Nonspecific binding of drugs to human liver microsomes

Abstract

Aims To characterize the nonspeci®c binding to human liver microsomes of drugs with varying physicochemical characteristics, and to develop a model for the effect of nonspeci®c binding on the in vitro kinetics of drug metabolism enzymes. Methods The extent of nonspeci®c 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 ®tted to a standard saturable binding model de®ned by the binding af®nity 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 nonspeci®c 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 x1 microsomal protein were 382 t 54 mM and 147 t 44 mM, respectively, and for amitriptyline were 375 t 23 mM and 178 t 33 mM, 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 i 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 Nonspeci®c 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.

Differential in vitro inhibition of M3G and M6G formation from morphine by (R)‐ and (S)‐methadone and structurally related opioids

“…Thus, plasma methadone concentration is less important in determining inhibition of morphine clearance compared with competitive inhibition. The apparent K i values reported here may also be an overestimation of actual K i values due to possible nonspecific binding of methadone to the microsomes, as is common for other basic amine drugs [48]. Also, in vivo methadone concentrations in the liver are likely to be larger than plasma concentrations, as …”

The glucuronidation of mycophenolic acid by human liver, kidney and jejunum microsomes

“…Based on these assumptions, extrapolated whole organ CL int values are 167.8, 7.9 and 4.4 l h x1 for liver, kidney and small bowel, respectively. Correction of kinetic data is necessary where nonspeci®c microsomal binding occurs [5], and hence the nonspeci®c binding of MPA to hepatic microsomes was determined. Ratios of MPA concentrations in the buffer and microsomal compartments of the equilibrium dialysis apparatus were 1.02 : 1, …”

“…Non-speci®c microsomal binding of MPA Non-speci®c binding of MPA to human liver microsomes was investigated by equilibrium dialysis, according to the procedure of McLure et al [5]. One compartment of the dialysis apparatus contained MPA (50, 250 or 1000 mM), human liver microsomes (0.05 mg ml x1 ; pooled microsomes from the ®ve livers used previously) and phosphate buffer (0.1 M, pH 7.4), and the other compartment phosphate buffer alone.…”

Effect of Albumin on Phenytoin and Tolbutamide Metabolism in Human Liver Microsomes: An Impact More Than Protein Binding

“…As a result, the decrease in reaction rates would be expected to be proportional to the extent of protein binding at substrate concentrations below K m . In addition, the apparent K m determined on the basis of the added concentration would then be higher than the "true" K m , but the V max should not be affected, as predicted by McLure et al (2000) and as observed for amitriptyline N-demethylation (Venkatakrishnan et al, 2000). However, Ludden et al (1997) reported a decreased K mu, whereas V max remained unchanged for PHT hydroxylation by human liver microsomes when BSA was added to decrease free fraction.…”