LITERATURE REVIEW
2.7 In vitro tools for predicting drug permeability
2.7.1 Cell-based methods
The first practical method for in vitro screening came in the form of cell monolayers. This system utilizes specific cell types that are grown on porous membranes, and which mimic the intestinal epithelial cell layer once differentiated. Cells are plated on a specialized two chamber insert which has an apical (A) side and a basolateral (B) side (Figure 2.6) between the porous membranes containing the monolayer. Test compounds are placed in buffer at the apical side whilst the basolateral side is exposed to buffer only. As time progresses, passive diffusion is verified by the transfer of the compound from the apical to the basolateral side through the cell monolayer. Analysis is done via HPLC or liquid chromatography/mass spectrometry (LC/MS) techniques using aliquots from both the compartments. This apical to basolateral (A>B) experiment quantifies permeability in the absorptive direction, which models absorption in the GI tract. This permeability assessment has been correlated by several research groups to absorption in the small intestine or to fraction of dose absorbed.
When such a correlation is established, it can assist medicinal chemists with predicting in vivo absorption of their compounds [54].
One important consideration when growing the cell monolayer is that the monolayer is kept intact (with no “spaces”). If this is not maintained, the compound could diffuse through the porous membrane, resulting in a false positive. Evaluation of the monolayer integrity is tested using trans-epithelial electrical resistance (TEER). The TEER value indicates if the cell layer is forming a continuous network of tight junctions and low TEER values represent a high level of paracellular permeation. Many devices to measure the TEER value are commercially available [54].
21 Figure 2.6 Cell permeability cell culture assay. Reproduced from Kerns and Di [54].
2.7.1.1 The Caco-2 cell line
The Caco-2 cell line was first established from a moderately well differentiated colon adenocarcinoma and was isolated from a 72-year old patient [55]. Caco-2 cell lines are said to be the gold standard when it comes to in vitro cell culture screening for permeability of new chemical entities. It is a polyclonal cell line, that is it consists of a heterogeneous population of cells, which means that the properties of the cells may change with time in culture [14].
They differentiate spontaneously in culture and remarkably mimic the functional and structural characteristics of mature human enterocytes [56]. The cells differentiate into a uniform monolayer with polarization, apical brush border microvilli, villi expression and dome formation [55, 56]. Furthermore the electrical properties (TEER values) and the ionic conductivity characteristics of these monolayers are similar to colonic crypt cells [55]. The time for these cells to reach full differentiation is approximately 10 days with confluency being reached at around 21 days [57]. The differentiated cells exhibit high levels of enzymes that are usually present in the brush border microvilli of human enterocytes (alkaline phosphatase, sucrose, amino peptidase etc.) [56]. Caco-2 cells produce tight junctions between their cells and contain many of the transporters that are found within the intestinal barrier. In addition, a distinctive property of this cell line is the expression of the P-gp protein. Although with certain research outputs, concentrating only on permeability, this does not represent a positive attribute of the cell line. However, its expression does open up research opportunities regarding this protein.
22 Numerous researchers have made use of this cell line due to its qualitative relationship with cellular transport and in vivo drug permeability [58]. There are extensive applications of Caco-2 cells which include studies regarding the transport mechanisms and analysing the permeability attributes of many amino acids, protein peptides, inhibitors and a series of drugs.
Extensive research using this cell line was done by Artusson and co-workers as well as Karlsson and co-workers who exploited the passive and active transport of these cells [19, 59-62]. The transport of drugs across the intestinal epithelium may occur by one or more of four different routes, namely the passive transcellular and paracellular routes, the carrier mediated route and by transcytosis. Caco-2 monolayers have been used to study drug transport by all four routes.
It has been reported however that the Caco-2 cell line does exhibit some characteristics of the colonic region of humans such as poor paracellular permeability and the high TEER values resemble colonic region properties which differs from the intestinal epithelium thus creating a disadvantage for this model [19, 20]. Other disadvantages are the extended time period that these cells take to differentiate (21 days) and the fact that many laboratories have reported diverse results when testing the same compounds. The heterogeneous nature of these cells explains the difference in morphology, paracellular permeability, and expression of enzymes and transporters reported by researchers [28, 63].
2.7.1.2 The MDCK cell line
Madin Darby canine kidney (MDCK) cells were derived from dog kidney by Madin and Darby in the 1950’s, and is thought to represent a distal tubular phenotype. They form monolayers that represent columnar epithelial cells with tight junctions when cultured on semi-permeable membranes. Ion channels and pumps are arranged on the membrane in a polarized fashion [64]. The MDCK cell line is amongst the best studied epithelial cell lines with respect to genetics, lipid composition, expression of proteins and other parameters. More recently, MDCK cells have also been used for transport studies [65, 66]. Although similar to Caco-2 cells in terms of morphology, MDCK cells have been proven more advantageous in high throughput screening (HTS) of a compound’s passive diffusion ability across the intestinal barrier. This is due to the fact that MDCK cells take a shorter time (2-7 days) to differentiate as well as display lower TEER values when compared to the Caco-2 cells. There are two distinct sub-clones of MDCK cells, MDCK strain I (MDCKI) that exhibit tight monolayers with high TEER values and MDCK strain II (MDCKII) that form monolayers with more “leaky” tight junctions with lower TEER values [67, 68]. The latter is preferred when dealing with drug transport studies because the “leakiness” resembles the human intestine more closely [69].
23 An attractive characteristic of MDCK cells is that they have a low expression of P-gp;
therefore the passive diffusion of drug transport can be studied without the hindering effects of P-gp. However they do express endogenous canine transporters due to the species difference, which is likely to be different from the transporters found in the human intestine, therefore it is not applicable to use these cells for active drug transport predicting active uptake or efflux mechanisms. However, there is still vast application using this cell line. One of the most studied applications apart from prediction of passive permeability is the genetic engineering of these cells to produce cells that overexpress P-gp (MDR1-MDCK).
Additionally these cells grow with more tight junctions and are generally used to predict permeability in blood brain barrier studies (BBB) [70].
2.7.1.3 The HT29 cell line
HT29 cells are also another well studied human colon carcinoma cell line. The wild type of this cell line when cultured under standard conditions (glucose containing media) grows in an unpolarised, undifferentiated multilayer which does not resemble enterocytes thus proving irrelevant in intestinal permeability studies. However when these cells are grown in a galactose supplemented medium they grow as differentiated polarized monolayers of absorptive and/or mucus secreting goblet cells [68]. Several mucus producing sub-lines have been established from human HT29 cells [71].The most popular clones are the HT29-H and the H29-MTX (methotrexate-induced cells), which produce mucus producing monolayers with enterocyte characteristics (highly polarized cells, which express microvilli, hydrolases and resemble goblet cells). These mucus-layer producing variants have attracted some interest for two main reasons; the mucus layer covering the intestinal epithelium in vivo may limit the absorption of some drugs and Caco-2 and MDCK cell lines do not secrete this barrier [68].With the exception of a few studies, the role of mucus on drug transport has been largely ignored. To remedy this issue, co-cultures of Caco-2 and H29-H and H29-MTX have been investigated, however these are not found in wide application in drug discovery [72-74].