Cytochrome P450 (P450) and uridine 5'-diphospho-glucuronosyltransferase (UGT) are major metabolizing enzymes in the biotransformation of most drugs. Altered P450 and UGT activities are a potential cause of adverse drug-drug interaction. A method for the simultaneous evaluation of the activities of five P450s (CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A) and four UGTs (UGT1A1, UGT1A4, UGT1A9, and UGT2B7) was developed using in vitro cocktail incubation and tandem mass spectrometry. The nine probe substrates used in this assay were phenacetin (CYP1A2), diclofenac (CYP2C9), S-mephenytoin (CYP2C19), dextromethorphan (CYP2D6), midazolam (CYP3A), 7-ethyl-10-hydroxy-camptothecin (SN-38) (UGT1A1), trifluoperazine (UGT1A4), mycophenolic acid (UGT1A9), and naloxone (UGT2B7). This new method involves incubation of two cocktail doses and single cassette analysis. The two cocktail doses and the concentration of each probe substrate in vitro were determined to minimize mutual drug interactions among substrates. Cocktail A comprised phenacetin, diclofenac, S-mephenytoin, dextromethorphan, and midazolam, whereas cocktail B comprised SN-38, trifluoperazine, mycophenolic acid, and naloxone. In the incubation study of these cocktails, the reaction mixtures were pooled and simultaneously analyzed using liquid chromatography-tandem mass spectrometry. The method was validated by comparing inhibition data obtained from the incubation of each probe substrate alone with data from the cocktail method. The IC50 values obtained in both cocktail and individual incubations were in agreement with values previously reported in the literature. This cocktail method offers a rapid and robust way to simultaneously evaluate phase I and II enzyme inhibition profiles of many new chemical entities. This new method will also be useful in the drug discovery process and for advancing the mechanistic understanding of drug interactions.

Testing for potential drug interactions of new chemical entities is essential when developing a novel drug. In this study, an assay was designed to evaluate drug interactions with 10 major human cytochrome P450 (P450) enzymes incubated in liver microsomes, involving 12 probe substrates with two cocktail incubation sets used in a single liquid chromatography-tandem mass spectrometry (LC-MS/MS) run. The P450 substrate composition in each cocktail set was optimized to minimize solvent effects and mutual drug interactions among substrates as follows: cocktail A was composed of phenacetin for CYP1A2, bupropion for CYP2B6, amodiaquine for CYP2C8, diclofenac for CYP2C9, S-mephenytoin for CYP2C19, and dextromethorphan for CYP2D6; cocktail B was composed of coumarin for CYP2A6, chlorzoxazone for CYP2E1, astemizole for CYP2J2, and midazolam, nifedipine, and testosterone for CYP3A. Multiple probe substrates were used for CYP3A owing to the multiple substrate-binding sites and substrate-dependent inhibition. After incubation in human liver microsomes, each incubation mixture was pooled and all probe metabolites were simultaneously analysed in a single LC-MS/MS run. Polarity switching was used to acquire the negative-ion mode for hydroxychlorzoxazone and positive-ion mode for the remaining analytes. The method was validated by comparing the inhibition data obtained from incubation of each individual probe substrate alone and with the substrate cocktails. The half-maximal inhibitory concentration values obtained from the cocktail and individual incubations were well correlated and in agreement with previously reported values. This new method will be useful in assessing the drug interaction potential of new chemical entities during new drug development. KEYWORDS drug interaction, high-throughput screening, IC 50 , liquid chromatography-tandem mass spectrometry, new chemical entities *These authors contributed equally to this work.

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Obesity is one of the metabolic diseases and is featured by excessive fat accumulation in the body and energy imbalance. Obesity is associated with metabolic diseases such as type 2 diabetes, hypertension, and cardiac disease. Recently, the incidence of obesity keeps increasing due to high caloric western‐style diet and lack of exercise. In order to explore food ingredient(s) with antiobesity activity, twenty kinds of fruits, vegetables, and medicinal plants were subjected to preliminary screening in which inhibitory activity against digestive enzymes and TG accumulation in 3T3‐L1 cells were measured. Among the samples tested, the seed extract of Oenothera biennis L. was found to have the highest inhibitory activity against TG accumulation in differentiated 3T3‐L1 adipocyte and regulate factors associated with adipogenesis.(Supported by a grant (R0002736) from Regional Industry R&D Assistance Program (2013) of Ministry of Trade, Industry and Energy, Republic of Korea)

Doenjang (fermented soybean paste) is one of the traditional fermented foods in Korea which has various physiological effects. In this study, we investigated the antioxidant and neuroprotective activities of doenjang prepared fresh and aged for 6 weeks. Freeze‐dried doenjang was extracted with 80 % (v/v) ethanol. The 80% ethanol extract of doenjang was subjected to determination of antioxidant activity by 2,2‐diphenyl‐1‐pycrylhydrazyl (DPPH) radical scavenging capacity, ferric ion reducing antioxidant power, 2,2'‐azinobis (3‐ehylbenzthiazoline‐6‐sulphonic acid) cation radical scavenging activity, and oxygen radical absorbance capacity assays. In addition, the neuroprotective effect of doenjang extract was investigated using MTT cytotoxicity and 2´,7´‐dichlorofluorescein diacetate (DCF‐DA) assays. It was found that the 80% ethanol extract of doenjang protected from neuronal cell death induced by ROS or glutamate. The underlying mechanism for neuroprotective effect of doenjang will be discussed. (supported by a grant from IPET (High Value‐added Food Technology Development Program, 2012, 112066‐3), Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea)