS method. [Journal Article]
- ABAnal Bioanal Chem 2018; 410(6):1833-1843
- Microsomes are an ideal medium to investigate cytochrome P450 (CYP450) enzyme-mediated drug metabolism. However, before microsomes are prepared, tissues can be stored for a long time. Studies about t...
Microsomes are an ideal medium to investigate cytochrome P450 (CYP450) enzyme-mediated drug metabolism. However, before microsomes are prepared, tissues can be stored for a long time. Studies about the stability of these enzymes in porcine hepatic and intestinal tissues upon storage are lacking. To be able to investigate CYP450 stability in microsomes prepared from these tissues, a highly sensitive and rapid HPLC-MS/MS method for the simultaneous determination of six CYP450 metabolites in incubation medium was developed and validated. The metabolites, paracetamol (CYP1A), 7-hydroxy-coumarin (CYP2A), 1-hydroxy-midazolam (CYP3A), 4-hydroxy-tolbutamide (CYP2C), dextrorphan (CYP2D), and 6-hydroxy-chlorzoxazone (CYP2E) were extracted with ethyl acetate at pH 1.0, followed by evaporation and separation on an Agilent Zorbax Eclipse Plus C18 column. The method was fully validated in a GLP-compliant laboratory according to European guidelines and was highly sensitive (LOQ = 0.25-2.5 ng/mL), selective, had good precision (RSD-within, 1.0-9.1%; RSD-between, 1.0-18.4%) and accuracy (within-run, 83.3-102%; between-run, 78.5-102%), and showed no relative signal suppression and enhancement. Consequently, this method was applied to study the stability of porcine hepatic and intestinal CYP450 isoenzymes when tissues were stored at - 80 °C. The results indicate that porcine CYP450 isoenzymes are stable in tissues at least up to 4 months when snap frozen and stored at - 80 °C. Moreover, the results indicate differences in porcine CYP450 stability compared to rat, rabbit, and fish CYP450, as observed by other research groups, hence stressing the importance to investigate the CYP450 stability of a specific species.