Aug 03, 2025
In-vitro Human or Mouse S9 intestinal stability assay
- Nick Lynch1,2
- 1Curlew Research;
- 2ASAP Discovery Consortium
- ASAP Discovery
Protocol Citation: Nick Lynch 2025. In-vitro Human or Mouse S9 intestinal stability assay. protocols.io https://dx.doi.org/10.17504/protocols.io.q26g79er8vwz/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: May 25, 2025
Last Modified: August 03, 2025
Protocol Integer ID: 218907
Keywords: ADME, DMPK, drug discovery, S9 fraction, intestinal stability, intestinal s9 stability assay, stability intestinal s9 human, mouse s9 intestinal stability, intestinal s9 human, mouse assay, intestinal stability, assay, mouse intestinal enzyme, metabolic stability, intestinal enzyme, mouse s9, enzyme, stability, test, small intestine
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Abstract
A stability intestinal S9 human or mouse assay, or intestinal S9 stability assay, is an in vitro test used to assess the metabolic stability of a compound in the presence of human or mouse intestinal enzymes. It simulates the breakdown of a compound by enzymes found in the small intestine, providing insights into how well it might be absorbed and metabolized in the body.
Troubleshooting
Safety warnings
Always wear appropriate PPE for this protocol
Refer to Material Safety Data Sheets for additional safety and handling information.
Summary
The Human S9 intestinal stability assay is run in 96-well plates on a Thermomixer set to 39°C and 700 rpm
This protocol can be used with the following S9 fraction material:
- Human
- Mouse
Sample Preparation
The test Compounds are diluted to 100µM using methanol in a separate plate
The quench plate is prepared by adding 400µL of cold methanol with internal standard to each
required well.
Assay
A 500mM MgCl₂ solution is made in phosphate buffer, then combined with alamethicin and sonicated before adding the S9 fraction to create the S9 incubation mix.
The S9 incubation mix (300µL) is added to the incubation plate and pre-incubated for 10 minutes.
Separately, 15mM NADPH and 30mM UDPGA are prepared and mixed to form a cofactor solution, with 46.5µL added to each well.
The reaction is initiated by adding 3.5µL of compound to column 1, immediately quenched by transferring 25µL to the cold quench plate (Time 0), and repeated for subsequent columns at defined time points (0, 5, 10, 15, 25, 35 min).
Throughout, mixing at 700rpm is maintained except during sampling.
After incubation, the quench plate is centrifuged at 4000rpm for 30 minutes at 4°C, and 150µL of the supernatant is transferred to a fresh LC-MS plate, avoiding disruption of the pellet.
Each well is then brought to 300µL with 30% methanol, and Method (MS) blanks are prepared by adding 300µL of 30% methanol to column 7.
Data Analysis
The Half-life (t1/2) and Intrinsic Clearance (CLint) are calculated
Acknowledgements
Grateful to Concept Life Sciences for supplying the original protocol summary