Liver microsomes (Human or CD-1 mouse) were diluted to 0.5 mg/mL in a solution containing 100 mM PBS buffer at pH 7.4, an NADPH regenerating system (Promega), and test compounds and control compounds were diluted from DMSO stock solutions to 1 µM, unless otherwise indicated. Control reactions were made by withholding the addition of the NADPH regenerating solution, which enabled us to assess whether compound stability was the result of NADPH-dependent metabolism. The mixture was incubated at 37 °C under gentle rotation and quenched by aliquoting 10 µL of solution into 90 µL ice-cold acetonitrile containing an internal standard. To evaluate the plasma stability of lead compounds, plasma (Human or CD-1 mouse) was warmed to 37 °C in a water bath, and test compounds were added to the plasma at 10 µM. Plasma samples were incubated at 37 °C under gentle rotation. At indicated time points, samples were quenched by aliquoting 10 µL of solution into 90 µL ice-cold acetonitrile containing an internal standard. Samples were collected and analyzed by LC-MS on a Waters H-Class Plus UPLC connected to a Xevo G2-XS QTOF mass spectrometer running in ESI+ mode. The method used a Waters Acquity UPLC BEH C18 (2.1 × 50 mm, 1.7 µm) column with a gradient of 23–100% B (A: water + 0.2% formic acid, B: 4:1 methanol/isopropanol).
Nadph regenerating system
Manufactured by Promega
Sourced in United States
The NADPH-regenerating system is a laboratory product designed to maintain the availability of NADPH, a critical cofactor in various enzymatic reactions. The system provides a continuous supply of NADPH to support the activity of enzymes that require this cofactor for their function.
Automatically generated - may contain errors
Lab products found in correlation
Mouse monoclonal anti or antibody, by Santa Cruz Biotechnology (3 mentions)
Mouse cd 1 liver microsomes, by Thermo Fisher Scientific (2 mentions)
High pressure liquid chromatography grade solvents, by Thermo Fisher Scientific (2 mentions)
Supersignal west pico trial kit, by Thermo Fisher Scientific (2 mentions)
Acquity uplc beh c18, by Waters Corporation (1 mentions)
Corresponding secondary antibodies, by Proteintech (1 mentions)
Ebastine, by Tokyo Chemical Industry (1 mentions)
Dextrose, by Merck Group (1 mentions)
Galactose, by Merck Group (1 mentions)
Anti cyp3a4 antibody, by Abcam (1 mentions)
Taq plus master mix, by Vazyme (1 mentions)
Telmisartan, by LGC (1 mentions)
Primestar hs dna polymerase and restriction enzymes, by Takara Bio (1 mentions)
4 hydroxydiclofenac, by LGC (1 mentions)
Bac to bac baculovirus expression system, by Thermo Fisher Scientific (1 mentions)
4 hydroxytolbutamide, by LGC (1 mentions)
Tianamp blood dna midi kit, by Tiangen Biotech (1 mentions)
7 protocols using nadph regenerating system
1
Liver Microsome and Plasma Stability Assay
2
Intrinsic Clearance of Compounds 1 and 2
3
Enzymatic Activity of CYP2J2 in Yeast
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The FinePure Universal DNA Purification Kit was purchased from GENFINE (Beijing, China). The CYP2J2 Human Tagged ORF Clone (RC207417) was purchased from Origene (United States). Saccharomyces cerevisiae strain YPH499 was obtained from ATCC (VA, United States). The yeast expression vector pESC-URA was purchased from Stratagene (United States). The following primary antibodies were used: rabbit polyclonal anti-CYP2J2 antibody (Abcam, United Kingdom), mouse monoclonal anti-OR antibody (Santa Cruz Biotechnology, United States) and corresponding secondary antibodies (Proteintech, China). Ebastine and terfenadine were purchased from Tokyo Chemical Industry. HydroxyEbastine, terfenadine alcohol and midazolam were purchased from Santa Cruz and TRC. The NADPH-regenerating system was purchased from Promega (United States). High-pressure liquid chromatography-grade solvents were purchased from Fisher Scientific Co., (United States). All the other chemicals and solvents were commercially available at analytical grade or the highest grade.
4
Silica Particle Inhibition of LIPA
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The inhibitory effects of silica particles at various concentrations (2, 10, 50, 200, and 800 μg/mL) and ketoconazole (200 nmol/L) were determined with HLMs (20 μg/mL) in the presence of NADPH Regenerating System (Promega). The incubation mixtures, which consisted of silica particles, ketoconazole, 10 μmol/L LIPA, and HLMs in potassium phosphate buffer (15 μL, respectively), were pre-incubated for 10 min at 37°C, and then the enzymatic reactions were initiated by the addition of 15 μL of NADPH. In addition, to determine whether LIPA was physically bound to the silica particles, we also started the reaction by adding 15 μL of LIPA after preparing a mixture of the silica particles, HLMs, buffer, and NADPH. Next, to determine whether the silica particles were physically bound to microsome proteins, we centrifuged a mixture of silica particles, LIPA, HLMs, and buffer at 1,000 × g or 5,000 × g for 20 min and then added NADPH (15 μL) to the supernatant (45 μL). For each of these procedures, the reactions were terminated after 10 min of incubation by the addition of reconstitution buffer (60 μL). Each plate was incubated at room temperature for 20 min, and then the luminescence was read with a luminometer for 1 s per well.
5
Yeast-based CYP3A4 Activity Assay
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The FinePure Universal DNA Purification Kit was supplied by GENFINE Biotech in Beijing, China. Taq plus master mix was purchased from Vazyme (Nanjing, China). Restriction enzymes, along with DO Supplement-Ura, all purchased from Takara Bio, Inc (Otsu, Shiga, Japan). Yeast strain YPH499 was obtained from the American Type Culture Collection (ATCC) in Virginia, USA. The growth mediums for yeast, specifically the yeast nitrogen base devoid of amino acids, along with dextrose, galactose, and MDZ, were supplied by Sigma-Aldrich (Missouri, USA). The rabbit polyclonal anti-CYP3A4 antibody was obtained from Abcam (Cambridge, UK) and the mouse monoclonal anti-OR antibody was from Santa Cruz Biotechnology in Dallas, Texas, USA.
The chemical 1-hydroxy Midazolam (1'-OH-MDZ) was obtained from Toronto Research Chemicals, Inc. (Toronto, Ontario, Canada). The NADPH-regenerating system was purchased from Promega in Madison, Wisconsin, USA. The Super Signal West Pico Trial Kit and highpressure liquid chromatography-grade solvents were acquired from Thermo Fisher Scientific Co.
in Waltham, Massachusetts, USA. All other chemicals and solvents used in our experiments were of the highest purity and analytical grade available on the market.
The chemical 1-hydroxy Midazolam (1'-OH-MDZ) was obtained from Toronto Research Chemicals, Inc. (Toronto, Ontario, Canada). The NADPH-regenerating system was purchased from Promega in Madison, Wisconsin, USA. The Super Signal West Pico Trial Kit and highpressure liquid chromatography-grade solvents were acquired from Thermo Fisher Scientific Co.
in Waltham, Massachusetts, USA. All other chemicals and solvents used in our experiments were of the highest purity and analytical grade available on the market.
6
Baculovirus-Mediated Recombinant Protein Expression
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Materials and kits were purchased from the following sources: TIANamp Blood DNA Midi Kit (TIANGEN, Beijing, China); PrimeSTAR HS DNA polymerase and restriction enzymes (Takara Bio, Inc.; Otsu, Shiga, Japan); Spodoptera frugiperda (Sf)21 insect cells, Sf-900TM III SFM insect culture medium and Bac to-Bac Baculovirus Expression System (Invitrogen, Carlsbad, CA, United States); Mouse monoclonal anti-OR antibody (Santa Cruz Biotechnology, Dallas, Texas, United States); Rabbit polyclonal anti-CYP2C9 antibody (Abcam, Cambridge, United Kingdom); Super Signal West Pico Trial Kit (Thermo Scientific, Rockford, IL, United States); 4-Hydroxytolbutamide, 4-hydroxydiclofenac, and telmisartan (Toronto Research Chemicals, Inc.; Toronto, Ontario, Canada); Diclofenac and chlorpropamide (Tokyo Chemical Industry Co., Ltd.; Tokyo, Japan); Tolbutamide, losartan and E-3174 (Sigma-Aldrich, St. Louis, MO, United States); NADPH-regenerating system (Promega, Madison, WI, United States); High-pressure liquid chromatography-grade solvents (Fisher Scientific Co.; Fair Lawn, NJ, United States). All other chemicals and reagents were of analytical grade or the highest commercially available quality.
7
Hepatic Clearance of Compounds 1 and 2
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The intrinsic clearance (CLint) of compounds 1 and 2 was determined using mouse (CD‐1) liver microsomes (Gibco). Briefly, microsomes (100 μg final protein concentration) and test compound in 0.1 M phosphate buffer, pH 7.4, were prepared. In parallel, an NADPH‐regenerating system (Promega) was prepared in 0.1 M phosphate buffer (pH 7.4). The solutions were pre‐incubated at 37°C for 10 min before assessment of the CLint was initiated by mixing the two solutions (50 μL of each; final compound concentration 1 μg·mL) at 37°C. After 0, 5, 10, 15, 20 and 30 min, the reactions were terminated by the addition of 100 μL of acetonitrile containing 1 μg·mL−1 verapamil and placed on ice for 30 min. The samples were then centrifuged at 12 000× g for 10 min and analysed by LC–MS to determine the quantity of the parent compound remaining over time. Carbamazepine (1 μg·mL−1) was used as a control for low CLint.
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