Reaction phenotyping

LC/MS/MS with selected reaction monitoring (SRM) offers a fast and simple means to analyze biological matrices, which is a key factor in high-throughput CYP inhibition screens using liver microsomes. Potentially, the LC/MS/MS technique is suitable for analyses of cocktail substrates in other in vitro drug metabolism evaluations such as CYP induction/activation assays, rapid analysis of pooled liver microsomes, rapid reaction phenotyping of tissue (hepatic and extrahepatic) samples, as well as evaluation of hepatocytes/tissue slice CYP activity. ° ... 

Drug-Drug Interactions Victims of Interaction Reaction Phenotyping 179... 

Reaction phenotyping is the semi-quantitative in vitro estimation of the relative contributions of specific drug-metabolizing enzymes to the metabolism of a test compound. It has been well documented that approximately two-thirds of marketed drugs are metabolized by members of the GYP superfamily, in particular the... 

Zhang, H., Davis, C.D., Sinz, M.W. and Rodrigues, A.D. (2007) Cytochrome P450 reaction-phenotyping an industrial perspective. Expert Opinion on Drug Metabolism and Toxicology, 3, 667-687. 

Williams, J.A., Hurst, S.I., Bauman, )., Jones, B.C., Hyland, R., Gibbs, J.P., Obach, R.S. and Ball, S.E. (2003) Reaction phenotyping in drug discovery moving forward with confidence Current Drug Metabolism, 4, 527-534. 

Grubb, M. F., Shipkova, P., Langish, R., and Josephs, J. L. (2005). Rapid determination of human liver metabolism reaction phenotype using LC/MS/MS. In Proceedings of the 53rd ASMS Conference on Mass Spectrometry and Allied Topics, San Antonio, TX. 

A recent paper by Chauret et al. described the discovery of a novel fluorescent probe that is selectively metabolized by CYP3A in human liver microsomes (32). This probe, DFB [3-[(3,4-difhiorobenzyl)oxy]-5,5-dimethyl-4-[4-(methylsulfonyl) phenyl] furan-2(5F/)-one], is metabolized to DFH [3-hydroxy-5,5-dimethyl-4-[4-(methylsulfonyl)phenyl]furan-2(5//)-one], which has fluorescent characteristics (Fig. 7). In vitro CYP reaction phenotyping studies (cDNA-expressed CYP proteins and immunoinhibition experiments with highly selective anti-CYP3A4 antibodies) demonstrated that DFB was metabolized primarily by CYP3A4 (Fig. 8). Furthermore, metabolism studies performed with human liver microsomes obtained from different donors indicated that DFB dealkylation and testosterone 6P-hydroxylation correlated well (Fig. 9). 

The use of positive controls is optional, but the FDA has nevertheless developed a list of preferred and acceptable inhibitors for use in reaction phenotyping studies that can be applied to CYP inhibition... 

Positive control inhibitors for each of the major CYP enzymes should also be included to further demonstrate that the test system is performing as expected. The direct-acting inhibitors used in our laboratory are summarized in Table 7, along with the IC50 values determined during assay validation and a comparison with literature values. It is worth noting that the positive control inhibitors used for CYP inhibition studies need not necessarily be CYP-selective inhibitors, in contrast to those used for reaction phenotyping, which should be CYP-selective inhibitors. 

III. IDENTIFICATION OF CYP ENZYMES INVOLVED IN A GIVEN REACTION REACTION PHENOTYPING... 

The regulatory perspective will be covered in greater detail in chapter 16. This section will briefly highlight the latest recommendations regarding in vitro reaction phenotyping studies provided by the FDA (1-3). The FDA notes that one way to approach such studies is to first determine the metabolic profile of a drug and estimate the relative importance of CYP enzymes. It is recommended that preliminary experiments be conducted with human hepatocytes (or liver slices) followed by LC/MS/MS analysis to directly characterize the metabolites formed, and their relative importance. The relative importance of CYP enzymes... 

If human metabolism studies with radiolabeled drug have not been performed prior to the conduct of reaction phenotyping studies, the initial experiments should use as complete an in vitro test system as possible, depending on the drug (e.g., tissue homogenates, liver slices, hepatocytes, etc.). 

CYP enzyme reaction phenotyping can be performed with human liver microsomes. 

If one or more major circulating metabolites contribute significantly to the pharmacological action of a drug or if there are safety issues associated with such metabolites, reaction phenotyping for the individual metabolites should be considered. 

Once an analytical method (e.g., LC/MS/MS) is established, it is necessary to qualify or validate the procedure from a regulatory GLP perspective. The desired criteria for method validation/qualification include determining the lower and upper LOQ, inter- and intraday precision, specificity of the method, and linearity of the calibration curves (166). Validation/qualification must be performed in the presence of the representative biological matrix that will be used in reaction phenotyping. For CYP reaction phenotyping studies, the matrix of choice is a pool of human liver microsomes (166). 

Step 2 involves an assessment of whether metabolite formation is proportional to incubation time and protein concentration under conditions that will be used for subsequent reaction phenotyping experiments. The goal is to establish in vitro... 

Correlation analysis is one of the four basic approaches to reaction phenotyping. It involves measuring the rate of drug metabolism by several samples of human liver microsomes (at least 10, according to the FDA) and correlating reaction rates with the variation in the level or activity of the individual CYP enzymes in the same bank of microsomal samples. This approach is successful because the levels of the CYP enzymes in human liver microsomes vary enormously from sample to sample (up to 100-fold), but with judicious selection of individual samples, they can vary independently from each other. 

Chemical and antibody inhibition represent the second and third approaches to reaction phenotyping. They typically involve an evaluation of the effects of known CYP enzyme inhibitors or inhibitory antibodies against selected CYP enzymes on the metabolism of a drug candidate by pooled human liver micro-somes. As in the case of correlation analysis, chemical and antibody inhibition experiments must be conducted with pharmacologically relevant concentrations of the drug candidate in order to obtain clinically relevant results. 

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