Measurement of Michaelis Constants for Cytochrome P450-Mediated Biotransformation Reactions Using a Substrate Depletion Approach

Abstract

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...

Contributions of Ionic Interactions and Protein Dynamics to Cytochrome P450 2D6 (CYP2D6) Substrate and Inhibitor Binding

“…Ternary and quaternary complexes are thought to underlie complex kinetics exhibited by P450s. The concentrations used for protein crystallization are ϳ1000-fold greater than the K m for mesoridazine formation from thioridazine determined by Obach and Reed-Hagen (40). These authors noted that thioridazine exhibited a small tendency to exhibit biphasic character suggestive of two binding sites with the population of the second binding site occurring at the highest concentration of thioridazine examined, 40 M. The authors concluded that this effect was unlikely to be significant for in vivo exposure to thioridazine.…”

Novel Binding Mode of the Acidic CYP2D6 Substrates Pactimibe and Its Metabolite R-125528

“…Although substitution of the N-octyl chain to the N-decyl chain did not change the stability against CYP2D6, the N-hexyl or N-dodecyl chain improved the CYP2D6 metabolic stability. For the determination of the K m values for imipramine, desipramine, amitriptyline, propranolol, dextromethorphan, and propafenone, the substrate depletion method (Obach and Reed-Hagen, 2002) was used. The final CYP2D6 protein concentration, substrate concentrations, and incubation times were set at 10 pmol/ml, 0.1, 0.3, 1, 3, 10, and 30 M, and 0, 3, 6, and 9 min, respectively.…”

“…Because of the absence of authentic samples for the metabolites of imipramine, desipramine, amitriptyline, propranolol, dextromethorphan, and propafenone, the substrate depletion method (Obach and Reed-Hagen, 2002) was used for determination of the Michaelis constant (K m ) values for typical CYP2D6 substrates. The final CYP2D6 protein concentration and substrate concentrations were set at 10 pmol/ml and 0.1, 0.3, 1, 3, 10, and 30 M, respectively.…”

Metabolism of fibrates by cytochrome P450s and UDP-glycosyltransferases in rat and human liver microsomes

“…In this study, we measured the metabolism of fibrates in RLM and HLM by applying the cofactors NADPH and UDPGA individually and simultaneously, allowing us to determine the contributions of both pathways using the same approach and detection platform. The substrate depletion approach has been shown to be comparable to the product formation approach and is increasingly being used in enzyme kinetics studies [23,27,30]. Considering the diversity of metabolites formed from the metabolism of fibrates and the requirement for semi throughput in this study, it is impractical to monitor product formation; thus the substrate depletion approach is more appropriate in this context.…”

“…(1) proposed by Obach and ReedHagen [27] with Origin software (version 7.5, MicroCal Software, Inc., Piscataway, NJ, USA).…”