Liquid chromatography/tandem mass spectrometric determination of inhibition of human cytochrome P450 isozymes by resveratrol and resveratrol-3-sulfate

trans-Resveratrol, a phenolic phytoalexin occurring in grapes, wine, peanuts, and cranberries, has been reported to both have anticarcinogenic, antioxidative, phytoestrogenic, and cardioprotective activities, and to be a weak inhibitor of cytochrome P450 (CYP)3A4, which might have significance for drug-drug interactions. Since trans-resveratrol is rapidly converted in vivo to primarily trans-resveratrol-3-sulfate, a rapid, selective, and sensitive method using liquid chromatography/tandem mass spectrometry (LC/MS/MS) was developed to investigate human cytochrome P450 inhibition by trans-resveratrol-3-sulfate. Effects of trans-resveratrol and trans-resveratrol-3-sulfate on the metabolism of selective cytochrome P450 substrates (CYP1A2/ethoxyresorufin, CYP2C9/diclofenac, CYP2C19/(S)-mephenytoin, CYP2D6/bufuralol, CYP3A4/testosterone) were monitored using cDNA-expressed human recombinant isozymes. For method validation, LC/MS/MS was used to measure the inhibition of various cytochrome P450 isozymes by different concentrations (0-50 microM) of known selective inhibitors. IC(50) values of 3.2, 1.4, 8.9, 0.2, and 0.3 microM were obtained for the standard isozyme inhibitors CYP1A2/furafylline, CYP2C9/sulfaphenazole, CYP2C19/tranylcypromine, CYP2D6/quinidine, and CYP3A4/ketoconazole, respectively, which were in good agreement with literature values. trans-Resveratrol showed IC(50) values of 11.6 microM for CYP2C19 and 1.1 microM for CYP3A4, but the IC(50) values exceeded 50 microM for all the other CYP isozymes, which indicated no inhibition. No enzyme inhibition was observed for trans-resveratrol-3-sulfate. Our results indicate that trans-resveratrol is a marginal inhibitor of CYP3A4 and a weak inhibitor of CYP2C19, but its major metabolite trans-resveratrol-3-sulfate is not an inhibitor of any of the cytochrome P450 isozymes investigated.     

Simultaneous determination of the inhibitory potency of herbal extracts on the activity of six major cytochrome P450 enzymes using liquid chromatography/mass spectrometry and automated online extraction

Here we describe a liquid chromatography/mass spectrometry (LC/MS) method with automated online extraction (LC/LC/MS) to simultaneously determine the in vitro inhibitory potency of herbal extracts on six major human drug-metabolising cytochrome P450 enzymes. Substrates were incubated with a commercially available mixture of CYP1A2/2C8/2C9/2C19/2D6 and 3A4 from baculovirus-infected insect cells and the resulting metabolites were quantified with LC/LC/MS using electrospray ionisation in the selected ion monitoring mode. Consistent inhibitory activities were obtained for known inhibitors and plant extracts using the enzyme/substrate cocktail and the individual enzymes/substrates. Popular herbal remedies including devil's claw root (Harpagophytum procumbens), feverfew herb (Tanacetum parthenium), fo-ti root (Polygonum multiflorum), kava-kava root (Piper methysticum), peppermint oil (Mentha piperita), eucalyptus oil (Eucalyptus globulus), red clover blossom (Trifolium pratense) and grapefruit juice (GJ; Citrus paradisi) could be identified as inhibitors of the applied CYP enzymes with IC(50) values between 20 and 1000 microg/mL.     

Analysis of frankincense from various Boswellia species with inhibitory activity on human drug metabolising cytochrome P450 enzymes using liquid chromatography mass spectrometry after automated on-line extraction

In our search for herbal remedies with inhibitory activity on cytochrome P450 (CYP) enzymes, we identified extracts of the gum-resin of Boswellia carteri, Boswellia frereana, Boswellia sacra and Boswellia serrata as equally potent, non-selective inhibitors of the major drug metabolising CYP enzymes 1A2/2C8/2C9/2C19/2D6 and 3A4. LC/LC/ESI-MS fingerprint analyses of the boswellic acids 11-keto-beta-boswellic acid, alpha-boswellic acid, beta-boswellic acid and their 3-O-acylated derivatives were used for the authentication of the commercially obtained frankincense samples. Although the boswellic acids could be identified as moderate to potent inhibitors of the applied CYP enzymes, they are not the major CYP inhibitory principle of frankincense.     

High-throughput cytochrome p450 inhibition assays by ultrafast gradient liquid chromatography with tandem mass spectrometry using monolithic columns

A generic method employing ultrafast liquid chromatography with tandem mass spectrometry (LC/MS/MS) was developed and employed for routine screening of drug candidates for inhibition of five major human cytochrome p450 (CYP) isozymes, CYP3A4, CYP2D6, CYP2C9, CYP2C19, and CYP1A2. The method utilized a monolithic silica rod column to allow fast flow rates to significantly reduce chromatographic run time. The major metabolites of six CYP-specific probe substrates for the five p450 isoforms were monitored and quantified to determine IC(50) values of five drug compounds against each p450 isozyme. Human liver microsomal incubation samples at each test compound concentration were combined and analyzed simultaneously by the LC/MS/MS method. Each pooled sample containing six substrates and an internal standard was separated and detected in only 24 seconds. The combination of ultrafast chromatography and sample pooling techniques has significantly increased sample throughput and shortened assay turnaround time, allowing a large number of compounds to be screened rapidly for potential p450 inhibitory activity, to aid in compound selection and optimization in drug discovery.     

High-throughput cytochrome P450 (CYP) inhibition screening via a cassette probe-dosing strategy. VI. Simultaneous evaluation of inhibition potential of drugs on human hepatic isozymes CYP2A6, 3A4, 2C9, 2D6 and 2E1

The inhibition potential of drugs towards five major human hepatic cytochrome P450 (CYP) isozymes (CYP2A6, 3A4, 2C9, 2D6, and 2E1) was investigated via cassette dosing of the five probe substrates (coumarin, midazolam, tolbutamide, dextromethorphan, and chlorzoxazone) in human liver microsomes using a 96-well plate format. After microsomal incubations had been terminated with formic acid, the five marker metabolites (7-hydroxycoumarin, 1'-hydroxymidazolam, 4-hydroxytolbutamide, dextrorphan, and 6-hydroxychlorzoxazone) were simultaneously quantified using direct injection/online guard cartridge extraction/tandem mass spectrometry (DI-GCE/MS/MS). Several advantages resulted from the use of a short C(18) guard cartridge (4 mm in length) for DI-GCE/MS/MS, including minimal sample preparation, fast online extraction, short analysis time (2.5 min), and minimal source contamination. In addition, this method demonstrated an inter-day accuracy range from -8.7 - 7.4% with a precision less than 8.3% for the quantification of all the marker metabolites. The inhibition assay for the five CYP isozymes was evaluated using their known selective inhibitors via individual and cassette dosing of the probe substrates. The IC(50) values measured via cassette dosing were consistent with those observed via individual dosing, which were all in agreement with the reported values. In addition, the validated assay was used to evaluate the inhibitory potential of 23 generic drugs (randomly selected) towards the five CYP isozymes. The results suggest the integration of the cassette dosing strategy and the DI-GCE/MS/MS method can provide a reliable in vitro approach to screening the inhibitory potential of new chemical entities, with maximal throughput and cost-effectiveness, in support of drug discovery and development.     

A UPLC-MS/MS assay of the "Pittsburgh cocktail": six CYP probe-drug/metabolites from human plasma and urine using stable isotope dilution

The efficiency of drug metabolism by a single enzyme can be measured as the fractional metabolic clearance which can be used as a measure of whole body activity for that enzyme. Measurement of activity of multiple enzymes simultaneously is feasible using a cocktail approach, however, analytical approach using different assays for drug probes can be cumbersome. A quantitative ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) based method for the rapid measurement of six cytochrome P450 (CYP) probe drugs and their relevant metabolites is described. The six specific probe substrates/metabolites are caffeine/paraxanthine (CYP1A2), flurbiprofen/4'-hydroxyflurbiprofen (CYP2C9), mephenytoin/4'-hydroxymephenytoin (CYP2C19), debrisoquine/4-hydroxydebrisoquine (CYP2D6), chlorzoxazone/6'-hydroxychlorzoxazone (CYP2E1) and dapsone/N-monoacetyldapsone (NAT2). These probes were quantified by stable isotope dilution from plasma and urine. The present workflow provides a robust, fast and sensitive assay for the "Pittsburgh cocktail", and has been successfully applied to a clinical phenotyping study of liver disease. A representative group of 17 controls and patients with chronic liver disease were administered orally caffeine (100 mg), chlorzoxazone (250 mg), debrisoquine (10 mg), mephenytoin (100 mg), flurbiprofen (50 mg) and dapsone (100 mg). Urine (0 through 8 h) and plasma (4 and 8 h) samples were analyzed for drug/metabolite amounts by stable isotope dilution UPLC-MS/MS. The phenotypic activity of drug metabolizing enzymes was investigated with 17 patient samples. Selected reaction monitoring (SRM) was optimized for each drug and metabolite. In the method developed, analytes were resolved by reversed-phase by development of a gradient using a water/methanol solvent system. SRM of each analyte was performed in duplicate on a triple quadrupole mass spectrometer utilizing an 8 min analytical method each, one with the source operating in the positive mode and one in the negative mode, using the same solvent system. This method enabled quantification of each drug (caffeine, chlorzoxazone, debrisoquine, mephenytoin, flurbiprofen, and dapsone) and its resulting primary metabolite in urine or plasma in patient samples. The method developed and the data herein demonstrate a robust quantitative assay to examine changes in CYP enzymes both independently or as part of a cocktail. The clinical use of a combination of probe drugs with UPLC-MS/MS is a highly efficient tool for the assessment of CYP enzyme activity in liver disease.     

Molecular docking,PKPD, and assessment of toxicity of few chalcone analogues as EGFR inhibitor in search of anticancer agents

In the present work, molecular docking of the chalcone analogues with receptor EGFR carried out using erlotinib as reference drug is reported. About 15 chalcone analogues were analyzed CHL(1–15). Molecules CHL2, CHL3, CHL9, CHL11, and CHL15 found strong affinity for receptor EGFR exhibiting binding energies ??7.7 kcal/mol, ??7.5 kcal/mol, ??7.6 kcal/mol, ??7.9 kcal/mol, and ??8.1 kcal/mol, respectively, when erlotinib a reference drug exhibits binding energy ??7.6 kcal/mol. Toxicity for molecules was assessed against the cytochromes P450 (CYP) and P-gp using Swiss ADMET. Molecule CHL9 could be a suitable lead compound inhibitor to CYP1A2 followed by CHL2 inhibitor of CYP1A2 and CYP2C9 and CHL15 with a most stable binding affinity of ??8.1 kcal/mol, inhibiting CYP1A2, CYP2C19, and CYP2D6. CHL3 has a binding affinity of ??7.5 kcal/mol, inhibiting all the 05 CYP enzymes (CYP1A2, CYP2C19, CYP2C9, CYP2D6, and CYP3A4). CHL11 has a binding affinity of ??7.9 kcal/mol, inhibiting CYP1A2, CYP2C19, and CYP2C9. Considering inhibition of CYP family enzymes by molecules, further here we have perform the enrichment analysis to these CYP family enzymes and reported the metabolic pathways which were probably affected by inhibition of these enzymes using EnrichR online enrichment analysis server. The current predictions over these 15 chalcone derivatives will be needed to further investigate in vivo and in vitro conditions to identify the optimum therapeutic efficacy and least toxicity.

     

Direct inhibition of Re Du Ning Injection and its active compounds on human liver cytochrome P450 enzymes by a cocktail method

The aim of this study was to investigate the direct inhibitory effects of Re Du Ning Injection (RDN) and its active compounds on the major cytochrome P450 enzyme (CYP) isoforms (CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A4) of human liver microsomes by ‘a cocktail method’. The activity of each CYP isform was represented as the formation rate of the specific metabolite from relevant substrate. Then a sensitive and specific ultra‐performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) method was developed and validated to simultaneously analyze the seven metabolites. RDN (0.035–2.26 mg/mL) showed a strong inhibitiory effect on CYP2C8, followed by CYP2C9, CYP2B6, CYP2C19, CYP1A2 and CYP3A4. The IC₅₀ value for each enzyme was 0.19, 0.66, 0.72, 1.27, 1.66 and 2.13 mg/mL, respectively. RDN competitively inhibited the activities of CYP1A2 (K _i = 1.22 mg/mL), CYP2B6 (K _i = 0.65 mg/mL) and CYP3A4 (K _i = 0.88 mg/mL); it also exhibited mixed inhibition of CYP2C8, CYP2C9 and CYP2C19 with a K _i value of 0.26, 0.64 and 0.82 mg/mL, respectively. However, the activity of CYP2D6 was not significantly inhibited even by 2.26 mg/mL RDN. Moreover, the data of nine active compounds on the CYPs showed that cryptochlorogenin acid, sochlorogenic acid B and sochlorogenic acid C were the major contributors to the inhibitory effect of RDN on CYP2C8, while the inhibitory effect of RDN on CYP2C9 might be caused by sochlorogenic acid A and sochlorogenic acid C. Moreover, neochlorogenic acid might be the major contributor to the inhibitory effect on CYP2B6. All of the findings suggested that drug–drug interactions may occur and great caution should be taken when RDN is combined with drugs metabolized by these CYPs.     

A high‐throughput inhibition screening of major human cytochrome P450 enzymes using an in vitro cocktail and liquid chromatography–tandem mass spectrometry

A sensitive and high‐throughput inhibition screening liquid chromatography–mass spectrometry (LC‐MS/MS) method was developed and validated for the simultaneous quantification of five probe metabolites (7‐hydroxycoumarin, CYP2A6; 4‐hydroxytolbutamide, CYP2C9; 4′‐hydroxymephenytoin, CYP2C19; α‐hydroxymetoprolol, CYP2D6; and 1‐hydroxymidazolam, CYP3A4) for in vitro cytochrome P450 activity determination in human liver microsome and recombinant. All the metabolites and the internal standard, tramadol, were separated on a Waters 2695 series liquid chromatograph with a Phenomenex Luna C₁₈ column (150 × 2.0 mm, 5 µm). Quality control samples and a positive control CYP inhibitor were included in the method. The IC₅₀ values determined for typical CYP inhibitors were reproducible and in agreement with the literature. The method was selective and showed good accuracy (99.13–103.37%), and inter‐day (RSD < 6.20%) and intra‐day (RSD < 6.13%) precision. Also, the incubation extracts of the sample were stable at room temperature (20 °C) for 48 h and for 96 h in the autosampler (4 °C). The presented method is the first HPLC‐MS/MS method of this combination for simultaneous detection of the five metabolites 7‐hydroxycoumarin, 4‐hydroxytolbutamide, 4′‐hydroxymephenytoin, α‐hydroxymetoprolol and 1‐hydroxymidazolam in a single‐run process. It is possible that the high‐quality and ‐throughput cocktail provides suitable information in drug discovery and screening for new drug entities. Copyright © 2013 John Wiley & Sons, Ltd.     

Effects of vindoline on rat cytochrome P450 isoform activities in vitro

A specific ultra-high-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UHPLC–Q-TOF–MS/MS) method has been described for the simultaneous determination of the metabolites of tacrine, bupropion, diclofenac, dextromethorphan and midazolam, which are the five probe drugs of the five cytochrome P450 (CYP450) isoforms CYP1A2, CYP2B, CYP2C11, CYP2D1 and CYP3A4. The inhibition degree was determined by calculating the IC₅₀. The chromatographic separation was performed on a C₁₈ column with a mobile phase consisting of 0.1% formic acid and acetonitrile. The mass spectrometric analysis was conducted in positive electrospray ionization mode. The IC₅₀ values of CYP1A2, CYP2B, CYP2C11, CYP2D1 and CYP3A were 113.4, 83.78, 22.50, 9.081 and 52.76 μmol L⁻¹, respectively. The in vitro results demonstrated that vindoline could inhibit CYP2D1 activity in rats, and weak inhibitory effect on CYP2C11 and CYP3A, but had no obvious effects on CYP1A2 and CYP2B.     

In vitro metabolism of a new cardioprotective agent, KR-32570, in human liver microsomes

KR-32570 (5-(2-methoxy-5-chlorophenyl)furan-2-ylcarbonyl)guanidine) is a new reversible Na+/H+ exchanger inhibitor for preventing ischemia-reperfusion injury. This study was performed to identify the metabolic pathway of KR-32570 in human liver microsomes. Human liver microsomal incubation of KR-32570 in the presence of NADPH and UDPGA resulted in the formation of six metabolites, M1-M6. M1 was identified as O-desmethyl-KR-32570, on the basis of liquid chromatography/tandem mass spectrometric (LC/MS/MS) analysis with the synthesized authentic standard. M2 and M3 were suggested to be hydroxy-KR-32570 and hydroxy-O-desmethyl-KR-32570, respectively. M1, M2, and M3 were further metabolized to their glucuronide conjugates, M4, M5, and M6, respectively. In addition, the specific P450 isoforms responsible for KR-32570 oxidation to two major metabolites, O-desmethyl-KR-32570 and hydroxy-KR-32570, were identified using a combination of correlation analysis, chemical inhibition in human liver microsomes and metabolism by expressed recombinant P450 isoforms. The inhibitory potency of KR-32570 on clinically major P450s was investigated in human liver microsomes. The results show that CYP3A4 contributes to the oxidation of KR-32570 to hydroxy-KR-32570, and CYP1A2 play the predominant role in O-demethylation of KR-32570. KR-32570 was found to inhibit moderately the metabolism of CYP2C8 substrates.     

流动注射-串联质谱法快速筛选植物提取物对药物代谢酶的抑制潜性

基于流动注射-串联质谱(FI-MS/MS)技术,综合运用微孔板低温孵育、低温样品相分离、六合一进样和多探针同时定量分析,建立了体外快速筛选复杂中草药组分抑制细胞色素P450同工酶活性的新方法。FI-MS/MS新技术不依赖色谱分离,也无需复杂的仪器设备和样品前处理,分析通量大幅提高。采用基质匹配标样和内标法定量,方法的准确度、精密度、灵敏度和线性范围均满足孵育体系中代谢物分析的需要。优化条件下测得各酶动力学参数和对照抑制剂的半抑制率(IC50)均与参考值一致;仅用两抑制剂浓度点即可获得满意的IC50近似值,两点IC50的测试重现性良好。结合两点IC50程序,FI-MS/MS方法成功应用于中药粗提液对人体主要P450酶抑制效应的快速筛选与分类,为多组分体系复杂药代相互作用的研究和评估提供了一种有效手段。     

乌头类生物碱组分在CYP450中的代谢指纹图谱及对CYP450活性的影响

利用超高效液相色谱-串联质谱(UPLC-MS/MS)的多反应监测(MRM)技术, 结合多探针底物方法, 对单酯型及双酯型乌头类生物碱组分对细胞色素P450(CYP450)亚型的活性影响进行了研究; 同时利用超高效液相色谱-四极杆-飞行时间串联质谱(UPLC-Q-TOF-MS/MS)对单酯型和双酯型乌头类生物碱组分在CYP450中的代谢指纹图谱进行了研究. 活性影响研究结果表明, 单酯型生物碱组分对CYP2C及2D的抑制能力较强, 其IC₅₀值分别为7.44和6.74 μmol/L; 双酯型生物碱组分对CYP1A2, 3A, 2C和2D均有较弱的抑制作用, 其IC₅₀值分别为39.48, 70.44, 17.36和86.04 μmol/L. 代谢指纹图谱显示, 双酯型生物碱组分在大鼠肝微粒体中有6个特异性产物可以作为该反应的特征峰.     

Cytochrome P450 reaction phenotyping and inhibition and induction studies of pinostrobin in human liver microsomes and hepatocytes

Pinostrobin (PI, 5‐hydroxy‐7‐methoxyflavanone) is a natural flavonoid known for its rich pharmacological activities. The objective of this study was to identify the human liver cytochrome P450 (CYP450) isoenzymes involved in the metabolism of PI. A single hydoxylated metabolite was obtained from PI after an incubation with pooled human liver microsomes (HLMs). The relative contributions of different CYP450s were evaluated using CYP450‐selective inhibitors in HLMs and recombinant human CYP450 enzymes, and the results revealed the major involvement of CYP1A2, CYP2C9 and CYP2E1 in PI metabolism. We also evaluated the ability of PI to inhibit and induce human cytochrome P450 enzymes in vitro . High‐performance liquid chromatography and liquid chromatography–tandem mass spectrometry analytical techniques were used to estimate the enzymatic activities of seven drug‐metabolizing CYP450 isozymes in vitro . In HLMs, PI did not inhibit CYP 1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 or CYP3A4 (IC₅₀ > 100 μm ). In the induction studies, PI had minimal effects on CYP1A2, CYP2B6and CYP3A4 activity. Based on these results, PI would not be expected to cause clinically significant CYP450 inhibition or induction.     

Use of stable isotope labeled probes to facilitate liquid chromatography/mass spectrometry based high‐throughput screening of time‐dependent CYP inhibitors

Inhibition curve shift is a commonly used approach for screening of time‐dependent CYP inhibitors which requires parallel paired incubations to obtain two inhibition curves for comparison. For the control incubation, a test compound is co‐incubated with a probe substrate in human liver microsomes (HLM) fortified with NADPH; for the time‐dependent incubation (TDI), the test compound is pre‐incubated with NADPH‐fortified HLM followed by a secondary incubation with a probe substrate. For both incubations, enzyme activity is measured respectively by liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis of the CYP‐specific metabolite, and a TDI inhibitor can be readily identified by inhibition curve shifting as a result of CYP inactivation by the test compound during the pre‐incubation. In the present study, we describe an alternative approach to facilitate TDI screening in which stable isotope labeled CYP‐specific probes are used for the TDI, and non‐labeled substrates are included in the control incubation. Because CYP‐specific metabolites produced in the TDI are stable isotope labeled, two sets of incubation samples can be combined and then simultaneously analyzed by LC/MS/MS in the same batch run to reduce the run time. This new method has been extensively validated using both a number of known competitive and TDI inhibitors specific to five most common CYPs such as 1A2, 2C9, 2C19, 2D6, and 3A4. The assay is performed in a 96‐well format and can be fully automated. Compared to the traditional method, this approach in combination with sample pooling and a short LC/MS/MS gradient significantly enhances the throughput of TDI screening and thus can be easily implemented in drug discovery to evaluate a large number of compounds without adding additional resource. Copyright © 2010 John Wiley & Sons, Ltd.     

Prediction of cytochrome P450 3A4, 2D6, and 2C9 inhibitors and substrates by using support vector machines

Statistical learning methods have been used in developing filters for predicting inhibitors of two P450 isoenzymes, CYP3A4 and CYP2D6. This work explores the use of different statistical learning methods for predicting inhibitors of these enzymes and an additional P450 enzyme, CYP2C9, and the substrates of the three P450 isoenzymes. Two consensus support vector machine (CSVM) methods, "positive majority" (PM-CSVM) and "positive probability" (PP-CSVM), were used in this work. These methods were first tested for the prediction of inhibitors of CYP3A4 and CYP2D6 by using a significantly higher number of inhibitors and noninhibitors than that used in earlier studies. They were then applied to the prediction of inhibitors of CYP2C9 and substrates of the three enzymes. Both methods predict inhibitors of CYP3A4 and CYP2D6 at a similar level of accuracy as those of earlier studies. For classification of inhibitors of CYP2C9, the best CSVM method gives an accuracy of 88.9% for inhibitors and 96.3% for noninhibitors. The accuracies for classification of substrates and nonsubstrates of CYP3A4, CYP2D6, and CYP2C9 are 98.2 and 90.9%, 96.6 and 94.4%, and 85.7 and 98.8%, respectively. Both CSVM methods are potentially useful as filters for predicting inhibitors and substrates of P450 isoenzymes. These methods generally give better accuracies than single SVM classification systems, and the performance of the PP-CSVM method is slightly better than that of the PM-CSVM method.     

Investigating the in vitro stereoselective metabolism of m‐nisoldipine enantiomers: characterization of metabolites and cytochrome P450 isoforms involved

m‐Nisoldipine, as a novel 1,4‐dihydropyridine calcium ion antagonist, was presented as a couple of enantiomers [(?), (+)‐m‐nisoldipine]. In this report, the in vitro metabolism of m‐nisoldipine enantiomers was investigated in rat liver microsomes (RLM) by the combination of two liquid chromatography mass spectrometric techniques for the first time. The metabolites were separated and assayed by ultra‐high performance liquid chromatography coupled to quadrupole time‐of‐flight mass spectrometry and further identified by comparison of their mass and chromatographic behaviors with reference substances. A total of 18 metabolites of (?)‐m‐nisoldipine and 16 metabolites of (+)‐m‐nisoldipine were detected, respectively, which demonstrated that (+)‐m‐nisoldipine is more metabolically stable than (?)‐m‐nisoldipine. In addition, the identified metabolic pathways of m‐nisoldipine enantiomers were involved in dehydrogenation, oxidation and ester hydrolysis. Afterwards, based on high‐performance liquid chromatography coupled to triple quadrupole linear ion trap mass spectrometry, various selective cytochrome P450 (CYP) enzyme inhibitors were employed to evaluate CYP isoforms. The results indicated that the inhibitors of CYP1A1/2, CYP2B1/2, 2D and 2C11 had no obvious inhibitory effects, yet the inhibitor of CYP 3A had a significant inhibitory effect on metabolism of m‐nisoldipine enantiomers. This showed that CYP 3A might primarily metabolize m‐nisoldipine in RLM. Copyright © 2015 John Wiley & Sons, Ltd.     

Biochemical and analytical development of the CIME cocktail for drug fate assessment in humans

Phenotyping based on drug metabolism activity appears to be informative regarding mechanism‐based interactions during drug development. We report here the first steps of the development of the innovative CIME cocktail. This cocktail is designed not only for the major cytochrome P450, with caffeine, amodiaquine, tolbutamide, omeprazole, dextromethorphan and midazolam as substrates of CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A, respectively, but also phase II enzymes UGT 1A1/6/9 with acetaminophen, P‐gp and OATP1B1 with digoxin and rosuvastatin, and renal function with memantine. An assay combining ultra‐performance liquid chromatography using a 1.7 µm particle size column with tandem mass spectrometry (UPLC/MS/MS) was set up for the simultaneous quantification of the 20 substrates and metabolites after extraction from human plasma using solid‐phase extraction. The method was validated in the spirit of the FDA guidelines. Mean accuracy ranged from 87.7 to 115%, the coefficient of variance (CV%) of intra‐ and inter‐run from 1.7 to 16.4% and from 1.6 to 14.9%, respectively, and for the limit of quantification (LOQ) with ten lots of plasma, accuracy ranged from 84 to 115% and CV% precision was <16%. Short‐term stability was evaluated in eluate (4 h, room temperature), plasma (24 h, room temperature), the autosampler (24 h, 4°C) and in three freeze/thaw cycles in plasma. All except three analytes were stable under these conditions. For the three others a specific process can be followed. This robust, fast and sensitive assay in human plasma provides an analytical tool for ten‐probe drugs of the CIME cocktail. Clinical samples will be assayed in the near future using this new assay method. Copyright © 2010 John Wiley & Sons, Ltd.