Human liver microsomes reaction phenotyping

Following the identification of metabolic pathways using human liver microsomes, reaction phenotyping studies may be conducted to determine which CYP isoforms are responsible for the metabolism. The active CYP isoforms can he identified using any of the following assay system.s, either alone or in combination ... 

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). 

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

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). 

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. 

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. 

The metabolism of the drug candidate is not measured under initial rate conditions. Prior to initiating reaction phenotyping studies, a pool of human liver microsomes should always be used to establish initial rate conditions (i.e., conditions under which metabolite formation is proportional to protein concentration and incubation time), and total amount of substrate consumed should be less than 10%. 

Rodriques AD. Integrated cytochrome P450 reaction phenotyping Attempting to bridge the gap between cDNA-expressed cytochromes P450 and native human liver microsomes. Biochem Pharmacol. 1999 57 465-480. 

A recent example of UGT reaction phenotyping is that for gemcabene, a Pfizer compound indicated for treatment of dyslipidemia, and cleared primarily via a single glucuronide in humans (Bauman et al., 2005). The strategy employed for UGT reaction phenotyping compound is outlined in Fig. 15.2. Although human liver microsomes were assayed in this example, the same experimental approach could also be used for human intestinal microsomes. A brief summary of the approach and key data follows. 

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. 

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