CYP Isoforms Research Articles

The Development and Validation of a Novel "Dual Cocktail" Probe for Cytochrome P450s and Transporter Functions to Evaluate Pharmacokinetic Drug-Drug and Herb-Drug Interactions.

This study was designed to develop and validate a 10 probe drug cocktail named “Dual Cocktail”, composed of caffeine (Cyp1a2 in rat and CYP1A2 in human, 1 mg/kg), diclofenac (Cyp2c11 in rat and CYP2C9 in human, 2 mg/kg), omeprazole (Cyp2c11 in rat and CYP2C19 in human, 2 mg/kg), dextromethorphan (Cyp2d2 in rat and CYP2D6 in human, 10 mg/kg), nifedipine (Cyp3a1 in rat and CYP3A4 in human, 0.5 mg/kg), metformin (Oct1/2 in rat and OCT1/2 in human, 0.5 mg/kg), furosemide (Oat1/3 in rat and OAT1/3 in human, 0.1 mg/kg), valsartan (Oatp2 in rat and OATP1B1/1B3 in human, 0.2 mg/kg), digoxin (P-gp in rat and human, 2 mg/kg), and methotrexate (Mrp2 in rat and MRP2 in human, 0.5 mg/kg), for the evaluation of pharmacokinetic drug–drug and herb-drug interactions through the modulation of a representative panel of CYP enzymes or transporters in rats. To ensure no interaction among the ten probe substrates, we developed a 2-step evaluation protocol. In the first step, the pharmacokinetic properties of five individual CYP probe substrates and five individual transporter substrates were compared with the pharmacokinetics of five CYP cocktail or five transporters cocktails in two groups of randomly assigned rats. Next, a pharmacokinetic comparison was conducted between the CYP or transporter cocktail group and the dual cocktail group, respectively. None of the ten comparison groups was found to be statistically significant, indicating the CYP and transporter substrate sets or dual cocktail set could be concomitantly administered in rats. The “Dual Cocktail” was further validated by assessing the metabolism of nifedipine and omeprazole, which was significantly reduced by a single oral dose of ketoconazole (10 mg/kg); however, no changes were observed in the pharmacokinetic parameters of other probe substrates. Additionally, multiple oral doses of rifampin (20 mg/kg) reduced the plasma concentrations of nifedipine and digoxin, although not any of the other substrates. In conclusion, the dual cocktail can be used to characterize potential pharmacokinetic drug–drug interactions by simultaneously monitoring the activity of multiple CYP isoforms and transporters.

Phenotypic analysis of human CYP2C9 polymorphisms using fluorine-substituted tolbutamide.

To investigate the effect of fluorine substitution on tolbutamide (TB) hydroxylation catalyzed by CYP2C9, the hydroxylation of TB and its fluorinated derivative 3'-fluoro-tolbutamide (3'-F-TB) by recombinant human CYP2C9*1, CYP2C9*2, and CYP2C9*3 was analyzed. In general, fluorine substitution near the metabolic site may decrease enzymatic oxidation owing to its electron-withdrawing nature. Fluorine substitution reduced the Michaelis-Menten-derived Km of 4'-hydroxylation of TB catalyzed by CYP2C9*1 from 115 (TB) to 77 (3'-F-TB) µM. In the case of TB hydroxylation catalyzed by CYP2C9*2, the Km value of TB was also reduced by fluorine substitution from 129 to 88 µM. The greatest effect of fluorine substitution on the Km in TB hydroxylation was observed in the catalysis by CYP2C9*3, in which the Km value decreased from 287 to 117 µM. When a mixture containing TB and 3'-F-TB was hydroxylated by CYP2C9, the hydroxylated metabolite ratio in CYP2C9*3 was significantly increased compared with that in CYP2C9*1 and CYP2C9*2 (p < 0.01, Tukey-Kramer test). These results suggest that obtaining the metabolite profiles of fluorine-substituted analogs of the key substrate molecule may be useful as a new tool for phenotyping polymorphic CYP isoforms.

Validated LC–MS/MS method for the determination of some prescribed CNS drugs: Application to an in vivo pharmacokinetic study of drug-herb metabolic interaction potential of khat

The consumption of khat (Catha edulis), a natural amphetamine-like psychostimulant plant, during co-administration of other conventional medications may alter treatment outcomes. Therefore, in this in-vivo study, we investigated the effect of concomitant co-administration of Khat on the pharmacokinetics of some prescribed CNS drugs: clomipramine, vilazodone, aripiprazole, sertraline in rats. A UPLC-MS/MS method was developed and validated for the simultaneous determination of clomipramine, vilazodone, aripiprazole, sertraline in rat plasma. using a mobile phase consisting of acetonitrile: 0.1% formic acid in water: 10 mM ammonium formate (10: 45: 45, v/v/v) pumped through Waters Sunfire-C18 column (5 µm, 4. 6× 50 mm), using escitalopram as internal standard. Detection was carried out using a triple-quadrupole tandem mass spectrometer using multiple reaction monitoring mode. Ionization was conducted using electrospray ion source in positive ionization mode, the method was linear over the concentration range (1–400 ng/ml), with good results for coefficient of variation at LLOQ and QCs for all the cited drugs; confirming method precision and accuracy. This method was successfully applied to a drug- herb pharmacokinetic study in rats to assess the potential effect of consumption of khat on the bioavailability of clomipramine, vilazodone, aripiprazole, sertraline. It was found that khat consumption significantly increase the bioavailability of these drugs this might be due to inhibition of their metabolic enzymes CYP450, CYP2D6 and CYP3A4 isoforms, with the consequent increase in their systemic exposure and potential adverse effects. thus, a potentially significant herb-drug interaction must be considered in patients chewing khat while prescribing one of these drugs.

What Do We Know About Remdesivir Drug Interactions?

The global pandemic of severe acute respiratory syndrome coronavirus 2 (SARS‐Co‐V‐2) has resulted in a critical need to rapidly develop new pharmacologic interventions and disseminate information. This has led to confusing and conflicting information on drug efficacy. Remdesivir has emerged as a promising treatment for SARS‐Co‐V‐2 infection yet published clinical pharmacology and drug interaction studies are limited. Additional studies of the disposition of remdesivir, its active metabolite (GS‐441524), and its triphosphate metabolite (GS‐443902) are needed.

NADPH-Independent Inactivation of CYP2B6 and NADPH-Dependent Inactivation of CYP3A4/5 by PBD: Potential Implication for Assessing Covalent Modulators for Time-Dependent Inhibition

Pyrrolo[2,1-c][1,4]benzodiazepine dimer (PBD) has shown broad antitumor properties and potential as a therapeutic agent for cancers. During a routine drug-drug interaction assessment, it was found that PBD is a reversible inhibitor of CYP2C8 (IC50 = 1.1 µM) but not CYP1A2, 2B6, 2C9, 2C19, 2D6, or 3A4/5. Additionally, PBD is a classic time-dependent inhibition (TDI) of CYP3A4/5, with >30-fold shift in IC50 after a preincubation with NADPH. All other CYPs tested did not show evidence for TDI, but potent inhibition of CYP2B6 (IC50 = 1.5 µM) was observed after a preincubation with or without (w/wo) NADPH, which was an unexpected observation given the fact that no inhibition was observed in the direct inhibition assay. No other CYP isoforms were susceptible to this apparent non-NADPH-dependent inhibition, suggesting that PBD may selectively inactivate CYP2B6 without metabolic activation. The washing of the human liver microsome pellet after incubation with PBD did not fully recover CYP2B6 activity, indicating that PBD is covalently bound to CYP2B6, leading to inactivation of the enzyme. To further investigate the mechanism of NADPH-independent inhibition, the IC50 shift was determined for several PBD analogs, and it was found that the compounds without both reactive imines did not show NADPH-independent inhibition of CYP2B6, implying that NADPH-independent inactivation was likely caused by direct covalent binding of PBD to the enzyme in a highly structure-specific manner. These data clearly highlight the need to assess direct and time-dependent inhibition w/wo NADPH to adequately characterize the in vitro CYP inhibitory properties of drug candidates with reactive moieties. SIGNIFICANCE STATEMENT: We described a very unique in vitro CYP inhibition profile of pyrrolo[2,1-c][1,4]benzodiazepine dimer as a potent reversible CYP2C8 inhibitor, an NADPH-dependent CYP3A4/5 time-dependent inhibition (TDI) inhibitor, and an NADPH-independent CYP2B6 TDI inhibitor, and inhibition of CYPs occurs through three distinct mechanisms: reversible drug-enzyme binding, enzyme inactivation via bioactivation, and enzyme inactivation by covalent binding via chemical reactions. Our results suggest that, for compounds with reactive functional moieties, false positives can be reported when the conventional TDI assay is utilized.

In vitro effects of peimine on the activity of cytochrome P450 enzymes

Peimine is a major component of Fritillaria ussuriensis, which is a widely used herb in pediatric. It is very common in Chinese traditional medicine to combine with two or more herbs in the clinic. To investigate the effect of peimine on the activity of cytochrome P450 enzymes (CYP450) is necessary for the clinical application of peimine.The effects of peimine on eight human liver CYP isoforms (i.e., 1A2, 3A4, 2A6, 2E1, 2D6, 2C9, 2C19 and 2C8) were investigated in vitro in human liver microsomes (HLMs) with the specific inhibitors as positive control and without peimine or inhibitors as negative control. The enzyme kinetic parameters were calculated.It was found that peimine inhibited the activity of CYP3A4, 2E1, and 2D6 in a concentration-dependent manner with the IC50 values of 13.43, 21.93, and 22.46 μM, respectively. The inhibition of CYP3A4 was performed in a non-competitive manner with the Ki value of 6.49 μM, and the inhibition of CYP2E1 and 2D6 was performed in a competitive manner with Ki values of 10.76 and 11.95 μM. Additionally, peimine inhibited the activity of CYP3A4 in a time-dependent manner with the KI/Kinact value of 6.17/0.049 min−1 μM−1.Peimine inhibited the activity of CYP3A4, 2E1, and 2D6, which indicated the potential interaction between peimine and drugs metabolized by CYP3A4, 2E1, and 2D6. Further studies are needed to verify the drug-drug interaction and the in vivo effects.

Assessment of in vitro inhibitory effects of novel anti MRSA benzoquinolizine fluoroquinolone WCK 771 (levonadifloxacin) and its metabolite on human liver cytochrome P450 enzymes

WCK 771 (INN: levonadifloxacin) is a novel antibacterial agent belonging to benzoquinolizine subclass of fluoroquinolones which is under clinical development as a parenteral formulation and its prodrug WCK 2349 (INN: alalevonadifloxacin) as an oral option. Both the drugs have been approved recently in India based on phase III trial completed for ABSSSI.In vitro CYP inhibition potential of levonadifloxacin and its sulfate metabolite (WCK 2146) were assessed in this study. The inhibitory effects of levonadifloxacin and its sulfate metabolite were assessed for seven key human liver CYP isoforms 1A2, 2B6, 2C8, 2C9, 2C19, 2D6 and 3A4 using human liver microsome (HLM) employing validated LC-MS/MS method.The results showed that levonadifloxacin and its metabolite did not inhibit enzyme activity of any of the key CYP isoforms (1A2, 2B6, 2C8, 2C9, 2C19, 2D6 and 3A4) even at supra therapeutic concentrations (12–24X, Clinical Cmax: 25–35µg/mL).These in vitro CYP inhibition studies of levonadifloxacin and its sulfate metabolite indicate lack of potential for pharmacokinetic drug interactions of levonadifloxacin when co-administered with drugs which are substrate of these isoforms. Therefore, further clinical studies evaluating CYP mediated drug–drug interactions are not warranted for levonadifloxacin and alalevonadifloxacin.

A Translational Physiologically Based Pharmacokinetics/Pharmacodynamics Framework of Target-Mediated Disposition, Target Inhibition and Drug-Drug Interactions of Bortezomib.

Bortezomib is a potent 20S proteasome inhibitor approved for the treatment of multiple myeloma and mantle cell lymphoma. Despite the extensive clinical use of bortezomib, the mechanism of the complex time-dependent pharmacokinetics of bortezomib has not been fully investigated in context of its pharmacodynamics (PD) and drug-drug interaction (DDI) profiles. Here, we aimed to develop a mechanistic physiologically based (PB) PK/PD model to project PK, blood target inhibition and DDI of bortezomib in patients. A minimal PBPK/PD model consisting of six compartments was constructed using a bottom-up approach with pre-clinical data and human physiological parameters. Specifically, the target-mediated drug disposition (TMDD) of bortezomib in red blood cells (RBC), which determines target inhibition in blood, was characterized by incorporating the proteasome binding affinity of bortezomib and the proteasome concentration in RBC. The hepatic clearance and fraction metabolized by different CYP isoforms were estimated from in vitro metabolism and phenotyping experiments. The established model adequately characterized the multi-exponential and time-dependent plasma pharmacokinetics, target binding and blood proteasome inhibition of bortezomib. Further, the model was able to accurately predict the impact of a strong CYP3A inducer (rifampicin) and inhibitor (ketoconazole) on bortezomib exposure. In conclusion, the mechanistic PBPK/PD model successfully described the complex pharmacokinetics, target inhibition and DDIs of bortezomib in patients. This study illustrates the importance of incorporating target biology, drug-target interactions and in vitro clearance parameters into mechanistic PBPK/PD models and the utility of such models for pharmacokinetic, pharmacodynamic and DDI predictions.

Binding and Metabolism of Brominated Flame Retardant β-1,2-Dibromo-4-(1,2-dibromoethyl)cyclohexane in Human Microsomal P450 Enzymes: Insights from Computational Studies.

The emerging brominated flame retardant, 1,2-dibromo-4-(1,2-dibromoethyl)cyclohexane (TBECH), has recently attracted strong interest due to its extensive detection in the environment and potential toxicological effects on humans. Previous in vitro experiments have shown that the technical mixture of TBECH and the pure β-isomer (β-TBECH) can be metabolized by cytochrome P450 enzymes (CYPs) into multiple metabolites, but the specific CYP isoforms involved in TBECH metabolism and the relevant metabolic regioselectivity remain unknown. Here, we, for the first time, investigated the binding patterns and affinities of β-TBECH in human CYPs 1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, and 3A4, through molecular dynamics (MD) simulations. The binding affinities of β-TBECH in CYPs, which are estimated by the calculated binding free energies, follow the order of 2A6 > 2C9 > 2B6 > 2E1 > 3A4 ≈ 2C19 ≈ 1A2 > 2D6. Although all CYPs are important β-TBECH receptors, only 2A6, 2C19, 2E1, and 3A4 are responsible for metabolizing β-TBECH. Specially, 2A6 and 2E1 may selectively hydroxylate the C1 and C7 sites of β-TBECH, while 2C19 and 3A4 show metabolic preference for C7- and C8-hydroxylations, respectively. The three hydroxylation routes proposed by the further density functional theory (DFT) calculations generate C1-, C7-, and C8-hydroxylated metabolites, while the latter two may further undergo debromination to yield the respective ketone and aldehyde as additional metabolites. The results provide meaningful insight into the binding and metabolism of β-TBECH by human CYPs, which is helpful for understanding the metabolic fate and toxicity mechanism of this chemical.

Mathematical Modeling of In Vitro‐In Vivo Atypical Kinetics of Diazepam

Cytochrome P450 (CYP) and uridine 5'‐diphospho‐glucuronosyltransferase (UGT) enzymes are responsible for metabolism of most marketed drugs. Due to the large binding sites of certain CYPs and UGTs, CYP and UGT substrates tend to show non ‐ Michaelis‐Menten kinetics (atypical kinetics). Although several in vitro data provide evidence for the existence of atypical kinetics, this characteristic is seldom observed with in vivo data. The purpose of this project is to characterize the atypical kinetics of diazepam and its sequential metabolism in in vitro and in vivo systems. Numerical methods will be used to obtain kinetic parameters, which will be used for in vitro‐in vivo correlations (IVIVC).Diazepam, which exhibits atypical kinetics in vitro, and its primary metabolites (temazepam, nordiazepam), and secondary metabolite (oxazepam) were characterized with in vitro studies with pooled male Sprague Dawley rat liver microsome (MRLM), pooled female Sprague Dawley (FRLM), and purified rat cyp isoforms (cyp3a1, cyp3a2, and cyp2c11) separately. Rat microsomal partition and rat plasma protein binding assays were conducted via equilibrium dialysis to determine the free fraction of the drug. Two substrate binding (ESS) models provided better fits than one substrate (ES) models, as determined from the resulting AICc values. Preliminary in vitro data from MRLM and cyp3a1 showed obvious sigmoidal saturation curves for temazepam formation. Only cyp3a1 show significant sigmoidal curves for nordiazepam formation. For cyp3a1, Km and Vmax values for temazepam and nordiazepam calculated with the EES model are significantly different from those with the ES model. The intrinsic clearance (CLint) values calculated from the MRLM and cyp3a1 ESS models is not constant within the observed range of diazepam plasma levels of rats. No obvious sigmoidicity of diazepam saturation curves was observed for cyp3a2 (male‐specific) and cyp2c11 (male‐specific) isoforms, which are other cyp isoforms responsible for diazepam metabolism in male rats. In order to characterize in vitro‐in vivo correlations for diazepam, in vivo PK studies with female and male rats are currently underway.Support or Funding InformationThis research was supported by NIGMS grants: R01GM114369 and R01GM104178.

In-Vitro CYP3A4, CYP2E1 and UGT Activity in Human Liver Microsomes by Strobilanthes crispus Leaf Extracts

Background: Strobilanthes crispus (L.) Bremek (Acanthaceae) leaves are used traditionally in Malaysia, Thailand, and Indonesia for anti-diabetic, anti-lytic, diuretic, and laxative purposes. Herb-drug interactions may potentiate or antagonize the absorption and metabolism of drugs which may result in potential toxicity. The aim of the present study was to investigate the effect of juice, hot aqueous, cold aqueous and methanol extracts of S. crispus leaves on phase I cytochrome 3A4 (CYP3A4) and Cytochrome 2E1 (CYP2E1) and phase II human liver enzyme UDP-Glucuronosyl Transferase (UGT). Methods: The herb-drug interactions of the leaf extracts and juice were determined by specific enzyme activity of CYP isoforms with specific probe substrate using spectrophotometry. CYP3A4 activity was measured for aminopyrine specific metabolite (formaldehyde) at 415 nm. CYP2E1 activity was determined using p-nitrophenol specific metabolite (p-nitrocatechol) at 535 nm. UGT activity was quantified through the consumption of p-nitrophenol by UGT at 405 nm. Results: All the S. crispus preparations showed significant inhibition of CYP3A4 activity. Only the methanolic extract showed a significant inhibition in CYP2E1. All the S. crispus extracts showed a significant effect on UGT activation at the higher concentration (1000 ng/ml). Only the cold aqueous extract and the juice showed UGT inhibition at lower concentration (1 ng/ml). Conclusion: S. crispus preparations showed in-vitro drug-herb interaction effects on human liver microsomes. Therefore, there is a possibility of drug-herb interaction could occur with S. crispus leaves through its effect on CYP3A4. Inhibition of the herb extracts on CYP2E1 could show anticarcinogenesis effects. The potency of drugs that metabolized via UGT pathway may be affected when co-administered with S. crispus leaf preparations.

Biological Evaluation and Molecular Docking with In Silico Physicochemical, Pharmacokinetic and Toxicity Prediction of Pyrazolo[1,5-a]pyrimidines.

Pyrazolo[1,5-a]pyrimidines 5a–c, 9a–c and 13a–i were synthesized for evaluation of their in vitro antimicrobial properties against some microorganisms and their immunomodulatory activity. The biological activities of pyrazolo[1,5-a]pyrimidines showed that the pyrazolo[1,5-a]pyrimidines (5c, 9a, 9c, 13a, 13c, 13d, 13e and 13h) displayed promising antimicrobial and immunomodulatory activities. Studying the in silico predicted physicochemical, pharmacokinetic, ADMET and drug-likeness properties for the pyrazolo[1,5-a]pyrimidines 5a–c, 9a–c and 13a–i confirmed that most of the compounds (i) were within the range set by Lipinski’s rule of five, (ii) show higher gastrointestinal absorption and inhibition of some CYP isoforms, and (iii) have a carcinogenicity test that was predicted as negative and hERG test that presented medium risk. Moreover, the molecular docking study demonstrated that the compounds 5c, 9a, 9c, 13a, 13c, 13d, 13e and 13h are potent inhibitors of 14-alpha demethylase, transpeptidase and alkaline phosphatase enzymes. This study could be valuable in the discovery of a new series of drugs.

Testing the Pharmacokinetic Interactions of 24 Colonic Flavonoid Metabolites with Human Serum Albumin and Cytochrome P450 Enzymes.

Flavonoids are abundant polyphenols in nature. They are extensively biotransformed in enterocytes and hepatocytes, where conjugated (methyl, sulfate, and glucuronide) metabolites are formed. However, bacterial microflora in the human intestines also metabolize flavonoids, resulting in the production of smaller phenolic fragments (e.g., hydroxybenzoic, hydroxyacetic and hydroxycinnamic acids, and hydroxybenzenes). Despite the fact that several colonic metabolites appear in the circulation at high concentrations, we have only limited information regarding their pharmacodynamic effects and pharmacokinetic interactions. Therefore, in this in vitro study, we investigated the interactions of 24 microbial flavonoid metabolites with human serum albumin and cytochrome P450 (CYP2C9, 2C19, and 3A4) enzymes. Our results demonstrated that some metabolites (e.g., 2,4-dihydroxyacetophenone, pyrogallol, O-desmethylangolensin, and 2-hydroxy-4-methoxybenzoic acid) form stable complexes with albumin. However, the compounds tested did not considerably displace Site I and II marker drugs from albumin. All CYP isoforms examined were significantly inhibited by O-desmethylangolensin; nevertheless, only its effect on CYP2C9 seems to be relevant. Furthermore, resorcinol and phloroglucinol showed strong inhibitory effects on CYP3A4. Our results demonstrate that, besides flavonoid aglycones and their conjugated derivatives, some colonic metabolites are also able to interact with proteins involved in the pharmacokinetics of drugs.

Prediction of seriniquinone-drug interactions by in vitro inhibition of human cytochrome P450 enzymes

Seriniquinone is a secondary metabolite isolated from a rare marine bacterium of the genus Serinicoccus. This natural quinone is highlighted for its selective cytotoxic activity toward melanoma cancer cells, in which rapid metastatic properties are still a challenge for clinical treatment of malignant melanoma. The progress of seriniquinone as a promising bioactive molecule for drug development requires the assessment of its clinical interaction potential with other drugs. This study aimed to investigate the in vitro inhibitory effects of seriniquinone on the main human CYP450 isoforms involved in drug metabolism. The results showed strong inhibition of CYP1A2, CYP2E1 and CYP3A, with IC50 values up to 1.4 μM, and moderate inhibition of CYP2C19, with IC50 value >15 μM. Detailed experiments performed with human liver microsomes showed that the inhibition of CYP450 isoforms can be explained by competitive and non-competitive inhibition mechanisms. In addition, seriniquinone demonstrated to be an irreversible and time-dependent inhibitor of CYP1A2 and CYP3A. The low inhibition constants values obtained experimentally suggest that concomitant intake of seriniquinone with drug metabolized by these isoforms should be carefully monitored for adverse effects or therapeutic failure.

Assessment of ionic homeostasis imbalance and cytochrome P450 system disturbance in mice during fumonisin B1 (FB1) exposure

Fumonisin B1 (FB1) is a mycotoxin frequently found in agricultural commodities, and poses a considerable risk for human and animal health. The aim of this study was to investigate the toxic effect of FB1 in mice intestine. Male Kunming mice (n = 40) were treated with FB1 diet for 42 days. Histopathological and biochemical analyses, including ion concentrations, transcription of ATPase subunits and mRNA expression of cytochrome P450s (CYP450s) analyses were performed on duodenum, cecum and colon of mice. The results revealed that FB1 caused histological alterations, including partial shedding of villous epithelial cells and inflammatory cell infiltration. Furthermore, a significant change in Na+, K+ and Ca2+ in serum, and the mRNA expression of ATPase subunits and CYP450s in intestinal tracts were observed in FB1-exposed mice. Our results suggested that FB1 exposure induce histopathological injury via disrupting CYP isoforms transcription and triggering ion homeostasis imbalance in mice intestinal tracts.

Pharmacokinetics of omeprazole in rats with dextran sulfate sodium–induced ulcerative colitis

Omeprazole is a commonly used drug in patients with ulcerative colitis (UC). This study investigated the pharmacokinetics of omeprazole in rats with UC induced by dextran sulfate sodium (DSS). The pharmacokinetics of intravenously administered omeprazole (20 mg/kg) was investigated in normal and UC rats using LC-MS/MS. The formation of 5-OH omeprazole, a main metabolite of omeprazole, in rat liver microsomes (RLMs) from normal and UC rats was compared. The protein levels of CYP1A2, CYP2D1, and CYP3A1 in the liver were measured by Western blot. Compared with normal rats, UC rats had increased plasma concentrations of omeprazole, resulting in an increased AUC0–240 min and decreased CL. DSS treatment decreased the formation rate of 5-OH omeprazole in RLMs but did not change the affinity of the enzymes. The Vmax and CLint of RLMs from UC rats were 62% and 48% those of RLMs from normal rats, respectively. The hepatic CYP1A2 and CYP3A1 protein levels in UC rats were 42.6 and 45.2% lower than those in normal rats, respectively; however, the protein levels of CYP2D1 in the two groups were similar. The activity and expression of some hepatic CYP450 isoforms were decreased by UC, leading to changes in the pharmacokinetics of omeprazole.

In vitro effect of pachymic acid on the activity of Cytochrome P450 enzymes

Pachymic acid is a wildly used traditional Chinese medicine with various pharmacological features. It also exists in many drugs which are wildly used in pediatric.The effect of pachymic acid on the activity of eight major CYP isoforms was investigated in human liver microsomes.The effects of pachymic acid on eight human liver CYP isoforms (i.e. 1A2, 3A4, 2A6, 2E1, 2D6, 2C9, 2C19 and 2C8) were investigated in vitro using human liver microsomes (HLMs), and the enzyme kinetic parameters were calculated.The activity of CP3A4, 2E1, and 2C9 was inhibited by pachymic acid in a concentration-dependent manner with IC50 values of 15.04, 27.95, and 24.22 μM, respectively. Pachymic acid is a non-competitive inhibitor of CYP3A4, with the Ki value of 6.47 μM. While the inhibition of CYP2E1 and 2C9 was performed in a competitive manner, with the Ki value of 11.96 and 10.94 μM, respectively. Moreover, the inhibition of CYP3A4 was in a time-dependent manner with the KI/Kinact value of 7.77/0.048 min−1 μM−1.The in vitro inhibitory effect of pachymic acid on the activity of CYP3A4, 2E1, and 2C9 indicated the potential drug-drug interaction with the drugs that metabolized by CYP3A4, 2E1, and 2C9. Further clinical and in vivo studies are needed to evaluate the significance of this interaction.

Di-(2-ethylhexyl) phthalate induced nephrotoxicity in quail (Coturnix japonica) by triggering nuclear xenobiotic receptors and modulating the cytochrome P450 system

Di-(2-ethylhexyl) phthalate (DEHP), a plasticizer that is mainly used in the production of polyvinyl alcohol-containing chloride products, has attracted attention due to potential threats to human health and the environment. Nevertheless, knowledge of DEHP-induced nephrotoxicity is still limited. To explore the mechanism of DEHP-induced nephrotoxicity, quail were treated with 0, 250, 500 and 1000 mg/kg DEHP by oral gavage for 45 days. Based on the results of histopathological analysis, DEHP exposure induced a disorganized renal structure, a partially dilated glomerulus and an atrophied Bowman’s space. Renal tubular epithelial cells were unclear, and swelling of columnar epithelial cells was observed, suggesting that DEHP exposure caused renal disease and renal injury. Notably, DEHP interfered with the homeostasis of cytochrome P450 systems (CYP450s) by increasing the activities or contents of CYP450s (total CYP450, Cyt b5, ERND, APND, AH and NCR). The expression levels of certain CYP450 isoforms (CYP1A, CYP1B, CYP2C, CYP2D, CYP2J and CYP3A) were significantly downregulated in the kidney in DEHP-treated quail. Furthermore, DEHP induced the expression of nuclear receptors (AHR, CAR and PXR) in a dose-dependent manner. The results of this study suggested that DEHP-induced nephrotoxicity in quail was associated with the induction of nuclear xenobiotic receptor (NXR) responses and interference with CYP450 homeostasis. In conclusion, all data indicated that DEHP induced nephrotoxicity by triggering NXRs and modulating the cytochrome P450 system. The results of this study provide a new basis for understanding the nephrotoxicity of DEHP.

Discovery of cytochrome P450 enzymes-inhibiting components in traditional Chinese medicine

With the widespread use of traditional Chinese medicine(TCM) and the integration of TCM and western medicine, drug-drug interaction(DDI) is considered as a major cause of therapeutic failures and side effects. Cytochrome P450 enzymes(CYPs) are responsible for large number of drug metabolism. CYP3 A4 and CYP2 D6, two important CYP isoforms, are responsible for about 80% drug metabolism of CYPs super family. The inhibition of CYPs is likely to be the most common factor leading to adverse DDI. Therefore, it is of great significance to predict potential CYP3 A4 and CYP2 D6 inhibitors to prevent the DDI. A fast and low-cost me-thod for calculating and predicting CYP inhibiting components was established in this paper, namely support vector machine(SVM) and molecular docking technology which are used to predict and screen drugs. Firstly, 12 qualitative models of two targets were established by using SVM, and the optimal model was selected to predict the compounds in traditional Chinese medicine database(TCMD). Then, molecular docking technology was used to establish docking model. By analyzing the key amino acids involved in drug-target interactions and combining with SVM model, potential inhibitors of CYP3 A4 and CYP2 D6 were found. From the computational results, astin D and epiberberine exhibited inhibition effect on CYP3 A4 and CYP2 D6, respectively. Astin D was only found in astins family from Aster tataricus, while epiberberine was considered to be the active constituent of Coptidis Rhizoma. Therefore, for the risk of DDI, extra attention should be paid to the source of these potential inhibitors, Asteris Radix et Rhizoma and Coptidis Rhizoma. This computational method provides technical support for discovering potential natural inhibitors of CYPs from Chinese herbs by using SVM and molecular docking model, and it is also helpful to recognize the CYPs-mediated DDI existing in TCM, providing research ideas for further pharmacovigilance of integrated therapy.

Association of CYP2C19*2 polymorphism with clopidogrel resistance among patients with high cardiovascular risk in Northeastern Mexico.

Oral antiplatelet drugs are a key to modern pharmacotherapy in cardiovascular atherothrombotic diseases. Clopidogrel (CLO) constitutes the main preventive treatment of atherothrombosis. However, a considerable inter-individual variation in CLO response has been documented, resulting in suboptimal therapy and an increased risk of recurrent adverse effects in some patients. The enzyme CYP2C19 has been reported to be the CYP isoform that activates CLO to its active metabolite. Several single nucleotide polymorphisms in the CYP2C19 gene have been identified as strong predictors of CLO-impaired pharmacological response. At least 16 variants have been associated with changes in CYP2C19 activity. The following research was composed of a total of 102 subjects with high cardiovascular risk in the northeast of Mexico, with a maintenance dose of 75 mg of CLO per day. The platelet reactivity was measured with VerifyNow P2Y12 assay, while the presence of CYP2C19*2 was identified by real-time polymerase chain reaction. Patients were categorized by CYP2C19 metabolizer status based on *2 genotypes using the common consensus star allele nomenclature as normal metabolizer (G/G), intermediate metabolizer (G/A), and poor metabolizer (A/A), respectively. The phenotype frequency for CYP2C19*2 was 74.5% (G/G), 21.6% (G/A), and 3.9% (A/A). The subjects with the A allele presented ≥235 P2Y12 reaction unit levels, classifying them how poor metabolizer. The prevalence of reduced CLO effectiveness was associated with the presence of CYP2C19*2 polymorphism among Mexican patients. The presence of the CYP2C19*2 allele is related to resistance to the antiplatelet effect of CLO (p = 0.003).