P450 Induction

Purpose

In the U.S. FDA Guidance for Industry: "Drug Metabolism/ Drug Interaction Studies in the Drug Development Process: Studies In Vitro", it is recommended that the ability of a new drug entity to induce cytochrome P450 isozymes be assessed. One method to accomplish this goal is to treat human hepatocyte cultures with the test article and to measure the expression and the activity of the individual isozymes compared to controls. Carrying out this assay during the discovery phase is one fast and cost-effective means of eliminating leads with low potential for success.

Assay Protocol

Human hepatocytes are obtained from commercial sources plated on collagen coated plates and maintained in Modified Williams' E medium supplemented with dexamethasone, insulin and gentamicin. The hepatocytes are treated in triplicate with rifampicin (50 mM), ß-napthoflavone (50 mM) and two concentrations (1 and 10 mM) of test article for three days. The cells are maintained at 37°C in a humid atmosphere containing 5% CO₂.

At the end of the treatment period, the hepatocytes are incubated with the probe substrates to determine the activity of the enzyme of interest (see Table 1 below for the list of probe substrates and the concentrations used) for 30 to 60 minutes. The medium is removed for quantification of the metabolite and the cells harvested for protein analysis. The samples are analyzed by HPLC or LC-MS/MS and the amount of metabolite formed is quantified by the use of a fourpoint standard curve.

Confirmation Data

The induction of various drug-metabolizing enzymes was characterized in four cultures of human hepatocytes. Representative data of the effect of rifampicin on CYP3A4 activity is shown below and is in agreement with the data published by Ramachandran et al., DMD 1999, 27, 1194-1199.