Factors of Concern Pharmacological or physiological relevance is always a
concern forin vitroexperiments, including microsomal stability assays. The
primary issue is the substrate concentration, but other issues, such as reaction
mixture constituents and the availability of these constituents (such as
NADPH) should also be considered. Microsomal incubations are carried out
either in PBS or in Tris–HCl buffer, while NADPH can be added directly in
excess or through regeneration from NADPin situ. The NADPH-regenerating
system is comprised of NADP+, G6PDH, and the substrate Glc-6-PO 4.
Cofactor regeneration can be more physiologically relevant when it is carried
out in a PBS-based reaction matrix. However, the influence of NADPH source
differences, the effect of different buffers, and the stability of G6PDH on the
rates of CYP-mediated biotransformation are not known (Adediran, 1991).
The selection of reaction conditions will depend on the circumstances. Direct
NADPH addition is a reasonable alternative to the regeneration at early
discovery stages, when the simplicity, reproducibility, and rigidity of the assay
system are usually predominant considerations. Relevant substrate concentra-
tions and the use of the relevant enzymes, in contrast, are essential for the
prediction of pharmacokinetics (Zhang and Wong, 2005). The anticipated drug
levels in the circulation of a patient on therapy frequently dictate the use of low
substrate concentrationsin vitro. The blood concentration, however, may not
always be consistent with that in the tissues, particularly in the liver,in vivo.It
is not uncommon for the hepatic concentrations of some of the therapeutic
agents to be much higher than those in the circulation (Levine et al., 2001;
Sugie et al., 2004). Apparently, pharmacologically relevant concentrations are
compound-dependent, and thus no general concentration standard is
applicable.
Nevertheless, 1 and 10mM are often arbitrarily selected as the initial
concentrations for metabolic stability assays (MacKenzie et al., 2002; Obach
and Reed-Hagen, 2002). The effects of organic solvents on enzyme activities
have been carefully investigated (Busby et al., 1999; Chauret et al., 1998;
Easterbrook et al., 2001). For the purpose of data comparability for enzyme
activities, the levels of organic vehicles should, in principle, be kept as low as
possible, and consistent in all incubations in a given experiment. Dimethyl
sulfoxide (DMSO) and methanol (or acetonitrile), the most commonly used
vehicle solvents, should be kept at levels equal to, or preferably less than, 0.2
and 2% (v/v), respectively (Easterbrook et al., 2001; Hickman et al., 1998).
Studies on metabolic stability using hepatocyte suspensions are not feasible for
automation/HTS, but these studies do provide rather complete profiles of hepatic
biotransformation without the supplements of cofactors and cosubstrates. The use
of S9 in metabolic stability studies can be evaluated in a manner similar to that
used for the microsomal assays, but with the possible addition of a broader panel
of cofactors or cosubstrates. These include NADPH for CYP/FMO-mediated
reactions, NADH for xanthine oxidoreductase and quinone oxidoreductase 2,
NADPH-dependent reductions by carbonyl reductases, and NADPH/NADH-
dependent reductions catalyzed by aldo–keto reductases, uridine 5
0
-diphosphateDETERMINATION OF METABOLIC STABILITY 417