In vitro CYP reaction

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). 

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

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)-... 

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... 

Irreversible inhibition of CYPs is particularly worrisome as its consequences cannot be predicted easily or quantified from in vitro data the in vivo effect of an irreversible inhibitor is usually greater than that predicted based on affinity alone. Moreover, irreversible inhibition is generally the consequence of the production of reactive metabolites (electrophiles), which can also bind covalently to endogenous proteins and, in rare cases, trigger serious autoimmune reactions [4]. 

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. ° ... 

Human Biologies International offers various in vitro data, screens (Hepato-Screen ), kits, and analysis software (HepatoSoft ) for CYP inhibition, reactions, and stability (323). 

The in vitro approaches feature human liver microsome incubations that contain drug candidates at a range of concentrations that span the anticipated maximum steady state plasma levels. The microsomal incubations also contain a specific probe substrate where the concentration closely approximates that Km value for the reaction under investigation. Quantitative analysis of the specific marker metabolites and internal standards using MRM provides a simple assessment of the potential inhibitory effects drug candidates have on the metabolism of specific CYP probe substrates. 

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... 

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. 

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