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, 49 (5), 453-61

Nonspecific Binding of Drugs to Human Liver Microsomes

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Nonspecific Binding of Drugs to Human Liver Microsomes

J A McLure et al. Br J Clin Pharmacol.

Abstract

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, fu(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 KD, and the maximum binding capacity Bmax. The derived binding parameters, KD and Bmax, 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 (fu(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. Bmax and KD values for nortriptyline at 1 mg ml-1 microsomal protein were 382 +/- 54 microM and 147 +/- 44 microM, respectively, and for amitriptyline were 375 +/- 23 microM and 178 +/- 33 microM, respectively. Bmax, but not KD, varied approximately proportionately with the microsome concentration. When KD is much less than the Km for a reaction, the apparent Km based on total drug can be corrected by multiplying by fu(mic). When the substrate concentration used in a kinetic study is similar to or greater than the KD (Km >/= KD), 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.

Figures

Figure 1
Figure 1
Nonspecific binding of nortriptyline to human liver microsomes. a) Dependence of fu(mic) on nortriptyline concentration at 1 mg ml−1 microsomal protein. b). Dependence of fu(mic) on microsomal protein concentration at 100 µm nortriptyline. Each point is the mean ± s.d. of triplicate determinations.
Figure 2
Figure 2
Binding of nortriptyline to human liver microsomes 1 mg ml−1. a) Binding plot according to equation 1. b) Scatchard plot.
Figure 3
Figure 3
Simulation showing the effect of nonspecific binding with varying KD on Michaelis-Menten kinetics (a), and on an Eadie Hofstee plot (b). The plots use total (added) substrate concentration, whereas the reaction velocities were calculated using free (unbound) substrate concentrations.
Figure 4
Figure 4
Simulation showing the effect of varying microsomal protein concentration on Michaelis-Menten kinetics (a), and on an Eadie Hofstee plot (b). Bmax was assumed to be proportional to microsomal protein concentration. Substrate concentration ranges used in kinetic studies are commonly 0.2 Km −3 Km.
Figure 5
Figure 5
Relationship between Km and KD. At the substrate concentration range used in kinetic studies when KmKD, the fu(mic) does not vary with substrate concentration (insert). When KmKD, the fu(mic) varies with substrate concentration over the range used in the kinetic study.

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