CYP450 Reaction Phenotyping
Identification of the specific CYP450 enzymes responsible for in vitro metabolism can be used to help predict the potential for drug interactions, the polymorphic impact on drug disposition, and the formation of toxic or active metabolites. Likewise, evidence that certain metabolic pathways are not important via in vitro studies may preclude the need for further clinical investigations.
The three proven methods to identify CYP enzymes responsible for a drug's metabolism use:
1. Specific chemical inhibitors or antibodies as specific enzyme inhibitors
2. Individual human recombinant CYP enzymes
3. Human liver microsomes prepared from individual donors livers and characterized for CYP activity
The USFDA recommends that at least two of the three methods be performed to identify the specific enzyme(s) responsible for a drug's metabolism.
At CellzDirect, all experiments are conducted under initial rate conditions when analytical conditions allow and at the appropriate test article concentrations.
Method 1
In this method, the kinetic parameters (Km and Vmax) for CYP450 metabolism are determined for the major CYP450s 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1 and 3A4 using pooled human liver microsomes and the test article in the presence and absence of specific chemical inhibitors.
Method 2
In method 2, the kinetic parameters are determined using cDNA expressed CYP isozymes for the major CYP450s 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1 and 3A4 and metabolism of the test article is monitored.
Method 3
For correlation analysis, a bank of characterized microsomes from individual livers are used and the kinetic parameters of metabolism are calculated. Statistical analyses are performed to establish a correlation between the metabolism rate of the test article and metabolism rate determined for each CYP enzyme in microsomes prepared from the individual donor livers.
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