Nonspecific binding of drugs to human liver microsomes

Aims  To characterize the nonspecific binding to human liver microsomes of drugs with varying physicochemical characteristics, and to develop a model for the effect of nonspecific binding on the in vitro kinetics of drug metabolism enzymes. Methods  The extent of nonspecific binding to human liver microsomes of the acidic drugs caffeine, naproxen, tolbutamide and phenytoin, and of the basic drugs amiodarone, amitriptyline and nortriptyline was investigated. These drugs were chosen for study on the basis of their lipophilicity, charge, and extent of ionization at pH 7.4. The fraction of drug unbound in the microsomal mixture, f u(mic) , was determined by equilibrium dialysis against 0.1  m phosphate buffer, pH 7.4. The data were fitted to a standard saturable binding model defined by the binding affinity K D , and the maximum binding capacity B max . The derived binding parameters, K D and B max , were used to simulate the effects of saturable nonspecific binding on in vitro enzyme kinetics. Results  The acidic drugs caffeine, tolbutamide and naproxen did not bind appreciably to the microsomal membrane. Phenytoin, a lipophilic weak acid which is mainly unionized at pH 7.4, was bound to a small extent ( f u(mic)  = 0.88) and the binding did not depend on drug concentration over the range used. The three weak bases amiodarone, amitriptyline and nortriptyline all bound extensively to the microsomal membrane. The binding was saturable for nortriptyline and amitriptyline. B max and K D values for nortriptyline at 1 mg ml −1 microsomal protein were 382 ± 54 µ m and 147 ± 44 µ m , respectively, and for amitriptyline were 375 ± 23 µ m and 178 ± 33 µ m , respectively. B max , but not K D , varied approximately proportionately with the microsome concentration. When K D is much less than the K m for a reaction, the apparent K m based on total drug can be corrected by multiplying by f u(mic) . When the substrate concentration used in a kinetic study is similar to or greater than the K D ( K m  ≥  K D ), simulations predict complex effects on the reaction kinetics. When expressed in terms of total drug concentrations, sigmoidal reaction velocity vs substrate concentration plots and curved Eadie Hofstee plots are predicted. Conclusions  Nonspecific drug binding in microsomal incubation mixtures can be qualitatively predicted from the physicochemical characteristics of the drug substrate. The binding of lipophilic weak bases is saturable and can be described by a standard binding model. If the substrate concentrations used for in vitro kinetic studies are in the saturable binding range, complex effects are predicted on the reaction kinetics when expressed in terms of total (added) drug concentration. Sigmoidal reaction curves result which are similar to the Hill plots seen with cooperative substrate binding.