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

of in vivo testing. The objective of in vitro screening of P450 inhibition
properties of drug candidates is to exclude potent inhibitors from
further development. P450 enzymes play a crucial role in the
metabolism of drugs and, therefore, P450 inhibition is the most
important mechanism for metabolic drug-drug interactions (41, 42).

Microsomes and cDNA-expressed enzymes are the preferred test
systems as they are more readily available than human hepatocytes
(Figure 2). Assays are based on the analysis of potential reductions
in the metabolism of an appropriate P450 probe substrate in the
presence of various concentrations of the tested compound (11, 13).
For the purpose of high throughput P450 inhibition screening, a
variety of strategies based on fluorescence, LC-MS and radiometry
approaches have been developed (43-45). Since several enzymes
may be involved in the metabolism of a compound, the use of
recombinant models expressing a single P450 may lead to an
overestimation of the inhibitory effect of a given drug (46). A major
limitation in making conclusive statements from assays in
microsomes or recombinant enzymes is that ultimately in vivo
metabolism is complicated by the role of processes missing in
subcellular models. Drug transport across membranes, further
metabolism by cytosolic enzymes, or binding to intracellular proteins
can be determinant in the actual concentration of the substrate and
inhibitor available to the enzyme (41, 47). Assays performed in intact
cells could be more predictive (47).

Drug-drug interactions can also occur as a consequence of P450
induction. Metabolic interactions due to enzyme induction are far
less frequent than those caused by inhibition; however, their
consequences can be clinically relevant. A drug with inductive
properties can accelerate its own metabolism or those of other
co-administered drugs, resulting in either therapeutic inefficacy or
in an exaggerated response. Screening of inducers cannot be done in
microsomes or recombinant models as it requires a cellular system
that is fully capable of expressing genes (Figure 2). Currently,
primary hepatocytes are still the unique in vitro model for global
examination of the inductive potential of drugs (14, 48, 49). After
24-72 h incubation with the test compound, P450 induction is
monitored as its increases in enzyme activity (using specific
substrates) or mRNA levels (by quantitative RT-PCR). Results are

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