Aug 03, 2025
In-vitro CYP inhibition pooled
- Nick Lynch1,2
- 1Curlew Research;
- 2ASAP Discovery Consortium
- ASAP Discovery
Protocol Citation: Nick Lynch 2025. In-vitro CYP inhibition pooled. protocols.io https://dx.doi.org/10.17504/protocols.io.81wgbkb1ygpk/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: 218909
Keywords: ADME, DMPK, drug discovery, cytochrome P450, cyp inhibition assay, multiple cytochrome p450 enzyme, cytochrome p450, cyp inhibition, multiple cyp enzyme, liver microsome, inhibitory profile across major drug, different cyp isoform, multiple cyp interaction, pooled assay, specific to different cyp isoform, rapid identification of potential drug, pooled enzyme source, cyp1a2, cyp, single test compound aliquot test, drug interaction, early drug development efficiency, test compound, predicting clinical drug, metabolizing enzyme, drug development, regulatory submissions in pharmaceutical development, human liver, inhibitory profile, cyp3a4, cyp2d6, drug interaction liability, cyp2c9, cyp2c19, pharmaceutical development, assay, potential drug, such as human liver, major drug, clinical drug, cocktail method, enzyme, liver, drug
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Abstract
This assay is crucial for understanding a compound's inhibitory profile across major drug-metabolizing enzymes, providing essential data for predicting clinical drug-drug interactions and supporting regulatory submissions in pharmaceutical development.
An in vitro CYP inhibition assay evaluates the potential of a test compound to inhibit multiple cytochrome P450 enzymes simultaneously using a cocktail approach. This pooled assay employs a mixture of substrates specific to different CYP isoforms (CYP1A2, CYP2C9, CYP2C19, CYP2D6 & CYP3A4) combined with a pooled enzyme source, such as human liver microsomes.
The cocktail method offers several advantages over individual isoform testing, including:
- Reduced material consumption - Single test compound aliquot tests multiple CYP enzymes
- Higher throughput screening - Multiple CYP interactions assessed simultaneously
- Early drug development efficiency - Rapid identification of potential drug-drug interaction liabilities
Troubleshooting
Safety warnings
Always wear appropriate PPE for this protocol
Refer to Material Safety Data Sheets for additional safety and handling information.
Summary
The pooled CYP inhibition is performed in 96-well plate format, the compounds are incubated at 0, 0.15, 0.5, 1.5, 5, 15 & 50μM concentration in a shaking incubator at 37ºC
Sample Preparation
CYPs are pooled in a ratio such that biotransformation of each probe substrate is specific for the particular CYP450
Substrates (at their Km) are also pooled within the same solution to create an enzyme-substrate stock and test compound is added to appropriate wells
Assay
The final solvent concentration is 1.0% DMSO
After equilibration to 37ºC, addition of NADPH and mixing initiates substrate biotransformation
Each concentration of test compound is assayed against five CYPs in the same well simultaneously.
The incubation is stopped at t = 10min by removal of the plate from the shaking incubator, followed by the addition of 200μL of ice cold methanol containing internal standard, and mixing.
The quenched samples are then centrifuged to precipitate the protein.
Analytical
The supernatants are analyzed by LC-MS/MS using generic analytical methods to measure metabolite formation.
Data Analysis
Clearance and half-life are calculated from the elimination rate constant derived from plotting response ratio versus time
IC50 (μM) of test compound against CYP1A2, CYP2C9, CYP2C19, CYP2D6 & CYP3A4 are calculated by plotting 6-point inhibition curves of the metabolite formation response.
Acknowledgements
Grateful to Concept Life Sciences for supplying the original protocol summary