Application of method suitability for drug permeability classification

Abstract

Experimental models of permeability in animals, excised tissues, cell monolayers, and artificial membranes are important during drug discovery and development as permeability is one of several factors affecting the intestinal absorption of oral drug products. The utility of these models is demonstrated by their ability to predict a drug's in vivo intestinal absorption. Within the various permeability models, there are differences in the performance of the assays, along with variability in animal species, tissue sources, and cell types, resulting in a variety of experimental permeability values for the same drug among laboratories. This has led to a need for assay standardization within laboratories to ensure applicability in the drug development process. Method suitability provides a generalized approach to standardize and validate a permeability model within a laboratory. First, assay methodology is optimized and validated for its various experimental parameters along with acceptance criteria for the assay. Second, the suitability of the model is demonstrated by a rank order relationship between experimental permeability values and human extent of absorption of known model compounds. Lastly, standard compounds are employed to classify a test drug's intestinal permeability and ensure assay reproducibility and quality. This review will provide examples of the different aspects method suitability for in situ (intestinal perfusions), ex vivo (everted intestinal sacs, diffusion chambers), and in vitro (cell monolayers, artificial membranes) experimental permeability models. Through assay standardization, reference standards, and acceptance criteria, method suitability assures the dependability of experimental data to predict a drug's intestinal permeability during discovery, development, and regulatory application.

Fig. 1

Demonstration of method suitability from a 21-day Caco-2 assay in 12-well format (30). Triangle HP drugs, Circle LP drugs, Square HP-IS, Dashed line LP/HP boundary, Dotted line 90% absorption

Fig. 2

Rat in situ perfusion assays from a Kim et al. (20) and b Zakeri-Milani et al. (45). Triangle HP drugs, Circle LP drugs, Dashed line LP/HP boundary, Dotted line 90% absorption, Square outlier drug

Fig. 3

Rat ex vivo diffusion assay from Ungell et al. (18) in the a jejunum, b ileum, and c colon. Triangle HP drugs, Circle LP drugs, Square outlier drugs, Dashed line LP/HP boundary, Dotted line 90% absorption

Fig. 4

Frog ex vivo everted gut sac assay from Trapani et al. (16). Triangle HP drugs, Circle LP drugs, Dashed line LP/HP boundary, Dotted line 90% absorption

Fig. 5

Caco-2 cell monolayers from a Bock et al. (14) in a 12-well, 21-day assay, b Lentz et al. in a six-well, 4-day assay (53), and c Withington (54) in a 24-well, 3-day assay. Triangle HP drugs, Circle LP drugs, Dashed line LP/HP boundary, Dotted line 90% absorption

Fig. 6

Caco-2 cell monolayers in a 96-well, 7-day assay from Alsenz et al. (55) with AP buffer at a pH 7.4 or b pH 6.5. Triangle HP drugs, Circle LP drugs, Square outlier drug, Dashed line LP/HP boundary, Dotted line 90% absorption

Fig. 7

MDCK-MDR1 cell monolayers in a 3- to 4-day, 24-well assay from Thiel-Demby et al. (56) at a pH 7.4 or b pH 5.5. Triangle HP drugs, Circle LP drugs, Square outlier drugs, Dashed line LP/HP boundary, Dotted line 90% absorption

Fig. 8

Artificial membrane assays from a Flaten et al. (vesicle membrane) (59) and b Corti et al. (diffusion cell) (60). Triangle HP drugs, Circle LP drugs, Square outlier drugs, Dashed line LP/HP boundary, Dotted line 90% absorption