Recombinant CYPs

Other assays for assessing CYP clearance are also employed, although often less widely or with lower compound throughput. Recombinant CYP enzymes allow the determination of the kinetic parameters for metabolism of individual compounds by individual CYPs. Recombinant CYPs also provide an avenue to assessing and understanding the potential for drug-drug interactions that may occur between two or more compounds. 

Comparative study of fluorescence CYPs assays of niclosamide with that obtained by conventional HPLC assay using human liver microsomes and recombinant CYPs was developed [72]. 

Several kinetic parameters can be measured on different experimental systems to account for the interaction of a compound with CYPs. For example when studying the metabolic stability of a compound, it could be measured in a recombinant CYP system, in human liver microsomes, in hepatocytes and so on. Each system increases in biological complexity. Although in the recombinant CYP system only the cytochrome under consideration is studied, in the case of the human liver microsomes, there is a pool of enzyme present that includes several CYPs, and finally in the hepatocyte cell system, metabolizing enzymes play an important role in the metabolic compound stability. In addition, transport systems are also present that could involve recirculation or other transport phenomena. The more complex the experimental system, the more difficult it is to extract information on the protein/ligand interaction, albeit it is closer to the in vivo real situation and therefore to the mechanism that is actually working in the body. 

XenoTech offers a selection of services for drug metabobsm-related research including liver and pulmonary microsomes and S9, cryopreserved hepatocytes from human and six other relevant species, antibodies directed against CYP enzymes, recombinant CYPs, and bDNA probe sets (322). 

Subsequent studies with DFP indicated that the human oxidative pathways were catalyzed by CYP3A and CYP1A, as demonstrated by turnover with recombinant CYP enzymes. Induction studies in the human hepatocyte model demonstrated that human CYP3A was not significantly induced by DFP. Therefore, despite the autoinduction observed in rats, this compound was carried forward into clinical trials. 

Table 4 Comparison of the Apparent Affinity (Km) with Which HLM and Recombinant CYP Enzymes (Bactosomes) Catalyze CYP-Selective Reactions...

As in the case of chemical inhibition, a lack of specificity can complicate the interpretation of antibody inhibition experiments. A lack of specificity and the nonspecific effects outlined above likely account for the majority of cases where the sum of the inhibitory effects of a panel of inhibitory antibodies adds up to greater than 100%. Another potential problem stems from the fact that many antibodies do not completely inhibit the activity of a microsomal CYP enzyme or the corresponding recombinant CYP enzyme. If an antibody inhibits the metabolism of a marker substrate by 80%, and if the same antibody inhibits the metabolism of drug candidate by 80%, there is uncertainty as to whether the inhibited enzyme contributes 80% or 100% to the metabolism of the drug candidate. This may seem like a trivial difference, but it has a... 

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. 

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). 

The FDA guidance document recognizes the difficulty of extrapolating the results obtained with recombinant enzymes to the situation in liver microsomes. Experiments with recombinant CYP enzymes provide valuable information on which CYP enzymes can and which ones cannot convert a drug candidate to a particular metabolite, and this information alone is particularly valuable in guiding the design or interpretation of correlation analysis, chemical inhibition, and antibody inhibition experiments. 

Compared with their microsomal counterparts, recombinant CYP enzymes may have a different K,n for marker substrates or a different K1 for chemical inhibitors. 

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. 

The X-ray structure of the complex of the D-Ser cholinyl ester of CsA and human recombinant CyP-B has been reported.212 Figure 10 compares this structure with that of CsA bound to CyP-A. The proteins are depicted in ribbon form. Overall, the structures of the two cyclophilins are closely similar. Both the N- and C-termini of CyP-B are extended relative to CyP-A. The N-terminal -strand of CyP-B bends toward the -barrel ... 

Individual drug metabolizing enzymes DMEs) like CYPs, FMOs, and UGTs are now routinely expressed, usually in insect cells using a baculovirus. Ultracentrifugation provides vesicles called microsomes, which contain these membrane-bound DMEs. One popular type of recombinant CYP preparation called Supersomes comes with CYP reductase, a necessary cofactor, co-expressed. Not only individual CYPs (2B6, 3A4, etc.) but also allelic variants like CYP2C9 2 are available for the study of possible pharmacogenomic effects on metabolism. 

Yamamoto T, Itoga H, Kohno Y, Nagata K, Yamazoe Y. Prediction of oral clearance from in vitro metabolic data using recombinant CYPs comparison among... 

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