Intracellular Retention of Three Quinuclidine Derivatives in Caco-2 Permeation Experiments: Mechanisms and Impact on Estimating Permeability and Active Efflux Ratio

Background: Three quinuclidine derivatives (FRM-1, FRM-2 and FRM-3) were subject to significant mass loss to cellular retention in Caco-2 permeation experiments. The apparent permeability coefficient (Papp) calculated with either ‘sink’ (Papp,sink) or ‘non-sink’ (Papp,nonsink) method was significantly biased. As a result, a simplified 3-compartmental distribution model was applied in this study to derive the ‘intrinsic’ Papp (Papp,int) and to understand the impact of cellular retention on estimating Papp and active efflux ratio (ER) values. Methods: Time-courses of the amount of test compounds in the donor, receiver and cells were determined in the presence and absence of bafilomycin A1 (BFA, 100 nM) and / or cyclosporine A (CsA, 10 μM). A mathematical model was constructed to describe the mass transfer of test compounds among three compartments. The temporal profiles of directional Papp,sink, Papp,nonsink and the corresponding of ER values were compared with the counterpart parameters derived from data-fitting to the mathematical model. Simulations were performed for a better understanding of experimental observations. Results: The mass recovery of test compounds deteriorated with incubation time and was direction dependent. Based on the directional Papp,sink values, the resulting ER is close to unity for FRM-1, and approximately 2 and 3.5 for FRM-2 and FRM-3. Treatment with BFA considerably enhanced mass recovery for FRM-1 and FRM-3 (by 5- and 2-fold) but elicited no impact on FRM-2, while ER values largely unchanged. Expectedly, Papp,nonsink was higher than Papp,sink, but the resulting ER was lower in most cases. In contrast, the model-derived Papp,int was much greater than the values of Papp,sink and Papp,nonsink. The model also quantitatively unveiled the respective contributions of lysosomal sequestration and nonspecific binding to the cellular retention of the compounds. Conclusion: Our work reveals the different mechanisms involved in cellular retention of these quinuclidine derivatives, and more importantly, demonstrates the value of kinetic analyses with mathematical modeling in minimizing the bias in Papp estimation when assumptions for conventional calculations are violated.