Modeling of telmisartan disposition in sandwich-cultured rat hepatocytes. Does cryopreservation change disposition kinetics?
Neel Deferm (1), Janneke Keemink (1), Peter de Witte (2), Pieter Annaert (1), Thomas Bouillon (1)
(1) Drug Delivery and Disposition, KU Leuven Department of Pharmaceutical and Pharmacological sciences, Leuven, Belgium (2) Laboratory for Molecular Biodiscovery, KU Leuven Department of Pharmaceutical and Pharmacological Sciences, Leuven, Belgium.
Objectives: To develop a physiological model which describes hepatic uptake, metabolism and biliary excretion of the angiotensin II type-1 receptor antagonist telmisartan and its glucuronide in sandwich-cultured rat hepatocytes (SCRH) and to utilize it to quantify differences in estimated kinetic parameters between cryopreserved and freshly-isolated hepatocytes.
Methods: In vitro disposition data were obtained by incubating SCRH (freshly-isolated or cryopreserved) with 1,3,10 or 20 µM telmisartan. Incubation buffers containing telmisartan and its glucuronide were collected, followed by cell lysis to determine intracellular levels. Additionally, biliary excretion of telmisartan and its glucuronide were calculated. An ordinary differential equation (ODE) model was fitted to concentration and amount-time profiles using NONMEM v7.3.0 [1]. The model includes compartments representing the buffers, cells, bile and cells+bile with compound distributed among them through both linear and non-linear processes. Cell source (freshly-isolated or cryopreserved) was implemented as a covariate in the dataset and its effect on parameter values was assessed by the difference in objective function values (log likelihood ratio test and Akaike criterion).
Results: A mechanistic eight-compartment model was developed which adequately described the in vitro disposition of telmisartan and its glucuronide. Passive diffusion clearance and kinetic parameters for active uptake of telmisartan from the medium compartment to the intracellular compartment were estimated to be 3 (±12%) µL/min (CLu,int,pass), 260 (±15%) pmol/min/well (Vmax,act) and 10 (±19%) µM (Km,act). The biliary excretion rate constants of telmisartan and its glucuronide were estimated at 3 (±10%) min-1 (Kbile) and 7 (±6%) min-1 (Kbile,glu), while estimates of metabolism kinetic parameters were 225 (±13%) pmol/min/well (Vmax,met) and 33 (±17%) µM (Km,app,met). Covariate analysis showed a significant effect (p < 0.01) of cryopreservation on Kbile (fresh: 3 (±18%) min-1, cryo: 2 (±10%) min-1), Vmax,met (fresh: 930 (±10%) pmol/min/106 cells, cryo: 1768 (±10%) pmol/min/106 cells), Kbile,glu (fresh: 7 (±18%) min-1, cryo: 6 (±10%) min-1) and Vmax,act (fresh: 1545 (±17%) pmol/min/106 cells, cryo: 2936 (±17%) pmol/min/106 cells).
Conclusions: Modeling of in vitro telmisartan disposition suggests that cryopreserving rat hepatocytes affects biliary excretion, metabolism and uptake of telmisartan and its glucuronide in SCRH.
References:
[1] Beal SL, Sheiner LB, Boeckmann AJ & Bauer RJ (Eds.) NONMEM Users Guides. 1989-2011. Icon Development Solutions, Ellicott City, Maryland, USA
