Dec 09, 2024
Microsomal stability assay for human and mouse liver microsomes - drug metabolism
- Lina Bortnichuk1,
- Anna Pashchenko1,
- Yurii Kheilik1
- 1Enamine Ltd
- ASAP Discovery
Protocol Citation: Lina Bortnichuk, Anna Pashchenko, Yurii Kheilik 2024. Microsomal stability assay for human and mouse liver microsomes - drug metabolism. protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvokdb9l4o/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 14, 2024
Last Modified: December 09, 2024
Protocol Integer ID: 106088
Keywords: Microsomal protein, Glucose-6-phosphate dehydrogenase, NADPH-cofactor , Intrinsic clearance , HPLC-MS/MS , Microsomes, microsomal stability, oral bioavailability, drug metabolism
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Abstract
Microsomal stability is a key parameter in drug discovery and development to support understanding of a compound's susceptibility to liver metabolism.
This assay assesses a compound's susceptibility to metabolism by liver enzymes, primarily cytochrome P450 (CYP) enzymes. Using either human or mouse microsomes.
The data analysis provides elimination constant, half-life, and intrinsic clearance . These are determined as results using linear regression analysis.
In this protocol, we measure (human or mouse) liver microsomal stability assay for 12 test articles in 96-well plate format. Microsomal incubations are carried out in 5 aliquots of 30 μL (one for each time point). The liver microsomal incubation medium comprises phosphate buffer (100 mM, pH 7.4), MgCl2 (3.3 mM), NADPН (3 mM), glucose-6-phosphate (5.3 mM), glucose-6-phosphate dehydrogenase (0.67 units/mL) with 0.415 mg of liver microsomal protein per mL. In the negative control reactions, the NADPH-cofactor system is substituted with phosphate buffer. Test compounds (2 μM, final acetonitrile concentration 1.6 %) are incubated with microsomes at 37°C, shaking at 100 rpm. Five time points over 40 minutes are analyzed (0, 7, 15, 25, and 40 min). The reactions are stopped by adding 5 volumes of acetonitrile with internal standard to incubation aliquots, followed by protein sedimentation by centrifuging at 5500 rpm for 5 minutes. Supernatants are analyzed using the HPLC-MS/MS system coupled with a tandem mass spectrometer.
The elimination constant (kel), half-life (t1/2), and intrinsic clearance (Clint) are determined in a plot of ln(AUC) versus time, using linear regression analysis:
Materials
Reagents and preparations:
- 100 mM (1x) Potassium phosphate buffer (PPB), pH 7.4
- 33 mM MgCl2 × 6Н2О solution
- Glucose-6-phosphate dehydrogenase (G6PDH) solution, 250 U/mL
- Human or mouse liver microsomes, 20 mg/mL
- Compounds stock solutions
Safety warnings
Always wear appropriate PPE for this protocol
Refer to Material Safety Data Sheets for additional safety and handling information.
Reagents and preparations
Reagents and preparations
Prepare 20 millimolar (mM) stocks of reference and test compounds in DMSO.
- Then, dissolve DMSO stock in acetonitrile (AcN) to 125 micromolar (µM) , and mix by vortexing. Place AcN stock solutions as shown in Fig. 1.
- Diclofenac and propranolol are used as reference compounds in the assay with human microsomes, imipramine and propranolol - with mice microsomes.
Fig. 1 Plate layout for compounds stock solutions in acetonitrile
Quenching solution with internal standards
Dissolve appropriate for LC-MS/MS analysis internal standards in ACN.
NADPH tetrasodium salt, D-Glucose-6-phosphate sodium salt
Weigh out 17.88 mg of NADPH tetrasodium salt (MW=833.35 g/mol) and 9.72 mg of D-Glucose-6-phosphate sodium salt (MW=282.12 g/mol) into separate tubes. Place tubes with powders at -20 °C .
Samples preparation
Samples preparation
10m
10m
Set the incubator-shaker at 37 °C and 100 rpm .
Place aliquots of microsomes and G-6-PDH On ice to thaw.
Prepare five 96-well plates and label them as «Preincubation», «K+/K-», «0+/0-», «40-», and «Incubation».
- Place microtubes into plates as shown in Fig. 2.
Fig. 2 Preparation of the 96-well plates with 0.75mL microtubes for the assay
Label tubes as «K-», «K+», and «Microsomes».
Prepare the following solutions:
- «K-». Mix 1021 µL 100 millimolar (mM) PPB and 325 µL 33 millimolar (mM) MgCl2 × 6Н2О.
- «K+». Mix 1388 µL 100 millimolar (mM) PPB and 650 µL 33 millimolar (mM) MgCl2×6Н2О solution.
- «Microsomes». Fill the tube with 5785 µL 100 millimolar (mM) PPB.
Aliquot 52 µL of «K-» solution into the corresponding microtubes of «K+/K-» plate, and then cover the microtubes with mates until starting the experiment.
Dispense the stop solution into the «0+/0-» plate, and then cover the microtubes with mates until the start of the experiment.
After thawing, add 216 µL of human or mouse liver microsomes into the tube labeled «Microsomes», mix manually, and place the tube On ice .
Aliquot 231 µL of «Microsomes» solution into the «Incubation» plate. Then, add 6.5 µL 125 micromolar (µM) AcN-stock solutions of compounds, and mix by pipetting.
- Cover microtubes with mates and place the plate in the incubator-shaker for 00:10:00 to pre-warm the solution before the experiment.
10m
Within 10 minutes perform the following steps:
Dissolve 17.88 mg of NADPH in 325 µL of 100 millimolar (mM) PPB, mix by pipetting;
Dissolve 9.72 mg of D-Glucose-6-phosphate in the same way.
Add 325 µL of both NADPH and D-Glucose-6-phosphate solutions into the «K+» tube;
Add 16 µL of G-6-PDH into the «K+» tube, gently mix by pipetting;
Aliquot 104 µL of «K+» solution into the corresponding microtubes of «K+/K-» plate.
Fill in the «0+/0-» plate with the desired amount of quenching solution.
Incubation
Incubation
45m
45m
After 10 minutes of pre-incubation, remove the «Preincubation» plate from the incubator-shaker, and mix samples by pipetting.
«K-» samples. Transfer 71 µL of pre incubation solution into the first parallel of «K-» microtubes, mix by pipetting, and aliquot solution into the corresponding «40-» and «0-» microtubes. Perform the same operation with another parallel.
«K+» samples. Transfer 142 µL of preincubation solution into the first parallel of «K+» microtubes, mix by pipetting, aliquot solution into the corresponding «0+» microtubes (start the timer from this moment), and into the microtubes of «Incubation» plate (7-, 15-, 25-, 40-min time points).
- Perform the same operation with another parallel and record the time shift between the 0-min time points of the first and second incubation parallels.
Cover «0+/0-» microtubes with caps, vortex plate and place in the refrigerator. Cover «7», «15», «25», «40», and «40-» microtubes with mats, and place in the incubator-shaker.
At each time point, remove corresponding microtubes from the incubator-shaker and add quenching solution to stop the reaction.
After the total incubation time, centrifuge the final plates at 5500 rpm, 00:05:00 . The final plates should look as shown in Fig. 3.
Fig. 3 Sample layout on the final plate
5m
Analyze the samples using HPLC-MS/MS in the following order (start the analysis from the samples with theoretically lower concentrations of the tested compound): 00:40:00 time point→25 min→15 min→7 min→0 min→40- min →0-.
40m
Data Analysis
Data Analysis
The elimination constant (kel), half-life (t1/2), and intrinsic clearance (Clint) are determined in a plot of ln(AUC) versus time, using linear regression analysis:
Equations used