Mouse and human liver microsomes

Manufactured by Xenotech

Sourced in United States

Mouse and human liver microsomes are subcellular fractions containing the endoplasmic reticulum and associated enzymes, primarily cytochrome P450 enzymes. These microsomes are commonly used in in vitro studies to assess drug metabolism and chemical interactions.

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Lab products found in correlation

Q exactive orbitrap mass spectrometer, by Thermo Fisher Scientific (1 mentions) Acquity uplc, by Waters Corporation (1 mentions) Xevo g2 q tof mass spectrometer, by Waters Corporation (1 mentions)

4 protocols using mouse and human liver microsomes

In Vitro Metabolism Profiling

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Test compounds in duplicate were dissolved in DMSO to obtain 0.2 mM solutions and pre‐incubated, at the final concentration of 1 μM, for 10 min at 37 °C in potassium phosphate buffer 50 mM pH 7.4, 3 mM MgCl₂, with mouse and human liver microsomes (Xenotech) at the final concentration of 0.5 mg/mL.
After the pre‐incubation period, the reaction was started by adding the cofactors mixture (NADP, Glc6P, Glc6P‐DH in 2 % sodium bicarbonate); samples (25 μL) were taken at time 0, 10, 20, 30 and 60 min added to 150 μL of a 0.02 μM acetonitrile solution of Verapamil used as Internal Standard (IS) to stop the reaction. After centrifugation the supernatants were analyzed by LC–MS/MS. A control sample without cofactors was added to check the stability of test compounds in the matrix after 60 min. 7‐ethoxycoumarin (7‐EC) and propranolol were added as reference standards. Sample and data analysis are reported in Supporting Information.

Bassanini I., Parapini S., Basilico N., Taramelli D, & Romeo S. (2022). From DC18 to MR07: A Metabolically Stable 4,4′‐Oxybisbenzoyl Amide as a Low‐Nanomolar Growth Inhibitor of P. falciparum. Chemmedchem, 17(21), e202200355.

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Metabolic Stability and CYP-Mediated Metabolism

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The metabolic stability was evaluated using mouse and human liver microsomes (Xenotech LLC, USA) as previously described.^{38 (link),39} For the cytochrome P450 (CYP)-mediated metabolism, the reaction was carried out with 100 mM potassium phosphate buffer, pH 7.4, in the presence of NADPH regenerating system (1.3 mM NADPH, 3.3 mM glucose 6-phosphate, 3.3 mM MgCl₂, 0.4 U/mL glucose 6-phosphate dehydrogenase, 50 μM sodium citrate). Reactions, in triplicate, were initiated by addition of the liver microsomes to the incubation mixture (compound final concentration was 1 μM; 0.2 mg/mL microsomes). Controls in the absence of cofactors were carried out to determine the specific cofactor-free degradation. After 60 min of incubation, aliquots of the mixture were removed, and the reaction was quenched by addition of three times the volume of ice-cold acetonitrile spiked with the internal standard. Compound disappearance was monitored over time using a liquid chromatography and tandem mass spectrometry (LC–MS/MS) method. Additionally, metabolite identification (compound 1) was performed by full scan analysis on Thermo Q Exactive Orbitrap mass spectrometer, and phase-I metabolites were monitored.

Šála M., Hollinger K.R., Thomas A.G., Dash R.P., Tallon C., Veeravalli V., Lovell L., Kögler M., Hřebabecký H., Procházková E., Nešuta O., Donoghue A., Lam J., Rais R., Rojas C., Slusher B.S, & Nencka R. (2020). Novel Human Neutral Sphingomyelinase 2 Inhibitors as Potential Therapeutics for Alzheimer’s Disease. Journal of medicinal chemistry, 63(11), 6028-6056.

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Microsomal Metabolism Assay for Drugs

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Human and mouse liver microsomes were obtained from XenoTech, LLC (Kansas City, KS). The reaction mixture was composed of liver microsomes (0.5 mg/mL), 20 mM Tris-HCl pH 7.4, 2.5 mM MgCl₂, and an NADPH generating system (0.1 mM NADP, 1.2 mM glucose-6-phosphate and 0.3 units/ml glucose-6-phophate dehydrogenase). Verapamil and disopyramide, which are highly and modestly metabolized, respectively, were used as comparator controls. Test compounds were added to the microsome reaction and loss of parent molecule was monitored after 1 h by LC/MS/MS.

Graci J.D., Michaels D., Chen G., Schiralli Lester G.M., Nodder S., Weetall M., Karp G.M., Gu Z., Colacino J.M, & Henderson A.J. (2017). Identification of benzazole compounds that induce HIV-1 transcription. PLoS ONE, 12(6), e0179100.

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Metabolic Stability Screening of Compounds

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The metabolic stability was assessed by incubating each test compound (0.5 μM) in duplicate with human and mouse liver microsomes (XenoTech, Kansas City, KS) at 37 °C and 0.4 mg ml⁻¹ microsomal protein. The metabolic reaction was initiated by the addition of an NADPH-regenerating system and quenched at various time points over a 60 min incubation period by the addition of acetonitrile containing diazepam as internal standard. Control samples (containing no NADPH) were included and quenched at 2, 30 and 60 min to monitor for potential degradation in the absence of cofactor. Samples were centrifuged and the supernatant analysed by LCMS. Analysis was conducted using a Waters Acquity UPLC coupled to a Waters Xevo G2 QTOF mass spectrometer operated in positive electrospray ionization MS^E mode with a cone voltage of 30 V. Samples (5 μl) were injected onto an Ascentis Express Amide column (50 × 2.1 mm, 2.7 μm) and eluted using a water/acetonitrile (both containing 0.05% formic acid) gradient over 4 min at a flow rate of 0.4 ml min⁻¹. Degradation rate half-lives and in vitro intrinsic clearance values were determined from the first order degradation profiles.

Tran A.T., Watson E.E., Pujari V., Conroy T., Dowman L.J., Giltrap A.M., Pang A., Wong W.R., Linington R.G., Mahapatra S., Saunders J., Charman S.A., West N.P., Bugg T.D., Tod J., Dowson C.G., Roper D.I., Crick D.C., Britton W.J, & Payne R.J. (2017). Sansanmycin natural product analogues as potent and selective anti-mycobacterials that inhibit lipid I biosynthesis. Nature Communications, 8, 14414.

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