IMB-YH-4py5-2H (purity>98%) was synthesized and purified by the Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences (Beijing, China). 3-(Dimethylamino)-1-phenyl-2-propen-1-one (Fig 1 ) was purchased from Sigma–Aldrich (Darmstadt, Germany) as an internal standard (IS, CAS: 1201-93-0, purity > 99%). HPLC-grade methanol and formic acid were purchased from Fisher Scientific (Fair Lawn, NJ, USA). Ultrapure water was obtained from a Millipore system (Bedford, MA, USA). All other reagents were of analytical grade. Pooled human liver microsomes, NADPH-regenerating system, and recombinant P450 enzymes (CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4 and CYP3A5) were purchased from Corning (Woburn, MA, USA).
Pooled human liver microsomes
Manufactured by Corning
Sourced in United States
Pooled human liver microsomes are subcellular fractions derived from pooled human liver samples. They contain the microsomal fraction of liver cells, which includes the endoplasmic reticulum and associated enzymes. Pooled human liver microsomes are used in in vitro studies to assess drug metabolism and pharmacokinetics.
Automatically generated - may contain errors
Lab products found in correlation
Methanol, by Thermo Fisher Scientific (2 mentions)
Acetonitrile, by Thermo Fisher Scientific (2 mentions)
Nadph, by Merck Group (2 mentions)
Phenacetin, by Merck Group (2 mentions)
Midazolam, by Merck Group (2 mentions)
Udpga, by Merck Group (2 mentions)
Alamethicin, by Merck Group (2 mentions)
Hplc grade methanol, by Thermo Fisher Scientific (1 mentions)
Formic acid, by Thermo Fisher Scientific (1 mentions)
Cyp3a4, by Corning (1 mentions)
Cyp1a2, by Corning (1 mentions)
Cyp2a6, by Corning (1 mentions)
Cyp2b6, by Corning (1 mentions)
Cyp2c9, by Corning (1 mentions)
Cyp2c19, by Corning (1 mentions)
Cyp2d6, by Corning (1 mentions)
Cyp2e1, by Corning (1 mentions)
Cyp3a5, by Corning (1 mentions)
Nadph regenerating system, by Corning (1 mentions)
Baculosomes, by Thermo Fisher Scientific (1 mentions)
8 protocols using pooled human liver microsomes
1
Synthesis and Characterization of IMB-YH-4py5-2H Compound
2
Solanidine Metabolism by Human CYP Enzymes
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The stock solution of solanidine from PhytoLab (Vestenbergsgreuth, Germany) was prepared at 1 mg·ml−1 in methanol. Solanidine 20 μM was then added to a reaction mixture (final volume 50 μl) containing 1 mg·ml−1 of human liver microsomes (HLM) or 100 pmol·ml−1 of CYP450 baculosomes (i.e. CYP1A2 , CYP2B6 , CYP2C9 , CYP2C19 , CYP2D6, CYP3A4 or CYP3A5 ) in phosphate buffer (pH 7.4, 0.1 M). Pooled human liver microsomes were obtained from Corning Gentest (Corning, NY, USA) and the baculosomes were purchased from Invitrogen, Life Technologies (Carlsbad, CA, USA). The mixture was preincubated for 3 min in a 37°C water bath, and the reaction was then initiated by the addition of a NADPH‐generating system (NADP 4 mmol·L−1, isocitrate 15 mmol·L−1, MgCl2 12.5 mmol·L−1 and isocitrate dehydrogenase 1.5 IU·ml−1 in phosphate buffer). Incubations were carried out at 37°C for 1 h and were performed in duplicates. In order to stop the reaction, 50‐μl methanol was added. The mixture was then centrifuged for 3 min at 10,000g, and the supernatant was diluted in water before being analysed in full scan mode with the same method as described above for metabolomic analysis using an LC system Vanquish coupled to a Q Exactive Focus system (Thermo Scientific, Bremen, Germany).
3
Quantitative Analysis of Cytochrome P450 Substrates
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Epimagnolin A, eudesmin, fargesin, and magnolin were obtained from PhytoLab GmbH & Co. (Vestenbergsgreuth, Germany). Dimethyllirioresinol was a gift from Natural Medicine Research Center in Korea Research Institute of Biology and Biotechnology (Ochang, Korea). Bufuralol hydrochloride, 1′-hydroxybufuralol maleate, d9-1′-hydroxybufuralol maleate, bupropion, hydroxybupropion, 4′-hydroxydiclofenac, 1′-hydroxymidazolam, 4′-hydroxymephenytoin, [S]-mephenytoin, and pooled human liver microsomes (catalog number 452161) were purchased from Corning Life Sciences (Woburn, MA, USA). Amodiaquine hydrochloride, N-desethylamodiaquine dihydrochloride, acetaminophen, coumarin, 7-hydroxycoumarin, diclofenac sodium, midazolam, phenacetin, and NADPH were obtained from Sigma-Aldrich (St. Louis, MO, USA). 13C2, 15N-acetaminophen was obtained from Toronto Research Chemicals (Toronto, ON, Canada). Methanol, acetonitrile, and water (liquid chromatography-mass spectrometry [LC-MS] grade) were purchased from Fischer Scientific (Fair Lawn, NJ, USA). All other chemicals were of the highest quality available.
4
Corylifol A Metabolism Assay
5
Quantification of Navtemadlin and Metabolite
6
Isolation and Characterization of Phytochemicals
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Verproside, picroside II, and isovanilloylcatalpol, isolated from Pseudolysimachion rotundum var. subintegrum as described previously [10 (link)], were gifts from KRIBB (Ochang, Korea). Alamethicin, PAPS, William′s E buffer, and UDPGA were obtained from Sigma-Aldrich (St. Louis, MO, USA). Pooled human liver microsomes, pooled human intestinal microsomes, pooled human liver S9 fractions, human cDNA-expressed UGT 1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B15, and 2B17 supersomes, cryopreserved human hepatocytes, and cryopreserved hepatocyte purification kits were obtained from Corning Life Sciences (Woburn, MA, USA). Human cDNA-expressed SULT 1A1*1, 1A1*2, 1A2, 1A3, 1B1, 1C2, 1C4, 1E1, and 2A1 supersomes were obtained from Cypex Ltd. (Dundee, UK). 4-Methylumbelliferone was obtained from Toronto Research Chemicals (North York, ON, Canada). Acetonitrile and methanol (HPLC grade) were obtained from Burdick & Jackson, Inc. (Ulsan, South Korea). The other chemicals were of the highest quality available. ProteoMass LTQ/FT-hybrid ESI Positive mode Cal Mix (MSCAL5) and Negative mode Cal Mix (MSCAL6) for calibration of Q-Exactive MS were obtained from Supelco (Bellefonte, PA, USA).
7
Profiling Human Liver Microsomal Metabolism
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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).
8
Microsomal Metabolism of Triazolam
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CLB and NCLB were purchased from Sigma-Aldrich Japan (Tokyo, Japan). Triazolam was purchased from Wako Pure Chemicals (Osaka, Japan). 1'-HydroxyTriazolam and 4-hydroxyTriazolam were gifted by Nihon Upjohn Co. (Tokyo, Japan). Pooled human liver microsomes (#452161) were purchased from Corning (Corning, NY). Pooled human intestine microsomes (#H0610.I) were obtained from Sekisui Medical (Tokyo, Japan). Other reagents were purchased commercially.
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