prediction of human metabolic clearance, such asKmandVmax,needtobe
accurately determined. The features of metabolic stability studies in both
discovery and development will be described in this chapter.
High throughput screening (HTS) through automation is generally desired
in pharmaceutical research, and is feasible for most steps of stability studies,
including reaction preparation, incubation, sample cleanup, and even sample
detection utilizing radiometric and fluorometric means (Crespi and Stresser,
2000; Jenkins et al., 2004). However, the desired reliable chromatography-
based detection may not be applicable to HTS, as the data analyses for
quantification are unlikely to be completely automated without human
intervention. This is particularly true for metabolic investigations during
development. Additionally, computational approaches are being increasingly
applied to delineate the complex kinetic behaviors of metabolic enzymes
(Mei et al., 2002; Zhang et al., 2002). Besides classic biochemical plots,
traditionally applied to characterize enzyme kinetics, nonlinear regression
analyses will be emphasized largely due to their unbiased nature and user-
friendly execution.
Finally, predictions of human pharmacokinetics usingin vitrometabolic
data, one of the ultimate goals forin vitrometabolic studies, are described, both
conceptually and in case studies.
13.2 DETERMINATION OF METABOLIC STABILITY
13.2.1 Aims
In early drug discovery, one of the properties required for lead candidates is
reasonable metabolic stability. The need for stability is based on the
presumption that metabolism or metabolic clearance tends to be the major,
if not the only determinant, for total body clearance when the pharmacokinetic
and pharmaceutical properties for the discovery compound are unknown. To
obtain metabolism data, liver subcellular fractions, microsomal preparations in
particular, are frequently chosen as standard enzyme preparations for
metabolic studiesin vitro, because of the abundance and the broad spectrum
of drug-metabolizing enzymes expressed in hepatocytes, principally localized in
the endoplasmic reticulum. On the contrary, lead candidate stability in blood
may also need to be studied, even prior to the microsomal assays, particularly
for compounds with potential hydrolytic susceptibilities, such as those
containing ester or amide bonds. Therapeutic agents, regardless of the route
of dosing, must be sufficiently metabolically stable in the blood to be able to
reach the intended biological targets. Determination of stability in blood (or
plasma), which is at risk from the hydrolytic activities, is straightforward, with
no requirements for supplements of any of the constituents required for
metabolism mediated by the majority of enzymes in liver subcellular fractions
(Liederer and Borchardt, 2005; Skopp et al., 2001). Moreover, hydrolytic
414 DETERMINATION OF METABOLIC RATES AND ENZYME KINETICS