15.2 Cytochrome P450 Reaction Phenotyping
Many enzyme systems exist that can contribute to the clearance of drugs from
the systemic circulation thereby influencing a NCE or its metabolite(s)’
systemic exposure and, therefore, its overall efficacy and adverse effect profiles.
Most commonly, it is the P450 class of enzymes governing drug clearance via
processing within the liver, the most important organ in the elimination of
drugs and other xenobiotics. This section will highlight perspectives on
identification strategies for determining the contribution that various P450
enzymes may have upon a drug’s disposition.
The enzymes in the P450 family are membrane proteins expressed in the
endoplasmic reticulum of mammalian cells with their most significant
location, based on expression levels, being hepatocytes. More than 50 P450
enzymes are known to exist in the human genome, however, fewer than 10
are known to be expressed sufficiently in human liver to have the potential
to play a significant role in xenobiotic metabolism. Two important factors
in governing the potential impact that a P450 enzyme has across drug
classes is its tissue expression level and substrate specificity. The most
important human P450 enzymes involved in drug metabolism are CYPs
1A2, 2C9, 2C19, 2D6, and 3A4 with an emerging understanding that CYPs
2B6, 2C8, and 3A5 may play a role in the metabolism of certain
therapeutics. CYPs 2A6 and 2E1 play only a minor role in xenobiotic
metabolism. While the CYPs 2D6, 3A4, and 2C9 constitute approximately
50% of total hepatic P450 protein, these three enzyme metabolize nearly
80% of therapeutic drugs (Shimada et al., 1994; Smith et al., 1998). In
certain situations larger than usual interindividual variability (greater than
twofold) in drug exposure may arise due to the polymorphic expression of
enzymes (CYPs 2C9, 2C19, 2D6) that drive a clearance-governing
elimination pathway for a drug. For example, clinical studies have shown
where CYP2D6 is estimated to contribute to more than 50% of a drug’s
clearance, reported exposure (area under the curve, AUC) ratios of
extensive/poor metabolizers can range as high as 50. For example, systemic
exposure to the proton pump inhibitors as expressed by the AUC (area
under the plasma level time profiles) is 5–12 times higher in poor
metabolizers than in extensive metabolizers (Klotz et al., 2004).
P450 metabolic reaction phenotyping to determine the relative contributions
for specific P450 enzymes for metabolic clearance should use (1) chemical
inhibition in collaboration with either (2) cDNA expressed systems, and/or (3)
the utilization of reaction rate determination across a phenotypically
characterized panel of microsomes from multiple individual donors (correla-
tion analysis) (Ring and Wrighton, 2000).
There exist some general considerations across in vitro reaction
phenotyping studies for P450s and non-P450s that need to be anticipated
in the process of defining incubation conditions. While protocol-specific
guidance (Method Sheets 1 and 2) is a part of the latter sections of this
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