In vitro screening for drugs that inhibit cytochrome P450 enzymes is well established as a means for predicting potential metabolism-mediated drug interactions in vivo. Given that these predictions are based on enzyme kinetic parameters observed from in vitro experiments, the miscalculation of the inhibitory potency of a compound can lead to an inaccurate prediction of an in vivo drug interaction, potentially precluding a safe drug from advancing in development or allowing a potent inhibitor to 'slip' into the patient population. Here, we describe the principles underlying the generation of in vitro drug metabolism data and highlight commonly encountered uncertainties and sources of bias and error that can affect extrapolation of drug-drug interaction information to the clinical setting.

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Previous labeling studies of abscisic acid (ABA) with 1802 have been mainly conducted with water-stressed leaves. In this study, 180 incorporation into ABA of stressed leaves of various species was compared with 18O labeling of ABA of turgid leaves and of fruit tissue in different stages of ripening. In stressed leaves of all six species investigated, avocado (Persea americana), barley (Hordeum vulgare), bean (Phaseolus vulgaris), cocklebur (Xanthium strumarium), spinach (Spinacia oleracea), and tobacco (Nicotiana tabacum), 180 was most abundant in the carboxyl group, whereas incorporation of a second and third 180 in the oxygen atoms on the ring of ABA was much less prominent after 24 h in 1802. ABA from turgid bean leaves showed significant 180 incorporation, again with highest 180 enrichment in the carboxyl group. The "80-labeling pattern of ABA from unripe avocado mesocarp was similar to that of stressed leaves, but in ripe fruits there was, besides high 180 enrichment in the carboxyl group, also much additional 18Q incorporation in the ring. In ripening apple fruit tissue (Malus domestica), singly labeled ABA was most abundant with more 180 incorporated in the tertiary hydroxyl group than in the carboxyl group of ABA. Smaller quantities of this monolabeled product (C-1'-180H) were also detected in the stressed leaves of barley, bean, and tobacco, and in avocado fruits. It is postulated that a large precursor molecule yields an aldehyde cleavage product that is, in some tissues, rapidly converted to ABA with retention of 180 in the carboxyl group, whereas in ripening fruits and in the stressed leaves of some species the biosynthesis of ABA occurs at a slower rate, allowing this intermediate to exchange 180 with water. On the basis of 180-labeling patterns observed in ABA from different tissues it is concluded that, despite variations in precursor pool sizes and intermediate turnover rates, there is a universal pathway of ABA biosynthesis in higher plants which involves cleavage of a larger precursor molecule, presumably an oxygenated carotenoid. carboxyl group, while smaller amounts of 180 are incorporated over extended periods in the ring oxygens of ABA. The fourth oxygen atom, in the carboxyl group, is derived from water (3). These results were interpreted to indicate that stressinduced ABA is derived from a large precursor pool which already contains two of the four oxygens present in ABA. As the primary precursor is depleted over time, other precursors containing fewer oxygen atoms feed into the pathway. The high degree of isotope enrichment in the carboxyl group suggests oxidative cleavage of a larger molecule, probably yielding an aldehyde. This intermediate would be further oxidized by a dehydrogenase and incorporate an oxygen atom from water into the carboxyl group of ABA. This idea is supported by the recent finding that ABA-aldehyde is the likely immediate precursor of ABA in higher plants (16,17).The objective of the present studies was to compare the biosynthesis of ABA that was either st...