The FDA issued a guidance on the safety testing of metabolites in February 2008, in which they stated that metabolites of concern are those that are detected at levels greater than 10% of the systemic exposure of the parent at steady state. This has presented many challenges in determining the circulating human metabolites at an early stage of development. The intention of this perspective is to address the question of how effective in vitro metabolism and early exploratory clinical data are in predicting the circulating metabolites from both a qualitative and a quantitative perspective. To this end, data were reviewed from 17 molecules in the Lilly portfolio for which there were in vitro data and a radiolabeled study in humans. Twelve example cases are presented in detail to demonstrate trends for when in vitro data adequately predicted in vivo (41%), when in vitro data underpredicted the circulating metabolites (35%), and when in vitro data overpredicted the circulating metabolites (24%). In addition, cases that present special challenges due to very low levels of the circulating parent or long half-lives of the parent and/or metabolites are presented. The trends indicate that the more complex the metabolism, the less likely the in vitro data were to predict the circulating metabolites. The in vitro data were also less predictive for N-glucuronidations and non-P450-mediated cleavage reactions. Although the in vitro data were better at predicting clearance pathways, the data set often failed to predict the quantity of metabolites, which is needed in consideration of whether or not a "disproportionate" metabolite may be circulating in human plasma.

In vitro assays using liver subcellular fractions or suspended hepatocytes for characterizing the metabolism of drug candidates play an integral role in the optimization strategy employed by medicinal chemists. However, conventional in vitro assays have limitations in their ability to predict clearance and generate metabolites for lowturnover (slowly metabolized) drug molecules. Due to a rapid loss in the activity of the drug-metabolizing enzymes, in vitro incubations are typically performed for a maximum of 1 hour with liver microsomes to 4 hours with suspended hepatocytes. Such incubations are insufficient to generate a robust metabolic response for compounds that are slowly metabolized. Thus, the challenge of accurately estimating low human clearance with confidence has emerged to be among the top challenges that drug metabolism scientists are confronted with today. In response, investigators have evaluated novel methodologies to extend incubation times and more sufficiently measure metabolism of low-turnover drugs. These methods include plated human hepatocytes in monoculture, and a novel in vitro methodology using a relay of sequential incubations with suspended cryopreserved hepatocytes. In addition, more complex in vitro cellular models, such as HepatoPac (Hepregen, Medford, MA), a micropatterned hepatocyte-fibroblast coculture system, and the HmREL (Beverley Hills, CA) hepatic coculture system, have been developed and characterized that demonstrate prolonged enzyme activity. In this review, the advantages and disadvantages of each of these in vitro methodologies as it relates to the prediction of clearance and metabolite identification will be described in an effort to provide drug metabolism scientists with the most up-to-date experimental options for dealing with the complex issue of low-turnover drug candidates.

Enolase catalyzes the interconversion of 2-phosphoglycerate and phosphoenolpyruvate. Kinetic isotope effects have been used to determine whether abstraction of the proton from C-2 and loss of hydroxide from C-3 of 2-phosphoglycerate occur in a concerted reaction or as sequential processes and whether these steps are kinetically significant for the enolase-catalyzed reaction. Enolase exhibits a significant primary deuterium isotope effect, as well as catalyzing the relatively rapid exchange of the C-2 proton with solvent water. Secondary C-3 deuterium isotope effects are also reported, both when the C-2 carbon carries a hydrogen and when this center is deuterated. These results provide information about the kinetic significance and timing of the transition state(s) associated with the loss of H+ and OH-. Strong evidence has been presented for a stepwise mechanism where both the rate of proton abstraction and one or both of the later transition states, i.e., those associated with hydroxide loss and product release, limit the overall reaction rate. If a concerted reaction were to be invoked, the presence of a small secondary 2H isotope effect in combination with the observed rate of exchange of the C-2 proton require the intrinsic secondary 2H kinetic isotope effect to be effectively unity. For the concerted mechanism, an intrinsic effect of unity would be consistent only with an extremely asymmetric transition state that is dominated by C-H bond cleavage.

Experimental data on the stereoselectivity of base-catalyzed 1,2-elimination reactions that produce conjugated carbonyl compounds are scarce in spite of the importance of these reactions in organic and biochemistry. As part of a comprehensive study in this area, we have synthesized stereospecifically-deuterated beta-tosyloxybutanoate esters and thioesters and studied the stereoselectivity of their elimination reactions under non-ion pairing conditions. With the availability of both the (2R*,3R*) and (2R*,3S*) diastereomers the innate stereoselectivity could be determined unambiguously. (1)H and (2)H NMR data show that these substrates produce 5-6% syn elimination, the usual amount for acyclic substrates undergoing E2 reactions. Contrary to earlier suggestions, activation by a carbonyl group has virtually no influence upon the stereoselectivity. Elimination of the (2R*,3R*) diastereomer of the beta-tosyloxyester and thioester produces 21-25% of the (Z)-alkene, much more than observed with a poorer beta-nucleofuge. A relatively large amount of (Z)-alkene product seems to be a good marker for an E2 pathway, in which the transition state is E1cB-like, rather than an E1cB(irrev) mechanism. Syn KIE values were higher than those for anti elimination for the esters as well as the thioesters. Experimental challenges to the synthesis of stereospecifically-deuterated beta-tosyloxyesters are discussed.