Relative activity factor

Evaluation of approach to predict the contribution of multiple cytochrome P450s in drug metabolism using relative activity factor effects of the differences in expression levels of NADPH-cytochrome P450 reductase and cytochrome b(5) in the expression system and the differences in the marker activities. Journal of Pharmaceutical Sciences, 91, 952—963. 

With this approach, the ratios of specific (or marker) catalytic activities in human liver microsomes to cDNA-expressed enzymes provide relative activity factors (RAFs). The RAF provides a means to relate the activity of the cDNA-expressed enzyme to the activity of the enzyme in its native environment. RAF is simply a ratio of enzyme activity or the two systems as used by the investigator. The RAF is calculated as ... 

Application of relative activity factors to NNK mutagenicity data prediction of the principal P450 for activation... 

An alternative approach to normalizing rates of drug metabolism by recombinant CYP enzymes is the application of a relative activity factor (RAF), in which the correction is not based on specific content but on specific activity, which requires a comparison of the rate of metabolism of a selective marker substrate by each recombinant CYP enzyme and human liver microsomes (75,194). The RAF is then multiplied by the observed rates of drug metabolism by each recombinant CYP enzyme before assessing the relative contribution of each enzyme to the metabolism of the drug. This approach has not been well validated. For example, it is not known whether the relative activity factor remains constant for several marker substrate reactions catalyzed by the same CYP enzyme. If the relative activity factor varies in a substrate-dependent manner, it would be difficult to know which RAF value to apply to the drug candidate under investigation. Another limitation of this approach is that the relative activity factor must be empirically determined for each lot of recombinant CYP enzyme (and preferably each batch of pooled human liver microsomes). 

Rates of metabolism by recombinant CYP enzymes can be normalized based on specific content or relative activity factor, but neither method reliably predicts the relative contribution of CYP enzymes to reactions catalyzed by human liver microsomes. 

Ohyama, K. Nakajima, M. Nakamura, S. Shimada, N. Yamazaki, H. Yokoi, T. A significant role of human cytochrome P450 2C8 in amiodarone N-deethyla-tion An approach to predict the contribution with relative activity factor. Drug Metab. Disp. 2000, 28, 1303-1310. 

First, the relative activity factor for individual isoforms (using isoform-specific substrates) is calculated. This is necessary as each lot of liver microsome would have different relative amounts of each P450 isoform. Ymax and Km values are determined for each isoform using isoform-specific substrates for both liver microsomes and rCYP. The Relative Activity Factor (RAF) is calculated using the following equation ... 

Venkatakrishnan, K., von Moltke, L. L., and Greenblatt, D. J. (1998) Relative quantities of catalytically active CYP 2C9 and 2C19 in human liver microsomes application of the relative activity factor approach. J. Pharm. Sci. 87 (7), 845-853. 

D.J. Greenblatt (2001). Application of the relative activity factor approach in scaling from heterolo-gously expressed cytochromes P450 to human liver microsomes Studies on amitriptyline as a model substrate. J. Pharmacol. Exp. Then 297, 326-337. 

Stormer E, von Moltke LL, Greenblatt DJ. Scaling drug biotransformation data from cDNA-expressed cytochrome P-450 to human liver a comparison of relative activity factors and human liver abundance in studies of mirtazapine metabolism. J Pharmacol Exp Ther 2000a 295 793-801. 

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