Cytochrome P450 Time dependent inhibition (IC50 shift)

Background: Time dependent inhibition (TDI) gains an increasingly greater attention as a predictor of the drug-drug interaction potential of clinical candidates. IC50 shift assay is a current standard approach for preliminary assessment of TDI. In addition to competitive inhibition of CYP450, some compounds display time dependent inhibition. CYP450 mediated transformation of these compounds results in metabolites which act as reversible inhibitors or modify chemically enzyme (e.g. via covalent bond formation). Their inhibitory potency increases with incubation time. TDI is manifested by difference in IC50 values measured under two different conditions: pre-incubation of a test article with an enzyme and a cofactor NADPH, which produces metabolites, and pre-incubation without NADPH. TDI can be divided into a number of mechanistic categories including irreversible (covalent) modification of the enzyme (mechanism based inactivation), quasi-irreversible (metabolite-intermediate complex), reversible (metabolite is more potent inhibitor than parent), etc. TDI inhibition is of particular importance because it may result in a long-lasting inhibition since usually the enzyme resynthesis is required to recover the CYP450 activity.

Deliverable: Dose-response inhibition curves (8 points, 3-fold serial dilution) of the test compound and the reference inhibitor starting from 100 µM concentration. All test points are performed in duplicates. The IC50 values are calculated using Microsoft Excel and GraphPad Prism software. Data include tables with inhibition % for each compound concentration and SD values, two dose-response inhibition curves (plus/minus NADPH), and IC50 shift value.

Sample Submission: A minimal accurately weighable quantity of dry compound (~2-3 mg or 5 µmol) or 100 µL of 40 mM stock DMSO solution is required for this assay. For multiple assays, lesser amount of compound per assay may be sufficient, which should be discussed for each particular project.