Cytoplasmic binding and disposition kinetics of diclofenac in the isolated perfused rat liver

Abstract

1. The binding kinetics of diclofenac to hepatocellular structures were evaluated in the perfused rat liver using the multiple indicator dilution technique and a stochastic model of organ transit time density. 2. The single-pass, in situ rat liver preparation was perfused with buffer solution (containing 2% albumin) at 30 ml min(-1). Diclofenac and [(14)C]-sucrose (extracellular reference) were injected simultaneously as a bolus dose into the portal vein (six experiments in three rats). An analogous series of experiments was performed with [(14)C]-diclofenac and [(3)H]-sucrose. 3. The diclofenac outflow data were analysed using three models of intracellular distribution kinetics, assuming (1) instantaneous distribution and binding (well-mixed model), (2) 'slow' binding at specific intracellular sites after instantaneous distribution throughout the cytosol (slow binding model), and (3) 'slowing' of cytoplasmic diffusion due to instantaneous binding (slow diffusion model). 4. The slow binding model provided the best description of the data. The rate constants for cellular influx and sequestration were 0.126+/-0. 026 and 0.013+/-0.009 s(-1), respectively. The estimated ratio of cellular initial distribution volume to extracellular volume of 2.82 indicates an almost instantaneous distribution in the cellular water space, while the corresponding ratio of 5.54 estimated for the apparent tissue distribution volume suggests a relatively high hepatocellular binding. The non-instantaneous intracellular equilibration process was characterized by time constants of the binding and unbinding process of 53.8 and 49.5 s, respectively. The single-pass availability of diclofenac was 86%. The results obtained with [(14)C]-diclofenac and [(3)H]-sucrose were not statistically different.

Figure 1

Schematic presentation of hepatocellular drug distribution. The well-mixed model (WM) assumes rapid diffusion (D_eff ≈ D_water) and almost instantaneous binding. The slow binding model (SB) assumes rapid diffusion but ‘slow' binding (D_eff/L²>>k_on) to various intracellular binding sites (which may be distributed heterogeneously). In the ‘slow' diffusion model (SD) the apparent diffusion coefficient D_eff is reduced due to almost instantaneous immobilization (D_water/L²<<k_on). Rapid diffusion in cell water is characterized by a distributional relaxation time of about 1 s (≈L²/D_water).

Figure 2

Typical [¹⁴C]-sucrose outflow profile with the fit obtained by equations (10) to (12) shown together with the corresponding diclofenac data collected simultaneously.

Figure 3

Comparison of experimental data and fitted diclofenac outflow curve in rat 1 for Model SB and Model WM (A). The early differences between Model SB and Model SD are only visible in a linear scale (B).

Figure 4

Normalized model sensitivities for Model SB based on the means of estimated parameter values. The sensitivity function S_pi (t) describes the relative change in the outflow curve predicted by the model in response to a change in the value of parameter p_i.