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Cytochrome P450 Time Dependent Inhibition Assay

Creative Animodel provides cytochrome P450 time dependent inhibition services as a part of in vitro ADME services. Our experienced scientists guarantee our clients the most reliable and cost efficient research services to best meet their research requirement in the drug development process.

What Is Time Dependent Inhibition?

Cytochromes P450 (CYP450 or CYPs) are hemoproteins which constitute a superfamily containing heme as a cofactor. And the human cytochrome P450 system is implicated in many drug interactions. Time-dependent inhibition (TDI) is a term which covers any phenomenon resulting in reducing of enzyme activity with incubation time. Potential mechanisms include the formation of inhibitory metabolites and mechanism-based inhibition (MBI). Moreover, TDI of CYP450 can lead to clinically relevant drug-drug interaction (DDI) or no-linear pharmacokinetics of a drug. It is reported that drugs that are time dependent inhibitors of CYP 450 cause severe drug-drug interactions, leading to prescription adjustments and have been withdrawn from the market. The draft FDA guidance and EMA guideline for drug interactions recommend assaying time dependent inhibition for investigational drugs. Therefore, it is important to assay time independent inhibition of drugs in vitro.

Available Approaches for CYP450 TDI Assay at Creative Animodel

• Single point assay.
The single point assay is often used in an early stage of ADME for many compounds. It is important to get a snapshot of the amount of time dependent inhibition at a top concentration relative to expected therapeutic concentrations in an early stage of drug development.
We use microsomes from human and various animal species as incubation system, adding a certain concentration of test compound with or without NADPH for pre-incubation. And then a portion of the solution is removed to the probe solution for the incubation. After the reaction is finished, the concentration of the substrate metabolite production in the incubation solution is measured, and the change of the enzyme activity in the case of adding and without NADPH is determined by the formula. While insufficient for detailed information, screening data in single point data allows rapid compound binding or rank order, which facilitates identification of structure activity relationships and efficient de-prioritization of potent inhibitors.

• IC50 shift assay.
It can determine the IC50 (half maximal inhibitory concentration) through pre-incubation within and without NADPH. We use drug-like probe substrates, human liver microsomes, NADPH and two pre-incubation times. The IC50 value is assayed under different conditions: 0 minute pre-incubation, 30 minutes pre-incubation with NADPH and 30 minutes pre-incubation without NADPH. The assay monitors the formation of the metabolites of the drug-like probe substrates in the absence and presence of a compound by LC-MS/MS following a pre-incubation period. This assay enables discrimination between compounds which cause reversible, irreversible, or both reversible and irreversible inhibition.

Figure1. IC50 shift data of a time dependent inhibitor

• Kinact/KI assay.
The Kinact is the maximal rate of enzyme inactivation at a saturating concentration of inhibitor and KI is the concentration at 50% Kinact. Kinact/KI assay is a later stage assessment of inhibitor kinetics. The detailed experimental conditions are determined according to the results of previously performed assays such as IC50 shift. For 7 different pre-incubation times (including 0 min) with human hepatocytes, we test the compounds by a range of concentrations to reach a scale from no inactivation to maximal inactivation.

Table1. The substrates and metabolites of CYP isoforms

CYP IsoformSubstrateMetabolite
2A6Coumarin7-OH coumarin
2C9Diclofenac4’-OH diclofenac
2C19S-mephenytoin4’-OH mephenytoin
2D6Bufuralol1’-OH bufuralol
2E1Chlorzoxazone6-OH chlorzoxazone
3A4Midazolam1’-OH midazolam
3A4Testosterone6β-OH testosterone

Creative Animodel can design studies to predict drug-drug interactions with appropriate methodologies. Comprehensive solutions and reliable results are provided for the entire process of drug discovery and development. We are confident to offer you CYP450 time dependent inhibition assay and other related services to promote your programs.

1. Riley R J. et al.; Time-dependent CYP inhibition, Expert opinion on drug metabolism & toxicology. 2007, 3(1): 51-66.
2. Fowler S., Zhang H.; In vitro evaluation of reversible and irreversible cytochrome P450 inhibition: current status on methodologies and their utility for predicting drug–drug interactions. The AAPS journal. 2008, 10(2): 410-424.

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