Cytochrome P450 (CYP) inhibition assay (DDI)

• Cytochrome P450 (CYP) represents a family of isozymes which play a major role in the metabolism of drugs. CYPs are located in the inner membrane of mitochondria and in the endoplasmic reticulum of cells in several tissues.
• Although more than 50 individual CYPs have been identified, the most important enzymes involved in drug metabolism are CYP3A4, CYP2C9, CYP2D6, CYP2C19, and CYP1A2.¹

• CYP450 enzyme polymorphism is responsible for observed variations in drug response among patients. Recognizing whether drugs act as enzyme substrates, inducers, or inhibitors can prevent clinically significant interactions from occurring.

• The human liver microsomal assay is accepted as the ‘gold standard’ for in vitro DDI assessment as it is felt to be closest to the native enzyme environment without the batch variability, potential transporter involvement, and cell penetration complications associated with human hepatocytes.² In vitro data are generally very predictive of DDI in vivo.³

Readout: % inhibition at single concentration, IC50 or Ki

Cyp Isoforms: 1A2, 2B6, 2C8, 2C9, 2C19, 2D6, and 3A4

Controls: Known isoform-specific inhibitors

CYP	Substrate	Inhibitor
1A2	Phenacetin	Naphthoflavone
2B6	Bupropion	Ticlopidine
2C8	Paclitaxel	Monteleukast
2C9	Diclofenac	Sulfaphenazole
2C19	Omeprazole	Tranylcypromine
2D6	Dextromethorphan	Quinidine
3A4	Testosterone	Ketoconazole

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).

Data Analysis:

IC50 or Ki determination.

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:

1. Slaughter, R. L.; Edwards, D. J.; “Recent advances: the cytochrome P450 enzymes”,   Pharmacother. 29, 619, (1995).
2. Fowler, S.; Zhang, H.; “In Vitro Evaluation of Reversible and Irreversible Cytochrome P450 Inhibition: Current Status on Methodologies and their Utility for Predicting Drug–Drug Interactions”, AAPS J. 10, 410, (2008).
3. Obach, R. S., et al. “The Utility of in Vitro Cytochrome P450 Inhibition Data in the Prediction of Drug-Drug Interactions”; J. Pharmacol. Exp. Ther. 316, 336, (2006).

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