NONSPECIFIC-BINDING TO MICROSOMES - IMPACT ON SCALE-UP OF IN-VITRO INTRINSIC CLEARANCE TO HEPATIC-CLEARANCE AS ASSESSED THROUGH EXAMINATIONOF WARFARIN, IMIPRAMINE, AND PROPRANOLOL

The nonspecific, noncovalent binding of three drugs, imipramine, warfa rin, and propranolol, to pooled human and animal liver microsomes has been determined using equilibrium dialysis in conditions where no cofa ctor (NADPH) was included in the incubation. The binding of warfarin w as dependent upon both protein and drug concentration, whereas the bin ding of propranolol and imipramine was also dependent upon protein con centration but generally independent of drug concentration, At a micro somal protein concentration of 1.0 mg/ml and a warfarin concentration of 10 mu M,the free fraction (f(u(mic))) was 0.85, The corresponding v alues for propranolol and imipramine were 0.41 and 0.16, respectively. Thus, although all three drugs exhibit high binding in plasma (f(u)<0 .1) the acidic drug warfarin differs from the basic drugs propranolol and imipramine in the extent to which each binds to microsomal protein , The binding of all three drugs to liver microsomes obtained from com monly studied animal species (rat, dog, and monkey) was almost identic al to that observed in human. Additionally, the binding of warfarin an d propranolol to microsomes obtained from insect cells used in baculov irus cytochrome P450 expression systems was similar to that exhibited in liver microsomes, when equal protein concentrations were compared, The enzyme kinetics of propranolol, imipramine, and warfarin oxidative metabolism were determined in pealed human liver microsomes, and the intrinsic clearance values obtained were used in scaling up to project human in vivo clearance. The values obtained by incorporating microso mal binding were compared with those in which this factor is ignored, The findings suggest that the parameter f(u(mic)) is important to obta in when attempting to relate in vitro intrinsic clearance to in vivo c learance. Also, this value is important to consider when comparing sub strates with respect to enzyme specificity, since measured apparent K- M values should be converted to true ''free K-M'' values by correcting for the free fraction in the in vitro incubation. Furthermore, the ex tent of nonspecific binding to microsomes is likely an important param eter to consider when attempting to relate K-i values measured in vitr o to observations of drug-drug interactions (or the lack thereof) in v ivo.