Cyp3a4

Manufactured by BD

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CYP3A4 is a cytochrome P450 enzyme that plays a crucial role in the metabolism of a wide range of drugs and other xenobiotic compounds. It is responsible for the oxidative biotransformation of a variety of substrates, including prescription medications, environmental pollutants, and endogenous compounds. CYP3A4 is an important tool for researchers and scientists working in the fields of pharmacology, toxicology, and drug development.

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Lab products found in correlation

Cyp2c9, by BD (8 mentions) Cyp2c19, by BD (7 mentions) Cyp2d6, by BD (7 mentions) Cyp2e1, by BD (6 mentions) Cyp1a2, by BD (5 mentions) Cyp2b6, by BD (5 mentions) Cyp1b1, by BD (4 mentions) Cyp2c8, by BD (4 mentions) Cyp3a5, by BD (4 mentions) Cyp2a6, by BD (3 mentions) Nadph, by Merck Group (3 mentions) Cyp1a1, by BD (2 mentions) Caco 2 cell line, by American Type Culture Collection (1 mentions) Hanks balanced salt solution (hbss), by Merck Group (1 mentions) Hepes, by Merck Group (1 mentions) Glucose, by Merck Group (1 mentions) Sodium bicarbonate, by Merck Group (1 mentions) Acetylthiocholine chloride, by Merck Group (1 mentions) Tetraisopropyl pyrophosphoramide, by Merck Group (1 mentions) Menadione, by Merck Group (1 mentions)

9 protocols using cyp3a4

Cytochrome P450 Enzyme Inhibition Assay

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CYP inhibition was performed with the recombinant enzymes CYP1A2, CYP2B6, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 obtained from BD Biosciences. Compounds for CYP inhibition testing were prepared as 10 mM stock solutions in DMSO. Isoform-specific substrates were incubated at 37 °C individually with P450 enzymes (Table 5) and a range of test compound concentrations (1.1, 3.3 and 10 μM) in duplicate. Each isoform was tested separately with one reference compound, a known positive control inhibitor. During preincubation, the 96-well black plates were scanned with a fluorescence plate reader to eliminate false results originating from the autofluorescence of the test compounds. At the end of the incubation, product formation was monitored with fluorescence detection. A decrease in the formation of the metabolite compared to the “no inhibition” control samples was used to calculate the IC₅₀ value.

Kaczorowska K., Stankiewicz A., Bugno R., Paluchowska M.H., Burnat G., Brański P., Cieślik P., Wierońska J.M., Milik M., Nowak M., Przybyłowicz A., Kozioł A., Hogendorf A., Hogendorf A.S., Kalinowska-Tłuścik J., Duszyńska B., Pilc A, & Bojarski A.J. (2023). Design and Synthesis of New Quinazolin-4-one Derivatives with Negative mGlu7 Receptor Modulation Activity and Antipsychotic-Like Properties. International Journal of Molecular Sciences, 24(3), 1981.

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In Vitro Characterization of RBx14255

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RBx14255 was synthesized at Ranbaxy Research Laboratories, India. The Caco-2 cell line (human colon carcinoma epithelial cell line) was obtained from ATCC (HTB-37, Manassas, USA) and cells were used at passage number 36. Corning Transwell® filters 12-well, HBSS, HEPES, glucose and sodium bicarbonate were obtained from Sigma Aldrich (India). Liver microsomes and purified recombinant CYP450 isozymes, CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 were purchased from BD Gentest (India). For the blood to plasma concentration ratio study, freshly collected mouse, rat and dog blood was obtained from in-house animals. Human blood was obtained from the Blood Bank (Gurgaon, India). For the protein binding study, a 96-well equilibrium dialyser with 150 μL half-cell capacity (HTDialysis®, Gales Ferry, USA) employing 12-14,000 Dalton molecular weight cutoff membranes was used. Standard substrates, metabolites, inhibitors, NADPH and deuterated analytical internal standards used for the CYP inhibition study were obtained from Sigma-Aldrich (Bangalore, India), BD Biosciences (Woburn, USA) and TRC (Toronto, Canada).

Chaira T., Barman T, & Samuel Raj V. (2020). In Vitro ADME, Preclinical Pharmacokinetics and Prediction of Human Pharmacokinetics of RBx14255, a Novel Ketolide with Pharmacodynamics Against Multidrug-Resistant Bacterial Pathogens. Journal of pharmacy & pharmaceutical sciences : a publication of the Canadian Society for Pharmaceutical Sciences, Societe canadienne des sciences pharmaceutiques, 23.

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Recombinant Human Cytochrome P450 Enzymes

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Baculovirus-insect
cell-expressed human CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2B6, CYP2C8,
CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, CYP3A5, and CYP3A7 were purchased
from BD Biosciences Discovery Labware (Woburn, MA, USA) and used according
to the manufacturer’s instructions.

Juvonen R.O., Ahinko M., Jokinen E.M., Huuskonen J., Raunio H, & Pentikäinen O.T. (2021). Substrate Selectivity of Coumarin Derivatives by Human CYP1 Enzymes: In Vitro Enzyme Kinetics and In Silico Modeling. ACS Omega, 6(17), 11286-11296.

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Cytochrome P450 Enzyme Assay Protocol

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Recombinant human acetylcholinesterase, superoxide dismutase, horseradish peroxidase, NADPH, menadione, acetylthiocholine chloride, 5,5′-dithiobis(2-nitrobenzoic acid), 7-ethoxyresorufin, tetraisopropyl pyrophosphoramide, and coumarin were purchased from Sigma (St. Louis, MO). Parathion, paraoxon, and diethylthiophosphate were from Chem Service Inc. (West Chester, PA). Amplex Red reagent was from Molecular Probes (Eugene, OR). Recombinant human CYPs (CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, CYP3A5 and CYP 3A7), and pooled human liver microsomes were purchased from BD Gentest (Woburn, MA). Recombinant human CPR, Vivid P450 substrates, 7-ethoxy-methyloxy-3-cyanocoumarin (EOMCC) and 7-benzyloxy-methyloxy-3-cyanocoumarin (BOMCC), 7-methoxy-4-trifluoromethyl coumarin and dibenzylfluorescein were from Life Technologies (Grand Island, NY). Glucose-6-phosphate and Glucose-6-phosphate dehydrogenase were from Roche Diagnostic (Indianapolis, IN).

Jan Y.H., Richardson J.R., Baker A.B., Mishin V., Heck D.E., Laskin D.L, & Laskin J.D. (2015). Vitamin K3 (menadione) redox cycling inhibits cytochrome P450-mediated metabolism and inhibits parathion intoxication. Toxicology and applied pharmacology, 288(1), 114-120.

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In vitro Liver Microsome and Cytosol Preparation

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Fluorochloridone, NADPH, and glutathione (GSH) were purchased from Sigma-Aldrich (St. Louis, MO). Human liver microsomes (HLM) from a pool of 33 donors (Catalog. 452161) were purchased from BD Gentest Corp (Woburn, MA). Human liver cytosols (HLC) from a pool of 46 donors (Catalog. HMCYPL) were purchased from GIBCO (ThermoFisher Scientific, MA). The recombinant enzymes CYP1A1, CYP1A2, CYP1B1, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, and CYP3A5, produced in baculovirus were purchased from BD Gentest. Pooled mouse liver microsomes (MLM) and cytosols (MLC) were prepared from male C57BL/6 mouse liver homogenates by differential ultracentrifugation.^{15 (link)}

Shi J., Xie C., Liu H., Krausz K.W., Bewley C.A., Zhang S., Tang L., Zhou Z, & Gonzalez F.J. (2016). Metabolism and Bioactivation of Fluorochloridone, a Novel Selective Herbicide, in Vivo and in Vitro. Environmental science & technology, 50(17), 9652-9660.

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CYP3A4 and CYP2C8 Mediated Metabolism

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Ammonium acetate was obtained from Mallinckrodt Baker (Phillipsburg, NJ), and LC/MS-grade methanol, acetonitrile (ACN) and ethyl acetate from Fisher Scientific (Fairlawn, NJ). CYP3A4 and CYP2C8 Supersomes™ coexpressed with cytochrome P450 reductase and cytochrome b5 were purchased from BD Biosciences (Woburn, MO). 4-OH-RA was synthesized as previously described [21 (link)]. atRA and NADPH were purchased from Sigma Aldrich (St. Louis, MO). atRA-d₅ and 4-oxo-13-cis-RA-d₃ were obtained from Toronto Research Chemicals (North York, ON).

Nelson C.H., Peng C.C., Lutz J.D., Yeung C.K., Zelter A, & Isoherranen N. (2016). Direct protein-protein interactions and substrate channelling between cellular retinoic acid binding proteins and CYP26B1. FEBS letters, 590(16), 2527-2535.

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Immunoassay of Cytochrome P450 Proteins

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A pool of human liver microsomes (HLM) from 50 donors, CYP2E1, CYP3A4, and CYP2C9 were purchased from (BD Biosciences, Missisauga, ON). CYP2E1 was modified by the reactive metabolite of INH in two ways: 1) by reacting CYP2E1 with INA-NHS, which we refer to as CYP2E1-INH, in a similar manner to the modification of lysozyme and 2) by incubation of CYP2E1 (0.5 mg/mL) with INH (500 μM) and a NADPH-generating system (Solutions A and B; BD Biosciences) for 1 hour at 37 °C.
Enzyme-linked immunosorbent assay (ELISA); 96-well microtiter plate (Bethyl Laboratories, Montgomery, TX) was coated with 400 ng of protein overnight. Proteins included HLM, CYP2E1, CYP2E1-INH, CYP3A4, CYP2C9, lysozyme or L-INH. Human serum diluted 1:1000 in TBST containing 4% milk was used as primary antibody and allowed to sit overnight with slight agitation. As secondary antibody, polyclonal goat anti-human IgG-peroxidase (AbD Serotec) was used. Color was developed using 3,3′,5,5′-tetramethylbenzidine (TMB) solution and absorbance was monitored at 450 and 540 nm.

Metushi I.G., Sanders C., Lee W.M, & Uetrecht J. (2014). Detection of Anti-Isoniazid and Anti-CYP Antibodies in Patients with Isoniazid-Induced Liver Failure. Hepatology (Baltimore, Md.), 59(3), 1084-1093.

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In Vitro Metabolic Profiling

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Ocedurenone and KBP-5314 (internal standard) were prepared at KBP Biosciences,. Pooled human plasma was collected from healthy participants (Shanghai Center for Drug Metabolism and Pharmacokinetics Research, China). Human liver microsomes (HLM, Lot No.: 34689), human recombinant CYP1A2 (Lot No.: 30974), CYP1B1 (Lot No.: 68977), CYP2A6 (Lot No.: 33769), CYP2B6 (Lot No.: 49736), CYP2C8 (Lot No.: 03459), CYP2C9 (Lot No.: 00621), CYP2C19 (Lot No.: 69449), CYP2D6 (Lot No.: 75311), CYP2E1 (Lot No.: 73026), CYP3A4 (Lot No.: 16022), CYP3A5 (Lot No.: 44743), and CYP4A11 (Lot No.: 31489) were all purchased from BD Gentest™ (USA). Human primary hepatocytes were purchased from XenoTech (Lots H949 and HC2-8, Kansas, USA) and In Vitro Technologies (Lot OHO, MD, USA). Suppliers of materials used in the in vitro assays for relevant drug-metabolizing enzymes or transporters are listed in Table S1. Other reagents were commercially available and of special reagent grade, high-performance liquid chromatography grade, liquid chromatography–mass spectrometry grade, or equivalent. Further details of in vitro materials and methods can be requested from KBP Biosciences.

Wang P., Liu J., Tan X., Yang F., McCabe J, & Zhang J. (2023). Pharmacokinetics and Drug–Drug Interaction of Ocedurenone (KBP-5074) in vitro and in vivo. European Journal of Drug Metabolism and Pharmacokinetics, 48(4), 397-410.

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Morusin Metabolism Kinetics Protocol

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Morusin was purchased from Shifeng Corp in Shanghai, China, and its purity was above 98%. Furafylline, xanthotoxin, quercetin, sulfaphenazole, omeprazole, quinidine, clomethiazole, ketoconazole, glucose-6-phosphate dehydrogenase, NADP⁺, D-glucose-6-phosphate, 4-methylumbelliferone (4-MU), 4-methylumbelliferone-β-D-glucuronide (4-MUG), Tris-HCl, 7-hydroxycoumarin, and uridine 5′-diphosphoglucuronic acid (UDPGA) (trisodium salt) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Recombinant human supersomes (CYP1A2, CYP2C9, CYP2D6, CYP2E1, CYP3A4, and CYP2C19) were obtained from BD Gentest Corp. (Woburn, MA, USA). All other reagents were of HPLC grade or of the highest grade commercially available.

Shi X., Mackie B., Zhang G., Yang S., Song Y., Su D., Liu Y, & Shan L. (2016). Identification of the Metabolic Enzyme Involved Morusin Metabolism and Characterization of Its Metabolites by Ultraperformance Liquid Chromatography Quadrupole Time-of-Flight Mass Spectrometry (UPLC/Q-TOF-MS/MS). Evidence-based Complementary and Alternative Medicine : eCAM, 2016, 9240103.

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