In vitro characterization of the erythrocyte distribution of methazolamide: A model of erythrocyte transport and binding kinetics

The rate and extent of binding of methazolamide to human erythrocytes was s tudied in vitro. All experiments were carried out at physiological temperat ure (37C) and pH (74). Methazolamide (MTZ) buffet concentrations were analy zed by HPLC. Distributional equilibrium between buffer and washed red blood cells was achieved after 1 hr. Results of equilibrium studies were consist ent with two classes of binding sites for MTZ within the erythrocyte: a low affinity, high capacity site (CA-I) and a high affinity, low capacity site (CA-II). A two-binding site model was fitted to experimental data generati ng estimates for binding parameters Ka(1) (0.0017 +/- 0.00022 mu M-1) nM(1) (636 +/- 5.23 mu M), Ka(2)(0.46 +/- 0.0083 mu M-1), and nM(2)(80.9 +/- 0.3 89 mu M). Based upon these findings, kinetic studies were performed in orde r to characterize the rate of drug drug distribution. The late of erythrocy te uptake of MTZ was mathematically modeled using a series of differential equations describing drug diffusion across the red blood cell membrane and subsequent complexation with intracellular binding sites. The model assumed that penetration of MTZ into the red blood cells was passive bur drug bind ing to the carbonic anhydrase isozymes was not instantaneous. Using a novel cur ve fitting technique, parameter estimates of RBC membrane permeability (0.0102 +/- 0.000618 cm/min), and binding rate constants k(-1) (0.254 +/- 0.0213 min(-1)), k(1) (0.0022 +/- 0.00020 ml/mu g-min), k(-2)(1.59 +/- 0.03 58 min(-1)), and k(2)(3.1 +/- 0.035 ml/mu g-min) were obtained. The model c haracterized the observed biphasic decline of MTZ buffer concentrations ove r time and may help explain the prolonged residence of MTZ in vivo.