Cytochrome P450 inhibition assay (2023)

Cytochrome P450 inhibition assay (1)

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).

Cytochrome P450 inhibition assay (2)

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)

Test ArticleConcentration0, 0,1, 0,25, 1, 2,5, 10, 25 µM
(various concentrations available)
Isoformas CYPCYP1A, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 (other isoforms available)
Test article requirementsDepending on the number of isoforms evaluated
controlsKnown isoform specific inhibitors
Analysis methodLC-MS/MS (except ethoxyresorufin for CYP1A)
data deliveryintegrated circuit50
The standard error of the integrated circuit50
Related services
Reversible CYP inhibition (Ki designation)
Time-dependent CYP inhibition (single point)
Time-dependent CYP inhibition (IC50 shift)
Time-dependent CYP inhibition (kinact/KI)


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.

Cytochrome P450 inhibition assay (3)

(Video) Mechanism of Cytochrome P450 (CYP) metabolism, induction, and inhibition

Figure 1
Cyprotex cytochrome P450 inhibition data for CYP3A4. The effect of 5 known CYP3A4 inhibitors (clotrimazole, ketoconazole, mibefradil, nicardipine and verapamil) on midazolam 1-hydroxylation has been studied 4 times. Error bars represent the standard deviation of 4 replicates in each experiment. The data show good compatibility of inhibitors with a wide range of inhibition potentials.

Cytochrome P450 inhibition assay (4)

Figure 2
Cyprotex IC Comparison50values ​​(mean ± standard deviation) for literature control inhibitors (2,3,4,5,6,7,8) values.

Questions and answers

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).

Cytochrome P450 inhibition assay (5)

Figure 3
Graph showing a typical braking profile.

(Video) Basics of Drug Interactions EXPLAINED | Inhibition & Induction

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;

hard brakingintegrated circuit50< 1 µM
moderate brakingintegrated circuit50between 1 and 10 µM
No or bad brakingintegrated circuit50> 10 µM

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.

isoformsubstrate reactionpositive control inhibitor
CYP1AEtoksyresorufinaOh- distillationα-Naftoflavona
CYP2B6bupropion hydroxylationTiclopidina
CYP2C86α-hydroxylation of paclitaxelMontelucaste
CYP2C94-hydroxylation of tolbutamideSulfaphenazole
CYP2C194-hydroxylation of S-mephenytoinTranylocipromine
CYP2D6DextrometorfanoOh- demethylationChinidyna
CYP3A41-hydroxylation of midazolamketoconazole
CYP3A46β-hydroxylation of testosteroneketoconazole

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?

(Video) CYP P450 Inhibition

The substrate concentration is equivalent to KM.

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 KEUfor 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.


1Obach RSI enter.(2006)JPET 316; 336-348
2this is hzI enter.(2001)Eur J Pharm Sci 12 (4); 447-52
3Turpeinen M.I enter.(2004)Drug Metab Distribution 32 (6); 626-631
4The Return of the DJI enter.(1988)Br J Clin Pharmacol 26 (1); 23-29
5Dierksa EAI enter.(2001)Drug Metab Distribution 29 (1); 23-9
6VA eagleI enter.(1998)Br J Clin Pharmacol 45 (2); 107-114
7GC temperamentalI enter.(1999)Xenobiotics 29 (1); 53-75
8nominate A.A.I enter.(2001)Drug Metab Distribution 29 (5); 748-53
9FDA Guidance to Industry - In Vitro Drug Interaction Studies - Cytochrome P450 Transporter and Enzyme Mediated Drug Interactions (2020)
10European Medicines Agency (EMA) guideline on drug interaction testing (adopted in 2012)
11Bjornsson TDI'm in. (2003)Lek Z Dispos 31; 815-832
12Stres DMI'm in. (2000)Drug Metab DistributionS28; 1440-1448

(Video) Cytochrome-P450 Interactions (Mnemonic)

To know more

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