chapter, it is important to generally consider the concentration of NCE, the
nature of the buffer system, the tolerance level of needed organic solvent,
and the duration of the incubation experiment. It is generally wise to
perform metabolic reaction phenotyping experiments using therapeutically
relevant concentrations. Concentration considerations may include plasma
and/or microsomal free versus bound drug, Cmax versus Cmin,systemic
versus portal drug concentrations (oral), and intrahepatic concentration (Ito
et al., 1998; Obach, 1997, 1999). Commonly employed buffers include Tris
or potassium or sodium phosphate at pH 7.4 to recreate conditions
representative of thein vivosituation regardless of the differing optimal
conditions for individual P450 enzymes. Organic solvents such as methanol
or acetonitrile may be needed to aid in compound dissolution, however,
sufficient levels of these solvents diminish enzyme activity so it is a good
practice to optimize solvent concentrations below 1% for microsomal
preparations (Busby et al., 1999; Chauret et al., 1998; Hickman et al., 1998).
Other commonly used solvents such as dimethyl sulfoxide (DMSO) should
be optimized for the specific enzyme system (generally less than 0.1–0.2%).
Experimental duration will depend upon time viability of the enzyme system
(45–60 min typical for hepatic microsomes) balanced against the time
needed to reliably detect changes in parent drug or metabolite
concentration.
Both microsomal and recombinant expressed systems are commercially
available and can be used in reaction phenotyping experiments as they contain
activities for the major P450s previously highlighted. The NCE is incubated in
the desired system and the concentration of parent (substrate depletion) or
metabolite (metabolite formation) concentrations determined as a function of
time. Linear initial rates, in regard to both enzyme concentration and time, are
desired for most appropriate kinetics but these are not always obtained as
multiple enzymes may be functioning in parallel or test article may be
sufficiently stable to prevent an accurate measure of disappearance. Therefore,
the substrate depletion method is most appropriate when sufficiently short
in vitrohalf-life compounds are being assessed and kinetics may be confidently
derived. A metabolite formation rate approach may be needed when relatively
stable compounds are evaluated and parent loss would not be detected with
statistical confidence (Obach and Reed-Hagen, 2002). Individual metabolite
formation methods work best when authentic metabolite standard or
radiolabel is available (again striving for linear initial reaction conditions);
this is often not available during the earlier drug discovery phases. The likely
contributions of each specific P450 enzyme to humanin vivoclearance may
then be estimated by combining typical hepatic expression levels of the
governing P450s and the respective enzyme kinetics of parent; this can then be
extended to anin vivoclearance prediction (Stormer et al., 2000b). If a bank of
phenotyped microsomes is available, the selection of donors representing a
range of activities can be used as additional confirmation (Williams et al.,
2003).
480 REACTION PHENOTYPING