Cytochrome P450 time dependent inhibition: IC50 shift assay

Aim: To determine time-dependent inhibition of test compound for cytochrome P450 in human liver microsomes by the IC50 shift assay.

Background information: Cytochrome P450 (CYP) inhibition is one of the most common types of drug-drug interactions (DDI). The inhibition mechanism can either be a reversible or irreversible (time dependent) interaction and IC50 shift allows to determine it. Irreversible inhibition is undesired since it usually implies the formation of a covalent bond between the enzyme and the metabolite.

The Cytochrome P450 time dependent inhibition assay determines the IC50 value under three different experimental conditions: 0 min pre-incubation and 30 min pre-incubation with/without NADPH. Following the pre-incubation, isoform-specific substrates are incubated individually with human liver microsomes in a range of test compound concentrations. At the end of the incubation, the formation of metabolite is monitored by UPLC-MS/MS at each of the test compound concentrations. A decrease in the formation of the metabolites compared to the vehicle control is used to calculate an IC50 value (test compound concentration which produces 50% inhibition).If the compound is a time-dependent inhibitor a shift to the left (increase in potency) occur between the 30 min pre-incubation without and with NADPH. The ratio of these values gives the IC50 shift.

Compound requirement: Dependent on number of isoforms assessed (2 mg / 1 CYP)

Turnaround time: 1 week / 1 compound / 1 isoform

Experimental design:

  • Test compound concentration: 8 different concentrations available
  • Number of replicates: 2
  • Pre-incubation time: 30 min (+/- NADPH) and 0 min
  • CYP isoforms: CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 (other isoforms available)
  • Controls: known isoform specific inhibitors
  • Analytical method: UPLC-MS/MS quantification

Delivered results:

  • IC50 shift for the CYP isoform tested

Dear colleagues, collaborators, and friends,

After more than 30 years of shared endeavours, in which we have grown personally and professionally, and with our emotions on the surface, we announce the closure of our company. We have travelled together through ups and downs, celebrated successes and overcome challenges as a team.

We extend our deepest gratitude to each of you for your dedication, passion, and commitment to a shared goal: discovering new treatments for the benefit and well-being of patients.

As we bid farewell, let’s cherish the memories we’ve created, the bonds we’ve formed, and the impact we’ve made together. Though our paths may diverge, the spirit of collaboration and camaraderie that defines our community will always endure.

We leave proud of the work accomplished and thank you for being an integral part of our journey, wishing you success in all your future challenges.

With heartfelt appreciation,

Welab’s team

We are proud to announce that Welab is partner of this new EU funded project.

In collaboration with 12 partners from 6 countries, Welab Barcelona is establishing the Antivirus Pandemic Preparedness EuropeAn pLatform (APPEAL), a European research initiative aimed at enhancing preparedness for future pandemics. This EU funded collaboration will establish a comprehensive program for the development of broad-spectrum antiviral drugs within a five year time frame ensuring drug affordability and accessibility to low income countries.
Link to the press release: https://lnkd.in/d-jmV2CS