1.Introduction2.General drug absorption mechanisms3.In vitro cell models4.Conclusions5.Expert opinionReviewCell-based in vitro models for predicting drug permeabilityBruno Sarmento, Fernanda Andrade, Sara Baptista da Silva, Francisca Rodrigues, Jose das Neves produce P-gp and establish tight junctions in vitro; differentiate and express several relevant efflux transporters6,11-13TC-7Caco-2 subcloneSimilar to Caco-2 cells but with higher brush-border enzymatic content15,16HT29-MTXHuman colorectal carcinoma cellsMucin-producing cells17-20 MDCKDog kidney epithelial cellsKidney cells from dog, with properties similar to Caco-2 cells25-27IEC-18Small intestinal crypt-derived rat cellsSize-selective barrier for paracellularly transported compounds22,23Caco-2/HT29 co-cultureHuman colorectal carcinoma cellsMimics the small intestinal epithelial layer containing both mucus-producing (HT29) and the columnar absorptive Caco-2 cells20,21 27,28Caco-2/Raji B co-cultureHuman colorectal carcinoma cells/ lmphocytesSimulates the human follicle-associated epithelium; Useful for nanoparticle internalization studies through M cells31-33,36Caco-2/HT29/Raji B co-cultureHuman colorectal carcinoma cells/ lmphocytesMimics different absorption pathways in the same model; useful for mucoadhesive nanoparticle internalization studies through M cells and mucin-producing cells37-39MDCK: Madin-Darby canine kidney.B. Sarmento et al.610Expert Opin. Drug Metab. Toxicol. (2012) 8(5)Expert Opin. Drug Metab. Toxicol. Downloaded from informahealthcare.com by University of Calgary on 05/24/12 For personal use only.of total P-gp in Caco-2 and MDCK cells and differences in the partitioning of substrates across the cell membrane bilayers27. Co-culture models comprising different cells present in the intestinal epithelium have been proposed, to more closely rep- resenting the heterogeneity of the intestinal epithelium at a cellular level5. Many of the co-cultured cell systems may be of greater interest in academic research but are still not common in industry. The majority of co-culture models are based on modifications and improvements of the Caco- 2 cell model. One example is the evaluation of cell monolayer models based on mixtures of Caco-2/HT29 co-culture for their use in screening of drug absorption and intestinal perme- ability in comparison with the properties of the respective monocultures21. The Caco-2/HT29 model offers the possi- bility of studying absorption and the effect of goblets cells simultaneously in drug transport and absorption enhance- ment, being well correlated with in vivo data28and reveals a paracellular permeability closer to the in vivo human situa- tion20. Moreover, it is possible to modify the permeability barrier of the cell monolayers both with respect to paracellular resistance and secretory transport via P-gp21, allowing more flexibility in adapting the in vitro system to the in vivo situa- tion as compared with the monocultures. Another clear advantage of this co-culture model is the evaluation of absorp- tion enhancers and mucoadhesive systems on the permeability of drugs and its high correlation with the in vivo situation, as recently reported by our group29,30. Another promising co-culture monolayer model based on Caco-2 cells and human Raji B lymphocytes has been devel- oped to mimic the human follicle-associated epithelium31-33. Raji B lymphocytes are able to convert enterocytes into M cells34. The Caco-2/Raji B co-culture model appears to be valuable for studying the absorption of large molecules and nanoparticles containing drugs, due to the high transcytotic capacities of M cells and their ability to transport a broad range of materials, including nanoparticles33,35. In fact, the impor- tance of M cells on nanoparticle transcytosis and drug absorption was demonstrated using this co-culture model36. Recently, the co-cultivation of Caco-2/HT29/Raji B cells allowed to reconstitute a cell culture monolayer system that better mimics the intestinal barrier, especially to investigate its interaction with nanoparticles37. Results demonstrated that the Caco-2/HT29/Raji B triple co-culture model may be reliable in obtaining a more physiological, functional and reproducible in vitro model of the intestinal barrier in order to study drug absorption both in solution and when associated with nanocarriers (Figure 1)38,39.3.2Vaginal and rectal models The use of in vitro cell models for studying rectal absorption of drugs has relied mainly on the Caco-2 monolayer model, which has been detailed above40,41. The similarity between these colon-derived cell monolayers and the epithelial layer at the colorectum provides the rationale for this option, althoughthe permeability features of drugs in the rectum are different from the intestine. Experimental work showed analogous results for the permeability of a low molecular weight (MW) heparin for the Caco-2 model and after rectal admin