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.
Cyp1a2
Manufactured by BD
Sourced in India, United States, Canada, Belgium
CYP1A2 is a laboratory equipment product designed for the detection and quantification of the CYP1A2 enzyme. CYP1A2 is a member of the cytochrome P450 enzyme family, which play a critical role in the metabolism of various drugs and other xenobiotic compounds. The core function of this product is to provide researchers and clinicians with a reliable tool to measure CYP1A2 levels, which can be useful in areas such as drug development, pharmacogenomics, and personalized medicine.
Automatically generated - may contain errors
Lab products found in correlation
Cyp2c19, by BD (7 mentions)
Cyp2c9, by BD (6 mentions)
Cyp2d6, by BD (6 mentions)
Cyp3a4, by BD (5 mentions)
Cyp2b6, by BD (4 mentions)
Cyp1b1, by BD (4 mentions)
Cyp2c8, by BD (4 mentions)
Cyp2e1, by BD (4 mentions)
Cyp1a1, by BD (3 mentions)
Cyp2a6, by BD (3 mentions)
Cyp3a5, by BD (3 mentions)
Nadph, by Merck Group (3 mentions)
Alamethicin, by Merck Group (3 mentions)
D glucose 6 phosphate sodium salt, by Merck Group (2 mentions)
Gentest human cyp1a1, by BD (2 mentions)
β nicotinamide adenine dinucleotide phosphate sodiumsalt nadp, by Merck Group (2 mentions)
Glucose 6 phosphate dehydrogenase, by Merck Group (2 mentions)
Prism 6, by GraphPad (2 mentions)
Caco 2 cell line, by American Type Culture Collection (1 mentions)
Hanks balanced salt solution (hbss), by Merck Group (1 mentions)
11 protocols using cyp1a2
1
Cytochrome P450 Enzyme Inhibition Assay
2
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).
3
Recombinant Human Cytochrome P450 Enzymes
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4
CYP1A1, CYP1A2, and CYP1B1 Supersomes Assay
5
Cytochrome P450 Enzyme Assay Protocol
6
In vitro Liver Microsome and Cytosol Preparation
7
Characterization of CYP-Mediated Metabolism
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Monkey liver S9 fraction was obtained from BD Bioscience Pharmingen (San Diego, CA). Trizma pre-set crystals, reduced β-nicotinamide adenine dinucleotide phosphate (NADPH), alamethicin, buspirone, potassium phosphate buffer, Krebs-Henseleit buffer powder, trypan blue, and uridine 5’-diphosphoglucuronic acid (UDPGA) were obtained from Sigma (St. Louis, MO). LC-MS grade acetonitrile (ACN), water, and methanol (MeOH) were purchased from EM Science (Gibbstown, N.J.). Deuterated acetonitrile and deuterated methanol were obtained from Cambridge Isotopes Laboratories Inc. (Andover, MA). NMR tubes were acquired from Kimble/Chase Life Science (Vineland, NJ). Human recombinant CYP (Supersomes) CYP3A4, CYP2C9, CYP2C19, CYP1A2, CYP2D6, CYP2C8, and CYP2E1 were purchased from BD Biosciences (San Jose, CA). Cryopreserved pooled mouse, rat, dog, monkey, and human hepatocytes together with hepatocyte thawing medium were obtained from Bioreclamation IVT (Baltimore, MD). The test article (diamine purine containing parent compound) was synthesized in Celgene.
8
Cytochrome P450 Enzyme Assay
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Gentest human
CYP1A1, CYP1A2,
CYP1B1, and CYP2A6 supersomes and rat CYP2B1 supersomes were purchased
from BD Biosciences (Franklin Lakes, NJ).d -Glucose-6-phosphate
sodium salt, β-nicotinamide adenine dinucleotide phosphate sodium
salt (NADP+), and glucose-6-phosphate dehydrogenase were
purchased from Sigma-Aldrich Corporation. Other reagents in bioassays
were purchased from Fisher Scientific International, Inc. Figures
were plotted with Prism 6 (GraphPad Software, Inc., La Jolla, CA).
CYP1A1, CYP1A2,
CYP1B1, and CYP2A6 supersomes and rat CYP2B1 supersomes were purchased
from BD Biosciences (Franklin Lakes, NJ).
sodium salt, β-nicotinamide adenine dinucleotide phosphate sodium
salt (NADP+), and glucose-6-phosphate dehydrogenase were
purchased from Sigma-Aldrich Corporation. Other reagents in bioassays
were purchased from Fisher Scientific International, Inc. Figures
were plotted with Prism 6 (GraphPad Software, Inc., La Jolla, CA).
9
Synthesis and Characterization of Novel Compounds
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IMM-H004 (purity >99%), M1, and M2 (purity >90%) (Li et al., 2010 (link)) were synthesized by Laboratory of Chemical Synthesis, and IMM-H004G (M3) (purity >99%) for animal study was provided by Prof. Dai (Laboratory of Biosynthesis of Natural Products, Institute of Materia Medica, Chinese Academy of Medical Science and Peking Union Medical College, Beijing, China). Propranolol (internal standard, IS), β-glucuronidase, sulfatase, 3′-phosphoadenosine-5′-phosphosulfate (PAPS), uridine 5′-diphosphoglucuronic acid (UDPGA), and alamethicin were obtained from Sigma-Aldrich (St. Louis, MO). Rat liver microsomes (RLMs) and cytosols were prepared by differential ultracentrifugation, and the protein concentration was determined by bicinchoninic acid assay (Beyotime Institute of Biotechnology, Jiangsu, China). Pooled mixed-gender human liver microsomes (HLMs), human liver cytosols, recombinant human cytochrome P450 enzymes (CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP2J2, CYP3A4, CYP4A11, CYP4F2, and CYP4F3), recombinant human UDP-glucuronosyltransferases (UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT12B4, UGT12B7, UGT12B10, UGT12B15, and UGT12B17), and recombinant human sulfotransferase (SULT1A1, SULT1A2, SULT1A3, SULT1B1, SULT1C2, SULT1C4, SULT1E1, and SULT2A1) were purchased from BD Gentest (Woburn, MA).
10
In Vitro CYP Enzyme Kinetics
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Citalopram, nortriptyline and melatonin were incubated with CYP1A2, CYP2C19 and 50-mixed gender pooled human liver microsome (HLM) were purchased from BD Biosciences (Mississauga, ON, Canada) with NADPH in 0.1M PBS buffer at 37°C for 30-60 min (40) . The incubation matrix was divided into 2 parts. One part was mixed with equal amount of methanol to de-nature the protein and was then analyzed by HPLC-DAD. The other part went through a liquid to liquid extraction with ethyl acetate and then analyzed by UPLC-MS. Each study was run in triplicate and the studies were repeated once.
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