The recent successes of in vivo-in vitro correlations for metabolically based drug interactions suggests that enzyme behavior is largely conserved. However, current approaches to the prediction of inhibition based interactions have remained essentially qualitative. For numerous inhibitors, inhibition constants determined in vitro do not adequately predict the extent of interaction in vivo. These inhibitors appear to be more potent in vivo than in vitro. A fundamental problem in the field of in vitro-in vivo correlations is the absence of a general methodology for obtaining the concentration of inhibitor at the enzyme site and the inhibition constant that actually prevails at the site. In the present application, we will test the """"""""free drug hypothesis,"""""""" i.e. the concept that inhibitor effect is governed by unbound inhibitor at the actual site of the enzyme. In the first two specific aims, we will demonstrate that the free drug hypothesis is valid for competitive inhibitors in in vitro systems so long as unbound concentrations are measured using an extensive list of inhibitors with different protein binding and lipophilicity characteristics.
Specific Aims 2 (a) and 2(b) will test the validity of the """"""""free drug hypothesis"""""""" in vivo. This will be accomplished using a new parameter, the """"""""inhibition constants ratio"""""""" (R/kI). Theory suggests that this ratio (defined as K/i/(pre) determined in a purified recombinant system divided by the K/i/iv determined in vivo) will equal one and that it will be independent of enzyme. This proposal will thus provide a useful framework to effectuate quantitative in vivo predictions from data derived in vitro.
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