Cytochrome P450 inhibition assay (2023)

Explore the potential risk of drug interactions with your compounds using our reversible cytochrome P450 (CYP450) inhibition assay for a variety of isoforms.

The Cytochrome P450 Inhibition Test is one of our productsin vitroADME inspection services. Cyprotex delivers consistent, high quality data with the cost-effectiveness of a highly automated approach.

Inhibition of cytochrome P450 enzymes (CYP450).

• Cytochrome P450 (CYP) is a family of enzymes that play an important role in drug metabolism.
• Assessment of a compound's potential to inhibit a specific cytochrome P450 enzyme is important because co-administration of the compounds can result in inhibition of the metabolism of one or both of the compounds. This may affect plasma concentrationsLiveand potentially lead to adverse drug reactions or toxicity.
• in vitroCytochrome P450 inhibition data are useful in designing study strategies for DDI clinical trials.
• Cyprotex cytochrome P450 inhibition assays utilize industry-accepted probe substrates and human liver microsomes.
• The Cyprotex Cytochrome P450 Inhibition Assay uses the reduction in metabolite formation compared to the vehicle control to calculate the IC50.₅₀value (concentration of test compound causing 50% inhibition).

The effects of new drugs on well-characterized drug metabolism reactions known to be specific to several human drug-metabolizing enzymes are routinely studied using:in vitrohe arrives.

¹Obach RS, Walsky RL, Venkatakrishnan K, Gaman EA, Houston JB e Tremaine LM (2006)JPET 316; 336-348.

Cytochrome P450 (CYP) Inhibition Assay Protocol (IC₅₀)

Cyprotex Cytochrome P450 (CYP) Inhibition Assay Data

Known cytochrome P450 (CYP) inhibitors were evaluated in the Cyprotex Cytochrome P450 Inhibition Assay in quadruplicate in 4 separate assays.

Questions and Answers on Cytochrome P450 (CYP) Inhibition.

Provide an overview of the Cyprotex cytochrome P450 inhibition assay.

Seven major isoforms of cytochrome P450 (CYP1A, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 - other isoforms available upon request) are tested in the Cyprotex Cytochrome P450 Inhibition Assay. Isoform-specific substrates are individually incubated with human liver microsomes and a range of test compound concentrations (typically 0.1 - 25 µM). At the end of the incubation, metabolite formation is monitored by LC-MS/MS (or fluorescence for CYP1A using ethoxyresorufin as substrate) at each concentration of test compound. An example of an integrated circuit₅₀the profile is shown in Figure 3.

The reduction in metabolite formation compared to vehicle control is used to calculate the IC50₅₀value (concentration of test compound causing 50% inhibition).

Why is it important to test for cytochrome P450 inhibition?

Cytochromes P450 are a family of enzymes that play an important role in drug metabolism. Assessment of a compound's potential to reversibly inhibit a specific cytochrome P450 enzyme is important because co-administration of the compounds can result in inhibition of the metabolism of one or both of the compounds. This may affect plasma concentrationsLiveand potentially lead to adverse drug reactions or toxicity.

Assessment of cytochrome P450 inhibition by an investigational new drug is recommended in the FDA guidelines for drug interaction studies (2020)⁹and EMA guidance on drug interaction testing (adopted in 2012)¹⁰.

How to interpret cytochrome P450 (CYP) inhibition assay data?

Typically, compounds can be divided into the following classification groups;

Braking power must always be considered in the context of expected effectsLiveconcentration of the test compound. Although the acceptance criteria are design andisoform specific, strong inhibition is considered unfavorable and may impede compound development.

What positive control substrates and inhibitors are used in the cytochrome P450 inhibition assay?

We used known specific positive control inhibitors for each of the isoform tests.

Table 1:Table showing positive control inhibitors for each cytochrome P450 isoform reaction

What is the concentration of the probe substrates relative to K?_M?

The substrate concentration is equivalent to K_M.

Are you testing each isoform as a cocktail or as separate incubations?

We don't incubate in cocktail form. All our cytochrome P450 inhibition reactions are incubated separately for each isoform.

Why are human liver microsomes used instead of a cDNA expressing enzyme to test for P450 inhibition?

Metabolism systems using recombinant enzymes are artificial because the enzyme is not present in its native environment and is often overexpressed. There are no competing enzymes and reactions in these systems. Human liver microsomes contain a full complement of P450 enzymes, making them more comparable to microsomesLivesituation.

Why do you use LC-MS/MS instead of fluorescent probes?

The conversion of ethoxyresorufin to resorufin is the only fluorescent reaction we decided to use. Ethoxyresorufin is selective for CYP1A and therefore can be used for liver microsomes. However, for the remaining isoforms, traditional probe substrates with LC-MS/MS detection are used.

There are several reasons why fluorescent probes should not be used for these isoforms listed below:

1. Developed fluorescent probes are not isoform specific and can only be used with individually expressed enzymes¹¹.
2. Inhibitor under test may cause interference which may lead to false negative results¹¹.
3. These substrates are not suitable for testingLive ¹¹.
4. fluorescent probes forin vitroreporting studies to regulatory authorities is not recommended¹¹.
5. There appears to be a weak correlation of inhibitory potential using different fluoroprobes¹².

Can I better characterize the type of inhibition found?

following the IC₅₀determination, we can then determine K_EUfor the test compound with respect to the appropriate isoform. This will provide information on braking force and type of braking (competitive, non-competitive, non-competitive or mixed) and can be used to estimate the impact of potentialLiveinteractions.