PBPK modeling to unravel nonlinear pharmacokinetics of verapamil to estimate the fractional clearance for verapamil N-demethylation in the recirculating rat liver preparation.

Abstract

We applied physiologically based pharmacokinetic (PBPK) modeling to study the dose-dependent metabolism and excretion of verapamil and its preformed metabolite, norverapamil, to unravel the kinetics of norverapamil formation via N-demethylation. Various initial verapamil (1, 50, and 100 μM) and preformed norverapamil (1.5 and 5 μM) concentrations, perfused at 12 ml/min, were investigated in the perfused rat liver preparation. Perfusate and bile were collected over 90 minutes, and livers were harvested at the end of perfusion for high-performance liquid chromatography analysis. After correction for the adsorption of 10%-25% dose verapamil and norverapamil onto Tygon tubing and binding to albumin and red blood cell, fitting of verapamil and formed and preformed norverapamil data with ADAPT5 revealed nonlinearity for protein binding, N-demethylation (V(max,met1)(VER --> NOR) = 96.6 ± 33.4 nmol/min; K(m,met1)(VER --> NOR) = 10.4 ± 4.1 μM), formation of other metabolites (V(max,met2(VER -->others) 288 ± 51 nmol/min; K(m.met2)(VER -->others )= 14.1 ± 4.9 μM), as well as biliary excretion (V(max,sec)(VER)= 0.911 ± 0.505 nmol/min; K(m,sec)(VER) = 4.75 ± 2.29 μM). The hepatic clearance of verapamil (CL(L)(VER) decreased with the dose (8.16-10.2 ml/min), with values remaining high relative to perfusate blood flow rate among the doses. The hepatic clearance of preformed norverapamil (11 ml/min) remained unchanged for the concentrations studied and approximated perfusate blood flow rate, suggesting a high norverapamil extraction ratio. The fractional formation of norverapamil and biliary excretion of verapamil based on fitted constants were 31.1% and 0.64% of CL(L)(VER), respectively. Enantiomeric disposition and auto-inhibition of verapamil failed to perturb these estimaties according to PBPK modeling, due to the low values of the Michaelis-Menten constant, Km, and inhibition parameter, kI.