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MARÍA JOSÉ GÓMEZ-LECHÓN y MARÍA TERESA DONATO AN. R. ACAD. NAC. FARM.

its concentration at the enzyme site. Two strategies underlying the
prediction of in vivo hepatic drug metabolism from in vitro data
have been defined (24, 25): the metabolite formation method, the
most widely used, and the more recently adopted substrate depletion
approach, where the intake of the parent drug is monitored over
time (25). From a biochemical point of view CLint, in vitro can be
considered in terms of the enzyme parameters of the Michaelis-
Menten equation (24, 26). Enzyme kinetics data must be obtained
under linear conditions with regard to the enzyme concentration
and incubation time, that is, the period when V0 is maintained. Once
CLint, in vitro is obtained from Michaelis-Menten constants (Km
and Vmax), reasonable estimates of in vivo hepatic clearance (CLint,
in vivo) can be obtained by using appropriate scaling factors and
modeling (24, 26, 27). Mathematical physiological models (well-
stirred, parallel tube, distributed and dispersion models) of hepatic
drug clearances, which use both anatomical and physiological data,
have been appraised in relation to their utility in predicting drug
removal by the liver (28-30).

IDENTIFICATION OF P450 ENZYMES INVOLVED IN DRUG
METABOLISM

Early identification of P450s responsible for the metabolism of
new molecules is important in drug discovery in order to minimize the
role of polymorphic enzymes leading to inter-individual variation and
potential drug-drug interactions. Pooled human liver microsomes are
the most frequently model used for this purpose (31, 32). A major
advantage of these in vitro systems is that P450 enzymes are present
in their physiological relative proportions and can interact with other
essential proteins required for P450-catalyzed oxidations (i.e. NADPH
cytochrome P450 reductase, cytochrome b5). By the use of selective
chemical inhibitors for individual P450s, the major metabolic
pathways for a new drug can be either readily demonstrated or ruled
out. The incubations in which the metabolism of the test drug is
reduced suggest the involvement of the P450 enzyme, whose activity
is affected by the inhibitor. One drawback that these assays present,
even when using potent inhibitors, is that it is not possible to
completely and selectively inhibit a single P450 activity.

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