β nicotinamide adenine dinucleotide phosphate nadp

Terpinen-4-ol (racemic), glucose-6-phosphate, glucose-6-phosphate dehydrogenase, and β-nicotinamide adenine dinucleotide phosphate (NADP⁺) were purchased from Sigma-Aldrich. High-performance liquid chromatography (HPLC) -grade acetonitrile was purchased from Fisher Scientific. HPLC-grade formic acid was purchased from Duksan Pure Chemicals. All of the other chemicals used were of the highest grade commercially available.
Originally, chromosomal DNA of B. megaterium KCCM 11745 for cloning of CYP102A1 wild type was obtained from Korean Culture Center of Microorganisms (KCCM, Republic of Korea). The gene of CYP102A1 wild-type (WT) was cloned and expressed in E. coli as described previously [57 (link)].

Standards of losartan (purity >98%) was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Losartan carboxylic acid (EXP3174) was purchased from Toronto Research Chemicals Inc., Canada. GLTs were purchased from Sichuan Kelun Pharmaceutical Co., LTD. β-Nicotinamide adenine dinucleotide phosphate (NADP) and lucifer yellow were provided by Sigma (St. Louis, MO). Rat liver microsomes were purchased from BD (Woburn, MA). Acetonitrile and methanol were purchased from Fisher Scientific (Fair Lawn, NJ). Formic acid was purchased from Anaqua Chemicals Supply Inc. Limited (Houston, TX). Ultrapure water was prepared with a Milli-Q water purification system (Millipore, Billerica, MA). All other chemicals were of analytical grade or better.

β-lapachone was obtained from Southeast Pharmaceuticals, Inc. (Jiangsu, China). Propofol, N-acetyl cysteine (NAC), dicoumarol (DIC), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), glucose 6-phosphate, glucose 6-phosphate dehydrogenase, β-nicotinamide adenine dinucleotide phosphate (NADP), uridine 5’-diphosphate-glucuronic acid (UDPGA), D-saccharic acid 1,4-lactone, β-D-glucuronidase and chlorzoxazone, were all purchased from Sigma Aldrich. Diazepam was obtained from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China).

The authentic standards of ellagic acid (EA), pyrogallol, and gallic acid (GA) were purchased from the Chengdu MUST Bio-Technology CO. Ltd. (Chengdu, China), phosphate-buffered saline (PBS, pH = 7.4, 0.01 M), HPD resin was purchased from Tianjin Baowen Chemical Technology CO. Ltd. (Tianjin, China), anaerobic bags (AnaeroPouch Anaero, Mitsubishi Gas Chemical Company Inc., Tokyo, Japan), GAM broth medium was bought from Qingdao Haibo Biology CO. Ltd. (Qingdao, China), β-nicotinamide adenine dinucleotide phosphate (NADP), glucose-6-phosphate (G-6-P), glucose-6-phosphate dehydrogenase (G-6-PD), dithiothreitol, and S-adenosyl-L-methionine (SAM) were obtained from Sigma Chemical CO. (St. Louis, MO, USA). Acetonitrile, methanol, and formic acid were of HPLC-grade, which were purchased from Thermo Fisher Scientific CO. Ltd. (Waltham, MA, USA). Purified water was prepared by using Milli-Q System (Millipore, Billerica, MA, USA). All other analytical-grade reagents were provided by the Sinopharm Chemical Reagent CO. Ltd. (Shanghai, China).

For the experiments, O-desmethyltramadol HCl (M1), glucose 6-phosphate, glucose 6-phosphate dehydrogenase, MgCl₂, β-nicotinamide adenine dinucleotide phosphate (NADP), chlorpropamide, Trizma^® base, Trizma^® hydrochloride, DMSO and formic acid were obtained from Sigma‒Aldrich (St. Louis, MO, USA). To evaluate the intrinsic clearance of M1 by CYPs, metabolic stability studies under NADPH system were conducted in human liver microsoms (HLM) [25 (link),26 (link)]. For details, NADPH-generating system (1.3 mM NADP⁺, 3.3 mM glucose 6-phosphate, 3.3 mM MgCl₂, and 0.4 unit/mL glucose-6-phosphate dehydrogenase) and HLM 0.25 mg/mL were added and preincubated at 37 °C for 5 min. Then, 20 μM M1 was added and reacted at 37 °C for 0, 1, 5, 10, 20, 30, and 40 min, respectively. The total volume of the reaction mixture was 100 μL. After each reaction, the reaction was terminated by addition of 150 μL of acetonitrile containing an internal standard (100 ng/mL chlorpropamide). All experiments were repeated duplicated and the samples were vortexed for 5 min and centrifuged (13,000 rpm, 4 °C) for 15 min. Then, supernatants were injected into LC-MS/MS and M1 were analyzed. The concentration of M1 (20 μM) at 0 min was used to evaluate the metabolism of CYP through the change of drug concentration over time.

The compounds methyl benzoate (1), n-propyl benzoate(3), n-butyl benzoate (4), methyl 2-naphthoate (7), phenylacetate (9), ethyl 2-bromobenzoate (10), ethyl 3-bromobenzoate (11), ethyl 4-bromobenzoate (12), methyl 8-fluoro-5-methyl-6-oxo-4H-imidazo[1,5-a][1,4]benzodiazepine-3-carboxylate (methyl flumazenil ester, 16), methyl 4-aminobenzoate (methyl benzocaine ester, 14), and methyl [1,1′-biphenyl]-4-carboxylate (17) were synthesised at the Laboratório de Avaliação e Síntese de Substâncias Bioativas (LASSBio®, UFRJ, Brazil) with degree of purity ≥98%. Flumazenil (13) was purchased from Cristalia® – Brazil. Formic Acid (96%), acetonitrile and methanol of HPLC grade were purchased from Tedia®-Brazil. Water used for the HPLC analysis was produced using a water purification system (Master System MS2000, Gehaka, Brazil). Ethylbenzoate (2), phenylbenzoate (5), benzylbenzoate(6), methyl 2-phenylacetate (8), ethyl 4-aminobenzoate (benzocaine, 13), bicinchoninic acid kit for protein determination, ethylenediamine-tetra-acetic-acid (EDTA), monobasic and bibasic potassium phosphate, bis(p-nitrophenyl) phosphate sodium salt, magnesium chloride hexahydrate, β-nicotinamide adenine dinucleotide phosphate (NADP), D-glucose-6-phosphate, glucose-6-phosphate dehydrogenase, tetrahydrofuran (THF) and lithium hydroxide (LiOH) were purchased from Sigma Aldrich (St. Louis, MO, USA).

Pooled human liver microsomes (n = 20) were obtained from Corning Gentest Corporation (Woburn, MA, USA) and stored at −150 °C until use. All the experimental procedures involving humans have been carried out in accordance with The Code of Ethics of the World Medical Association (Declaration of Helsinki) guidelines. Phenacetin, bupropion, tolbutamide, dextromethorphan, midazolam, 3-acetamidophenol (internal standard), chlorpropamide (internal standard), glucose 6-phosphate (G6P), glucose 6-phosphate dehydrogenase (G6PDH), β-nicotinamide adenine dinucleotide phosphate (NADP) and tris (hydroxymethyl) aminomethane hydrochloride (Tris-HCl) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Chlorzoxazone was purchased from Alfa Aesar (Massachusetts, USA). 4-acetamidophenol, hydroxybupropion, 4-hydroxytolbutamide, dextrorphan, 6-hydroxychlorzoxazone and 1-hydroxymidazolam were obtained from Toronto Research Chemical (North York, Canada). Mebendazole (internal standard) was obtained from Aladdin Industrial Co. (California, USA). Plumbagin (purity > 98%) was purchased from Dalian Meilun Biotech Co., Ltd (Dalian, China). Acetonitrile and methanol (all HPLC grade) were purchased from Fisher Chemicals (Leicester, UK). Formic acid (HPLC grade) was purchased from TEDIA (Ohio, USA). Distilled water was purified in a Millipore system Milli Q (Millipore Corp., Bedford, MA, USA).