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CYTOCHROME P450 INHIBITION STUDIES USING FDA RECOMMENDED
PROBE SUBSTRATES IN HUMAN LIVER MICROSOMES
Many drug-drug interactions are metabolism based and mediated primarily via the Cytochrome P450 (CYP) family of enzymes. Ten
CYP isoforms are expressed in a typical human liver (CYP1A2, CYP2A6, CYP2B6, CYP2C8/9/18/19, CYP2D6, CYP2E1, and
CYP3A4). The inhibition of these enzymes may have important clinical consequences as inhibition of a CYP isoenzyme(s) by a
xenobiotic may decrease the metabolic clearance of a co-administered drug resulting in elevated blood concentrations of the drug
leading to adverse drug effects or toxicity. Early assessment of an NCE’s ability to inhibit the activity of a particular CYP subtype can
be achieved using human liver microsomes.
In accordance with the FDA Draft Guidance for Drug-Drug Interactions Definitive CYP Inhibition Studies are carried out as follows: •
Assays are performed in 96-well microtiter plates with pooled human liver microsomes.
12 concentrations of test compound are incubated together with probe substrate in the presence of NADPH, and the inhibition of metabolite formation by each CYP subtype is determined.
A known inhibitor for each CYP subtype is run in parallel as a positive control.
All incubations are performed in triplicate.
Metabolite formation for each CYP subtype in the presence and absence of test compound is measured by validated LC/MS methods.
Where possible, the IC50 value (concentration which reduces the metabolism of the probe substrate by 50%) will be determined for the test compound for each CYP subtype.
As an example, to assess the inhibition of CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4 activity, the known selective inhibitors were incubated together with their respective probe substrates, in pooled human liver microsomes in the presence of NADPH. The IC50 of each reference inhibitor for the CYP 450 enzyme subtype was determined as a
measure of the inhibition of probe substrate metabolite formation. The IC50 of each reference compound is presented in the graphs
below and in the table above. These values are consistent with data reported in the literature2.
USFDA (2006) Draft Guidance for Industry: Drug Interaction Studies-Study Design, Data Analysis, and Implications for Dosing and Labeling, U.S. Food and Drug Administration Publication
2. Walsky and Obach, Drug Metabolism and Disposition, 2004; 32: 647-660
NoAb also offers a complementary activity and mRNA based CYP450 induction assay, to evaluate the CYP1A2, CYP2B6, and CYP3A4
induction potential of NCEs. All of these services are examples of NoAb’s commitment to providing the best drug discovery tools for our
clients, helping to shape drug discovery.
For more information please contact us at:
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