ABSTRACT:The Michaelis constant (K M ) for cytochrome P450-mediated drug biotransformation reactions can be an important parameter in understanding the potential for a drug to exhibit saturable metabolism in vivo and nonlinear dose-exposure relationships. K M values were measured for several drug biotransformation reactions using recombinant heterologously expressed human enzymes. These determinations were made using an approach of monitoring substrate loss ("in vitro t 1/2 " method) at multiple substrate concentrations, with the objective of comparing K M values determined by this approach with K M values determined using the conventional approach of measuring product formation rates at several substrate concentrations. The reactions examined were CYP2C9-catalyzed diclofenac 4-hydroxylation, CYP2D6-catalyzed dextromethorphan O-demethylation and thioridazine S-oxidation, CYP2C19-catalyzed imipramine N-demethylation, CYP3A4-cata- The determination of enzyme kinetic parameters, K M , V max , and CL int Ј , for cytochrome P450-catalyzed reactions is an important aspect in drug discovery and development (Obach, 2001). In drug discovery, these in vitro data can be used in compound design to optimize the human pharmacokinetic behavior of new chemical entities. Such parameters can be used to predict human clearance prior to human administration (Iwatsubo et al., 1996;Obach et al., 1997;Houston and Carlile, 1997;Lave et al., 1999). In drug development, enzyme kinetic data for drug biotransformation reactions can be used to better understand the mechanism of clearance of new chemical entities in humans to assess potential determinants of interindividual variability in pharmacokinetics such as drug-drug interactions, genetic polymorphisms of drug clearance mechanisms, and nonlinear dose-exposure relationships.The K M value (Michaelis constant) is a primary descriptor of the enzyme kinetic behavior of a biotransformation reaction. In simple kinetic systems, it represents the substrate concentration that will yield a reaction velocity that is half of the theoretical maximum reaction velocity that would occur at an infinite substrate concentration (V max ). In a practical sense, the K M value can be reflective of the potential of a drug to saturate, or partially saturate, an enzyme involved in its metabolic clearance. The lower the K M value, the greater the possibility that the drug concentrations attained in vivo will saturate the metabolic clearance pathway. When this phenomenon occurs, it can be problematic because the drug will exhibit a nonlinear, supraproportional dose-exposure relationship (Ludden, 1991). For drugs with low-therapeutic indices, small increases in dose can potentially introduce toxicity as drug concentrations in vivo will increase to a much greater extent than that expected from the increase in dose. Such pharmacokinetic behavior has been observed for numerous compounds spanning several therapeutic indications, some of which are listed in Table 1. The K M value will not solely be responsible for supra...