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Cytochrome P450 (CYP) inhibition assay (DDI)

 

 

Readout: % inhibition at single concentration, IC50, or Ki

Cyp Isoforms: CYP3A4, CYP2C9, CYP2D6, CYP2C19, CYP1A2

Controls: Known isoform-specific inhibitors

 

Assay Description:

CYP isoform-specific substrates (see table below) are incubated with human liver microsomes at a single concentration or at a range of test compound concentrations (typically 0.1 – 50 µM).  At the end of the incubation, the amount of parent remaining relative to each substrate is monitored by LC-MS/MS at each of the test compound concentrations. Typical experiments for determining IC50 values involve incubating the substrate at concentrations below its KM. For Ki determinations, both the substrate and inhibitor concentrations are varied to cover ranges above and below the drug’s KM and inhibitor’s Ki.

The MS detection is performed by using a Sciex API 4000 Q trap instrument. Each compound is analyzed by reversed phase HPLC using a Kinetex 2. C18 100Å column (3.0 mm X 30 mm, Phenomenex). Mobile phase – solvent A: water with 0.1% formic acid,  solvent B: acetonitrile with 0.1% formic acid. The amount of parent compound is determined on the basis of the peak area ratio (compound area to IS area).

 

CYPSubstrateInhibitor
1A2PhenacetinNaphthoflavone
2C9DiclofenacSulfaphenazole
2C19OmeprazoleTranylcypromine
2D6DextromethorphanQuinidine
3A4TestosteroneKetoconazole

 

Data Analysis:

IC50 or Ki detemination.

Typically compounds can be categorized as follows:

Potent inhibition: IC50 < 1 µM

Moderate inhibition: 1 µM < IC50 < 10 µM

No or weak inhibition: IC50 > 10 µM

 

Abbreviations:

DDI                          Drug-drug interaction

DMSO                     Dimethylsulfoxide

HPLC                       High-performance liquid chromatography

LC                             Liquid chromatography

MS                           Mass Spectrometry

 

Literature: