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).
Nadph regenerating system
Manufactured by Corning
Sourced in United States
The NADPH regenerating system is a laboratory tool used to facilitate the production of NADPH, a key cofactor required for various enzymatic reactions in biochemical research and applications. This system provides a reliable and consistent source of NADPH, enabling researchers to conduct experiments and assays that rely on NADPH-dependent processes.
Automatically generated - may contain errors
Lab products found in correlation
Formic acid, by Merck Group (3 mentions)
Formic acid, by Thermo Fisher Scientific (2 mentions)
Nifedipine, by Merck Group (2 mentions)
Benzydamine hydrochloride, by Merck Group (2 mentions)
Glucose 6 phosphate dehydrogenase, by Corning (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 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)
Hplc grade methanol, by Thermo Fisher Scientific (1 mentions)
Pooled human liver microsomes, by Corning (1 mentions)
Terbutaline, by Merck Group (1 mentions)
Lovastatin, by Merck Group (1 mentions)
Cisapride, by Merck Group (1 mentions)
16 protocols using nadph regenerating system
1
Synthesis and Characterization of IMB-YH-4py5-2H Compound
2
Hepatocyte Stability Assay Protocol
3
Characterization of Drug Metabolism Assays
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Uridine 59-diphosphoglucuronic acid trisodium salt (UDPGA), NADPH, alamethicin from Trichoderma viride, adenosine 3-phosphate 5-phosphosulfate, diclofenac sodium salt, midazolam hydrochloride, felodipine, S-(+)-mephenytoin, raloxifene hydrochloride, lovastatin, nisoldipine, nifedipine, methadone, quinidine, testosterone, benzydamine hydrochloride, cisapride, cyclosporin, terbutaline, sildenafil, verapamil, trazodone, atorvastatin, simvastatin, buspirone, rifabutin, saquinavir, terfenadine, zolpidem, repaglinide, indinavir, alprazolam, carbazeran, enalapril, ramipril , and dabigatran etexilate were purchased from Sigma-Aldrich (St. Louis, MO). NADPH-regenerating system containing NADP + , glucose-6phosphate, and glucose-6-phosphate dehydrogenase was purchased from Corning (Wiesbaden, Germany). Organic solvents were of LC-mass spectrometry (MS) or higher quality grade and were acquired from VWR International (Radnor, PA) or Fisher Scientific UK Ltd (Loughborough, UK). Purified water was obtained from Milli-Q Integral 5 Water Purification System (Merck KGaA, Darmstadt, Germany). Phosphate buffer, HQM Hepatocyte/Enterocyte Incubation Medium, and Co-Factor N for MetMax were purchased from In Vitro ADMET Laboratories (Columbia, MD).
4
Rat Liver Microsome Incubation Assay
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Rat liver microsome (RLM; Corning Gentest; Tweksbury, MA, USA) was thawed and diluted with potassium phosphate buffer (100 mM, pH 7.4, final concentration) that contained an NADPH regenerating system (Corning Gentest) with moracin M, KW02, or KW06 (1.0 μM, final concentration). Reaction mixtures were shaken at 37°C for 30 min. These reaction mixtures (50 μL) were sampled at 0 and 30 min. Reactions were stopped with ice cold acetonitrile (150 μL). These mixtures were centrifuged at 13,000 rpm for 5 min at 4°C. After separation of the supernatant, 5 μL aliquots were injected into an LC-MS/MS instrument.
5
Comprehensive Analytical Characterization of Ivermectin
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IVM (IVM‐B1a > 95%, IVM‐B1b < 2%) was purchased from Sigma‐Aldrich. IVM‐B1a (95% purity) was purchased from Toronto Research Chemicals Inc. IVM‐B1b (99.27% purity) was purchased from Clearsynth Labs Ltd. Pooled human liver microsomes (50 donors, Gibco™) were purchased from Thermo Fisher Scientific. Cryopreserved primary human hepatocytes (M00995‐P, UBV donor) and hepatocyte culture medium (InVitroGRO™ CP Medium) were purchased from BioIVT LLC. cDNA‐expressed human CYP isoenzymes 1A2, 2B6, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, 3A4, and 3A5 (Corning® Supersomes™), insect cell control microsomes, human P450 cytochrome b5 oxidoreductase, and NADPH regenerating system were purchased from Corning Inc. LC–MS grade water and acetonitrile were purchased from J.T Baker. LC–MS grade ammonium acetate, formic acid, and HPLC grade organic solvent for NMR analysis were purchased from Sigma‐Aldrich. CD3CN 99.8% and DCOOD 98% were purchased from Deutero GmbH. Water for NMR was prepared from a Milli‐Q purification system from Merck.
6
Quantification of Deuterium-Labeled Ibrutinib and Metabolites
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Ibrutinib (free base; I‐3311) was purchased from LC Laboratories. Deuterium‐labeled ibrutinib ([2H5]ibrutinib, d5‐ibrutinib, 22,561) and PCI 45227 (M37) were purchased from Cayman Chemical. Midazolam (M‐908), 1′‐hydroxymidazolam (H‐902), and d4‐1′‐hydroxymidazolam (H‐921) were purchased from Cerilliant Corporation. The 4β‐HC (700036) and cholesterol (700000P) were purchased from Avanti Polar Lipids. Deuterium‐labeled 4β‐HC ([2H7]4β‐HC, d7‐4β‐HC, H917982) and deuterium‐labeled cholesterol ([2H7]cholesterol, d7‐cholesterol, C432503) were purchased from Toronto Research Chemicals. Dimethyl sulfoxide (DMSO), Optima liquid chromatography mass spectrometry LC–MS grade water, Optima LC–MS grade acetonitrile, Optima LC–MS grade methanol were obtained from Fisher Scientific. All other chemicals and reagents were of analytical grade or higher.
As described previously,23 an NADPH‐regenerating system, consisting of solution A (26 mM NADP+, 66 mM glucose 6‐phosphate, 66 mM MgCl2 in water) and solution B (40 U/ml glucose 6‐phosphate dehydrogenase in 5 mM sodium citrate), was purchased from Corning Life Sciences. InVitroGRO Krebs–Henseleit buffer (KHB) medium (product no. Z99074) and thawing (HT) medium (product no. 990006) were purchased from BioIVT.
As described previously,
7
Cytotoxicity and CYP3A4 Enzymatic Assays
8
Midazolam Metabolism Experimental Protocol
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Midazolam was purchased from Wako Pure Chemicals (Osaka, Japan). α-HydroxyMidazolam solution, α-hydroxyMidazolam-d4 solution and TRI reagent were purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA). Zero Blunt TOPO PCR Cloning Kit was purchased from Invitrogen (Carlsbad, CA, USA). NADPH Regenerating System was purchased from Corning Inc. (Corning, NY, USA). TaKaRa Ex Taq, 10 × Ex Taq buffer, dNTP mixture and Trans IT-LT1 were purchased from Takara Bio Inc. (Shiga, Japan). High capacity cDNA synthesis kit was purchased from Applied Biosystems (Foster City, CA, USA). KOD Plus and KOD FX Neo were purchased from TOYOBO (Osaka, Japan). Rabbit anti-rat CYP3A2 antibody was purchased from Nosan Corporation (Kanagawa, Japan). Goat anti-mouse CYP3A antibody (L-14) was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Rabbit Anti-Goat IgG H&L (HRP) was purchased from abcam (Tokyo, Japan). Anti-Rabbit IgG, HRP-Linked Whole Ab Donkey and enhanced chemiluminescence system (ECL) plus Western blotting detection reagents were purchased from GE Healthcare (Chalfont St. Giles, UK). All of the other reagents were of the highest commercially available grade.
9
In Vitro Metabolism Assay Protocol
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The reagents (formic acid, acetonitrile, ethyl acetate, sodium phosphate, sodium hydrogen phosphate, potassium carbonate, potassium hydrogen carbonate) all of analytic grade, were from Sigma-Aldrich (Milan, Italy). Water was ultra-purified using a Milli-Q system (Millipore, Vimodrone, Milan, Italy). The human liver microsomes (HLMs, from 20 Caucasian male and female donors of different ages), the cDNA-expressed CYPs (CYP1A2, CYP3A4, CYP3A5, CYP2C9, CYP2C19, and CYP2D6), and all the reagents used for the in vitro metabolism (i.e., sodium phosphate buffer, NADPH regenerating system containing NADP+, glucose-6-phosphate, and glucose-6-phosphate dehydrogenase) were supplied by Corning Incorporated (Milan, Italy). The enzyme β -glucuronidase from E. Coli was purchased from Roche (Monza, Italy).
10
Efavirenz Metabolic Kinetics Assay
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Liver and brain microsomes from pooled human donors (0.5 mg/ml) were incubated at 37°C for 0, 5, 15, 30, 60, 90, 120, 150, and 240 minutes with 100 mM potassium phosphate buffer (pH 7.4; Corning), NADPH regenerating system (Corning), UGT reaction mixture (Corning), and 5 μM EFV (Toronto Research Chemicals). Samples were prewarmed at 37°C for 5 minutes before initialization of reaction via the addition of microsomes. At each incubation time point, an aliquot was removed from the reaction tube and quenched using ice-cold 100 nM EFV-d4 (Toronto Research Chemicals) in acetonitrile. Samples were stored on ice until they were centrifuged at 10,000 × g and 4°C for 5 minutes. The supernatant was dried in a vacufuge and reconstituted in 100% methanol. Ionized metabolites and internal standard EFV-d4 (m/z = 318.0452) were detected via the ultra high performance liquid chromatography high-resolution mass spectrometry (uHPLC-HRMS) method described above. Peak area values were obtained in QualBrowser (Thermo Fisher) using a 5-ppm mass error tolerance. Data visualization and analysis were carried out in GraphPad Prism (version 9.3.1).
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