ABSTRACT
OBJECTIVE：To study the inhibitor y effects of cajanonic acid A on 5 kinds of cytochrome P 450（CYP）enzyme，in human liver microsomes in vitro . METHODS ：By Cocktail probe substrate method ，50.0，15.0，5.0，1.5，0.5，0.15，0.05 μmol/L cajanonic acid A were added into liver microsomes ， and incubated with mixed probe substrates [including phenacetin ， dextromethorphan，omeprazole，testosterone and toluenesulfonbutylurea （probe substrates of CYP 1A2，CYP2D6，CYP2C19， CYP3A4，CYP2C9，respectively）]. On the basis of setting up blank group and positive control group [ α-naphthalene brass ， quinidine，（+）-N-3-benzyl vanillin ，ketoconazole and sulfabendazole （specific inhibitors of CYP 1A2，CYP2D6，CYP2C19， CYP3A4，CYP2C9，respectively）]，using puerarin as internal standard ，UPLC-MS/MS method was adopted to determine the contents of corresponding metabolites （acetaminophen， dextrophane， 5-hydroxy omeprazole ， 6 β-hydroxytestosterone， hydroxytolbutamide）. The determination was performed on ACQUITY UPLC ® BEH C 18 column，with mobile phase consisted of 0.01％ formic acid aqueous solution- 0.01％ acetonitrile formic acid （gradient elution ）at the flow rate of 0.4 mL/min. The column temperature was 40 ℃，and the sample size was 2 μL. An electrospray ionization source was used to conduct positive and negative ion scanning in the multiple reaction monitoring mode. The data acquisition range was m/z 100-1 200，the collision gas was argon ， the atomized gas was nitrogen ，the gas flow rate of the cone hole was 50 L/h，the desorption gas flow rate was 800 L/h，the capillary voltage under positive and negative mode was 2.0， 1.5 kV，and the ion source temperature was 120 ℃，110 ℃， respectively. The desolvent temperature were 400 ℃ and 450 ℃ ， respectively. Non linear regression analysis was performed by using Graphpad Prism 5.0 software and IC 50 wascalculated. RESULTS ：The linear ranges of above metabolifes were 0.26-8.35，0.36-34.56，0.10-3.09, 3.67-117.37，0.15-4.88 μmol/L（R2＞0.99）. The limits of quantitation were 0.26，0.36， 0.10，3.67，0.15 μmol/L，respectively. The IC 50 values of specific inhibitors in positive control group to CYP 1A2，CYP2D6， CYP2C19，CYP3A4 and CYP 2C9 in human liver microsomes were all within the acceptable range reported in the literature. The IC50 values of cajanonic acid A to CYP 1A2，CYP2D6 and CYP 3A4 in human liver microsomes were all more than 50 μmol/L，and the IC 50 values of CYP 2C9 and CYP 2C19 were 4.94 and 18.00 μmol/L，respectively. CONCLUSIONS ：Cajanonic acid A has no inhibitory effect on CYP 1A2，CYP2D6 and CYP 3A4，but has a certain inhibitory effect on CYP 2C9 and CYP 2C19.
ABSTRACT
Mammalian catechol-O-methyltransferases(COMT)are an important class of conjugative enzymes,which play a key role in the metabolism and inactivation of catechol neurotransmitters,catechol es-trogens and a wide range of endobiotics and xenobiotics that bear the catechol group.Currently,COMT inhibitors are used in combination with levodopa for the treatment of Parkinson's disease in clinical practice.The crucial role of COMT in human health has raised great interest in the development of more practical assays for highly selective and sensitive detection of COMT activity in real samples,as well as for rapid screening and characterization of COMT inhibitors as drug candidates.This review summarizes recent advances in analytical methodologies for sensing COMT activity and their applications.Several lists of biochemical assays for measuring COMT activity,including the probe substrates,along with their analytical conditions and kinetic parameters,are presented.Finally,the challenges and future perspec-tives in the field,such as visualization of COMT activity in vivo and in situ,are highlighted.Collectively,this review article overviews the practical assays for measuring COMT activities in complex biological samples,which will strongly facilitate the investigations on the relevance of COMT to human diseases and promote the discovery of COMT inhibitors via high-throughput screening.
ABSTRACT
Cytochrome P4502J2 (CYP2J2) is widely distributed in various human tissues and takes a part in the metabolism of endogenous compounds and drugs. CYP2J2 can convert arachidonic acid (AA) to expoxyeicosatrienoic acids (EETs), which have various biological effects, implying the important role of CYP2J2 in the regulation of cardiovascular system and promotion of tumor progression and metastasis. Additionally, CYP2J2 plays an indispensable role in the intestinal metabolism of various drugs, such as astemizole, terfenadine and ebastine. In this review, the metabolic function, characteristic of catalysis and tissue distribution of CYP2J2 are discussed with the latest literatures both in China and abroad. The state-of-the-art methods for characterization of CYP2J2 and current trend of substrate discovery as well as its relationship with disease are highlighted. This review gives in-depth understanding of the function of CYP2J2 and its role in disease advance. The information of ligand (substrate and inhibitor) will provide the theoretical guidance and reference to the development of novel drugs for CYP2J2.
ABSTRACT
The study was conducted to evaluate the pharmacokinetics of midazolam (MDZ) under different oral dosages in rats, and determine the optimum oral dosage of MDZ, a CYP3A probe substrate in vivo. Male Sprague-Dawley rats were given a single oral dosages of MDZ at 1, 2, 5, 10, 15 or 20 mg·kg-1. Plasma concentrations of MDZ and its major metabolite 1-hydroxyl midazolam (1-OH MDZ) were determined at different time points using a validated LC-MS/MS method. Pharmacokinetic parameters were calculated using non-compartmental model. The Cmax, AUC0-t and AUC0-∞ of MDZ and 1-OH MDZ were linearly increased over the dose range of 1-5 mg·kg-1 (r > 0.99), but not at the dose of 15 or 20 mg·kg-1. The AUC/Dose at 1-10 mg·kg-1 were not significant different, but that of 15 or 20 mg·kg-1 were significantly higher. No significant sedative reaction was observed in all the rats at dosages of 1-5 mg·kg-1, however loss of righting reflex was observed in rats receiving dosages of 10-20 mg·kg-1. In conclusion, the optimized oral dose of MDZ was 1-5 mg·kg-1 when MDZ is used as the CYP3A probe substrate in rats.
ABSTRACT
Analysis of metabolic activities of cytochrome P450 isoenzyme is a crucial index to study drug/toxicant metabolism, drug-drug interaction, polymorphism and et al. Due to this practice, it is important to use the proper probe substrate and to conduct the experiment under optimal conditions. The validation information in literatures on the most common and newest in vitro probe substrates have been reviewed.