Recombinant Human CYP Enzymes

VmaxHLM (nmol/min/mg HLM) VmaxthCYP (nmol/min/pmol rhCYP) 

Ho vever this approach does not address inter-individual variability in CYP expression nor the apparent substrate specificity of RAFs. This may be overcome through the use of intersystem extrapolation factors (ISEFs) vhich compare the intrinsic activities of rCYP versus liver microsomes and provide CYP abundance scaling by mathematical means. This employs the RAF approach and adjusts for the actual amount of liver microsomes CYP present (measured by immunochemistry) rather than a theoretical amount (Equation 8.4). Such corrections can be made using nominal specific contents of individual CYP proteins in liver microsomes or more appropriately employ modeling and simulation software (e.g., SIMCYP www.simcyp.com) which takes into account population-based variability in CYP content. 

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Human liver microsomes and recombinant human CYP enzymes are the preferred test systems because kinetic measurements are not confounded by cellular uptake and other metabolic processes present in hepatocytes and liver slices. 

Table 5 Direct Inhibition of CYP Enzymes in Human Liver Microsomes and Two Different Preparations of Recombinant Human CYP Enzymes... 

Recombinant human CYP enzymes expressed in bacteria (Bactosomes from Cypex) Recombinant human CYP enzymes expressed in insect cells (Supersomes from BD-Gentest) Chloromethyl fluorescein diethylether (216). 

In experiments with recombinant human CYP enzymes, the rate of formation of a metabolite by multiple CYP enzymes does not provide adequate information on the relative importance of the individual pathways. 

Determining Km values with recombinant human CYP enzymes can be used to differentiate high and low affinity enzymes involved in the metabolism of a drug candidate. 

Metabolism by purified or recombinant (cDNA-expressed) human CYP enzymes, which can establish whether a particular CYP enzyme can or cannot metabolize a drug candidate, but it does not address whether that CYP enzyme contributes substantially to reactions catalyzed by human liver microsomes. The information obtained with purified or recombinant human CYP enzymes can be improved by taking into account large differences in the extent to which the individual CYP enzymes are expressed in human liver microsomes, which is summarized in Table 8, and by determining the... 

A combination of at least three approaches (correlation analysis, recombinant human CYP enzymes, and either chemical or antibody inhibition) is highly recommended to identify which human CYP enzyme or enzymes are responsible for metabolizing a drug candidate. 

Cytochrome b5 affects the kinetics of drug metabolism by certain CYP enzymes hence, coexpression of this microsomal hemoprotein (together with NADPH-CYP reductase) can affect the catalytic efficiency of certain recombinant CYP enzymes (76,109). For example, the presence of cytochrome b5 tends to increase Fmax for reactions catalyzed by CYP3 A4, whereas it tends to decrease Km for reactions catalyzed by CYP2E1. In both cases, cytochrome b5 increases Vmax/Km, which is a measure of in vitro intrinsic clearance. The fact that some commercially available recombinant CYP enzymes are expressed with cytochrome b5 while others are not complicates the interpretation of results of studies performed with recombinant human CYP enzymes. 

Figure 27 Metabolism of DB289 (antiparasitic prodrug) by a panel of recombinant human CYP enzymes. Abbreviation CYP, cytochrome P450.

Recent work on the epoxidation of EPA and DHA using recombinant human CYP enzymes has shown that the predominant products were 17,18-EETeTr and 19,20-EDP, respectively. Interestingly, a concomitant reduction in AA-derived epoxidase derivatives was observed, suggesting that some of the EPA and DHA observed effects may be attributed to a shift in the generation of such metabolites (Van Rollins et al. 1988 Fer et al. 2008). 

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