Cytochrome P450 Inhibition Assay

Human cytochrome P450 (CYP450) enzymes play an essential role in drug metabolism. Even subtle changes in the activities of CYP enzymes may cause severe drug-drug interactions. Creative Bioarray aims to help our clients with evaluating the interaction potential of your compounds.

CYP450 Inhibition Assay Introduction

  • Why CYP450 inhibition assay?
    • Cytochrome P450s (CYPs) are a family of enzymes that play a significant role in drug metabolism.
    • A common consequence of multiple medications is an increase in adverse drug events, mainly from drug-drug interactions. Most currently available drugs are metabolized by CYP450 enzymes. Interactions caused by CYP450-mediated metabolic pathways due to the sharing of multiple drugs are common, especially through reversible or irreversible CYP450 inhibition.
    • The inhibition of CYPs is associated with an increase in the incidence of clinical drug-drug interactions (DDI). CYP450 inhibition data can be used to design strategies to study clinical DDI studies.
  • CYP450 inhibition mechanisms

CYP450 enzymes are one of the main enzymes for drug clearance. Drugs can occupy many active sites of drug enzymes or weaken the activity of drug enzymes, slow down the metabolism of other drugs or themselves, increase blood drug concentration, and improve drug efficacy, but may enhance side effects.

Drug interactions due to drug metabolism inhibition are frequent since (1) CYP450-mediated metabolism is the primary route of elimination for a large number of drugs, and (2) multiple drugs can compete for the same CYP450 active site. Mechanisms of CYP450 inhibition can be categorized as:

  • Reversible Inhibition
    • Competitive
    • Non-competitive
  • Irreversible/quasi-irreversible (mechanism-based inhibition)

CYP450 inhibition mechanismsFigure 1. CYP450 inhibition mechanisms(Deodhar et al.,2020).

Brief Protocol

Various cytochrome P450 isoforms have been studied in the CYP450 inhibition test. The isoform-specific substrate is incubated with human liver microsomes and a series of test compound concentrations separately. At the end of the incubation, the formation of metabolites was monitored by LC-MS/MS at each test compound concentration.

The reduction in metabolite formation compared to the vehicle control is used to calculate the IC50 value (the concentration of test compound that produces 50% inhibition).

Test PlatformCryopreserved human hepatocytes (3 donors)
CYP IsoformsCYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 (other isoforms are available on request)
Measurement MethodLC-MS/MS
DataIC50, Standard error of IC50

Quotation and Ordering

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  1. Deodhar; et al. "Mechanisms of CYP450 Inhibition: Understanding Drug-Drug Interactions Due to Mechanism-Based Inhibition in Clinical Practice." Pharmaceutics, vol. 12, no. 9, Sept. 2020, p. 846.
  2. EMA. Guideline on the investigation of drug interactions (June 2012)

For research use only. Not for any other purpose.