CYP reaction phenotyping

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). 

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). 

Table 10 Advantages and Disadvantages of the Various Approaches to In Vitro CYP Reaction Phenotyping...

Ideally, definitive CYP reaction phenotyping should be available before the initiation of clinical development. Unfortunately, more accurate data can only be obtained once clearance pathways are identified in human subjects, and human radiolabeled studies are generally not conducted as the first set of clinical studies for NCEs. In this context, CYP reaction phenotyping, performed using various human in vitro systems is expected to be as complete as possible (Bjornsson, 2003). 

FIGURE 5.4 Proposed clinical drug interaction studies based on in vitro CYP reaction phenotyping data. It is assumed that the drug in question is cleared via CYP-dependent metabolism (/m LO)-... 

Reaction phenotyping is the study that measures quantitative contribution of a DME to total clearance of a given drug. The CYP reaction-phenotyping process requires the integration of data obtained from various in vitro systems... 

DDI CYP inhibition (rCYP, HLM) (see Section 6.3.2.3) CYP induction (PXR-TA, Fa2N-4, hepatocytes) (see Section 6.3.2.3) CYP reaction phenotyping Assessing in vitro CYP inhibition and induction potentials Predicting in vivo DDIs Assay (HLM CYP inhibition) ° Human hve microsomes ° 0.5-100 rM probe concentration depending on each isozyme Analysis ° PPT followed by LC-MS/MS or onhne SPE-MS/MS... 

Metabolic stability assays can also be utilized to perform CYP reaction phenotyping, namely the identification of specific CYP isozymes responsible for the metabolic reaction [74], Two approaches are commonly used to perform reaction phenotyping by metabolic stability assays. One approach tests the metabolic stability of NCEs in individual human recombinant CYP enzymes, whereas the other incubates NCEs with regular human liver microsomes (HEM) along with specific inhibitors for each isozyme. The reaction phenotyping results are useful in determining whether the test compounds are substrates of specific CYP isozymes, and therefore, whether they are likely to be susceptible to DDIs mediated by those isozymes. 

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. ° ... 

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... 

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

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. 

The FDA-approved and acceptable chemical inhibitors for reaction phenotyping are included in Table 2. Many of the inhibitors listed in Table 2 are metabolism-dependent inhibitors that, in order to inhibit CYP, require preincubation with NADPH-fortified human liver microsomes for 15 minutes or more. In the absence of the metabolism-dependent inhibitor, this preincubation of microsomes with NADPH can result in the partial, spontaneous loss of several CYP enzyme activities (see sec. II.C.7.c). Furthermore, the organic solvents commonly used to dissolve chemical inhibitors can themselves inhibit (or possibly activate) certain CYP enzymes, as discussed in section II.C.4. Therefore, appropriate solvent and preincubation controls should be included in all chemical inhibition experiments. 

One of the complicating factors with chemical inhibitors is that a chemical that inhibits one CYP enzyme may activate another enzyme. If both enzymes contribute to metabolite formation, the inhibitory effect of the chemical on one enzyme may be offset by its activating effect on the other enzyme. a-Naphthoflavone is an inhibitor of CYP1A2 but an activator of CYP3A4, whereas quinidine is an inhibitor of CYP2D6 but, in certain cases, an activator of CYP3 A4 (184-186). a-Naphthoflavone and quinidine both appear on the list of FDA preferred and acceptable chemical inhibitors, so their ability to inhibit one enzyme but activate another are relevant to reaction phenotyping. 

---