Determination of drug absorption parameters in Caco-2 cell monolayers with a mathematical model encompassing passive diffusion, carrier-mediated efflux, non-specific binding and phase II metabolism

摘要

Intestinal absorption is required for a sufficiently high bioavailability of drugs administered by the peroral route. Several molecular mechanisms are involved in intestinal absorption and can profoundly influence its magnitude including permeation of the mucosa by passive diffusion, transport across the intestinal wall by carrier mediated processes, chemical and enzymatic alteration of the molecule in the intestinal lumen and/or in the enterocyte, dissolution behaviour of the drug and interaction with food ingredients or coadministered drugs at the dissolution and the transport level. These mechanisms typically act simultaneously and each depends on drug molecule and epithelium related chemical and biological factors whose effect is not definitely established. This makes intestinal absorption a rather complex process, which, despite recent advances, is fundamentally still poorly understood. Therefore, experimental verification of drug absorption remains a must in current industrial drug development practice. Prediction of in vivo absorption based on in vitro methodology may help reduce the volume of necessary clinical investigations. Cell culture techniques predominantly employing the Caco-2 cell line have been established in the last decade as a screening and study tool of intestinal absorption. This technique, although widely used in industrial and academic settings, still poses a number of challenges. These include artefacts like adsorption to container surfaces and cellular accumulation, which might lead to an erroneous estimation of the permeability, poor recovery, and faulty mass balance. The estimation of independent parameters for parallel processes (e.g. passive permeability and active efflux) is still not the common procedure in the analysis of permeation data and the usually employed apparent permeability coefficient Papp and efflux ratio (ER) are afflicted with limitations. The objective of the present work was to establish a methodology for determining the contribution of passive diffusion, carrier-mediated transport, enzymatic degradation and non-specific binding/adsorption to drug absorption measured in the Caco-2 cell system and investigating drug-drug interactions for absorption in this system. Transport experiments across the cell monolayer were conducted in bi-directional modus using model drug compounds (Amentoflavone, Verapamil, Digoxin and Quinidine) that are known to be subject to more than one of the above molecular mechanisms. In order to delineate the contribution of these mechanisms, a model for analysing the experimental data was introduced. This model encompassed quantitative expressions based on biophysical or physicochemical principles of the effect of all these mechanisms on transport and described the variation of drug concentration in the different compartments of the cell system as a result of the simultaneous action of these mechanisms. Theududresulting system of differential equations was fitted to the experimental data usingudregression analysis following numerical integration and relevant parameters reflecting theudquantitative effect of each mechanism involved in absorption were deduced. Theseudparameters were the passive permeability coefficient, first and zero order carrierudmediated transport rate, first order metabolic rate constant and binding constant. Thisudwas done for the above drugs used individually and for selected binary mixtures.udAmentoflavone exhibited strongly asymmetric transport, which was almost not detectableudin the apical to basal direction and pronounced in the basal to apical direction. Thisudsuggested that Amentoflavone is subject to apical efflux in the Caco-2 cells. This wasudpartly reversed by GF120918 and Vinblastine which are known inhibitors of Pglycoproteinud(P-gp) and P-gp and MRP2, respectively, indicating that Amentoflavone wasudsubstrate of at least one of these efflux transporters. The active apical efflux was almostudabolished at low temperature. Amentoflavone also underwent phase II metabolism in theudCaco-2 cells. At least two glucoronides and one sulfate were detected by HPLC-MS,udwhich were hydrolysable by specific enzymes. These metabolites exhibited also apicaludefflux. Finally, Amentoflavone was strongly adsorbed to the surface of the Transwelludplates used in cell culture and transport studies. The adsorption and desorption rateudconstant and the total number of surface binding sites was determined in blankudexperiments using the plates without cells. For this, a model describing the timeuddependent concentration decrease of the drug due to adsorption was employed. Theseudsurface adsorption parameters were subsequently used in the model describing theudabsorption of Amentoflavone in Caco-2 cells. This procedure allowed the determinationudof absorption parameters that were not biased by non-specific binding effects.udAmentoflavone was found to have a rather low passive cell permeability, which combinedudwith its substantial apical efflux resulted in its marginal absorption in the apical to basaludtransport direction. The metabolic rate constant was smaller than the efflux rate constantudbut still sufficiently large to produce relevant amounts of metabolite in the course of theudcell absorption experiment.udVerapamil, Digoxin and Quinidine were studied in a wide concentration range individuallyudand in binary mixtures to determine the significance of the interplay of passive diffusionudand apical efflux on absorption and how this is affected by concomitant administration ofuda second drug. Passive permeability coefficients were independent of concentration andudvaried among the three drugs over at least a ten-fold range. The rate of apical massudefflux varied between the three drugs, increased with concentration and seemed to leveludoff for at least one of the three drugs in the studied concentration range. The dominanceudof one mechanism over the other depended on concentration in a different pattern for theududthree compounds. Thus, the outcome of the combination of passive permeation andudapical efflux for apical to basal absorption can only be predicted if the passiveudpermeability coefficient and the concentration dependence of the carrier mediated effluxudrate are known. All three drugs were substrates of apical efflux carriers. In binaryudmixtures, they commonly reduced the efflux rate of the concomitant compound. Thisudreduction was mutual yet its extent varied between the compounds. Hence, these apicaludefflux carrier substrates may also act as inhibitors and vice versa exhibiting at least audpartially overlapping specificity.udIn conclusion, the introduced model approach and data analysis provide a quantitativeudinsight into the process of drug absorption which can be used for a better understandingudand potentially as a means supporting the prediction of in vivo absorption based on celludculture data and the delineated effect of the involved mechanisms.