Subtype Inhibition Studies
Background
Many drug-drug interactions are metabolismbased and of these, most involve Cytochrome
P-450 (CYP) enzymes. Eleven xenobiotic
metabolizing CYPs are expressed in a typical
human liver (CYP1A2, CYP2A6, CYP2B6,
CYP2C8/9/18/19, CYP2D6, CYP2E1 and CYP
3A4/5). Of these, six principal enzymes (CYP1A2,
CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A4)
appear to be the most commonly responsible for
the metabolism of most drugs and the associated
drug-drug interactions. This is primarily due to
the preference of these enzymes to bind and/or
metabolize chemical structures commonly found
in drugs, and due to the mass abundance of some
of these enzymes in human liver.
Key Features of the Assay
• available for the following nine enzymes:
CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9,
CYP2C19, CYP2D6, CYP2E1 and CYP3A4
• rapid throughput fluorescence-based assay
Assay Applications
• identifies which metabolizing enzyme a drug is
interacting with
• predicts the potential for a new drug to interact
with co-administered drugs
Assay Principle
The assays use microsomes prepared from
insect cells, each expressing an individual CYP
subtype (CYP1A2, CYP2A6, CYP2B6, CYP2C8,
CYP2C9*1, CYP2C19, CYP2D6*1, CYP2E1 or
CYP3A4) expressed from the corresponding human
CYP cDNA using a baculovirus expression vector.
The assays monitor, via fluorescence detection,
the formation of a fluorescent metabolite following
incubation of the microsomes with a specific CYP
substrate. Because inhibition constants are substrate
selective for CYP3A4, at least two substrates are
commonly examined for this enzyme. This rapid
throughput assay does not distinguish between a
substrate and inhibitor. Further studies can be
performed to characterize substrates and inhibitors.
Assay Protocol
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