Transporter Uptake Studies Using CACO-2 Cells

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II.D.6 Transporter Uptake Studies Using CACO-2 Cells... 

Cell lines, such as the Caco-2 and MDCK cells [27, 35, 47, 49, 57, 67, 128-133], have been used frequently to study different transporters in the GI tract. These cell lines have been evaluated for transport both in absorptive and secretory direction and in addition also been transfected with specified transporter systems of interests to yield new clones [23, 31, 72, 79, 80, 134] or co-cultures [135], Some of the uptake transporters belonging to the organic cation transporter (OCT) family have also been identified in cell lines such as the pig kidney cell line LLC-PK1, and MDCK [67, 136]. In fact, its presence in Caco-2 cells needs to be further elucidated as reports have shown both the absence and presence of transporters from this family of transporters [136-138],... 

Both active and passive transport occur simultaneously, and their quantitative roles differ at different concentration gradients. At low substrate concentrations, active transport plays a major role, whilst above the concentration of saturation passive diffusion is the major transport process. This very simple rule can be studied in an experimental system using cell culture-based models, and the concentration dependency of the transport of a compound as well as asymmetric transport over the membrane are two factors used to evaluate the presence and influence of transporters. Previous data have indicated that the permeability of actively absorbed compounds may be underestimated in the Caco-2 model due to a lack of (or low) expression of some uptake transporters. However, many data which show a lack of influence of transporters are usually derived from experiments... 

Clearly, genome-wide studies contribute to a better understanding why differences in absorption along the intestine, and between in vitro and in vivo situations, can be expected. These studies could on the one hand confirm that the most widely used in vitro system to study drug absorption, namely, the Caco-2 cells, may not always be very accurate in predicting uptake rates for a new compound. On the other hand, it has to be noted that compared to other in vitro systems, which are being used to study transport in liver and kidney, the Caco-2 cells correlate certainly the best with the in vivo system with respect to the mRNA expression of transporters [3],... 

Some of the advantages of cell monolayer models include the ability to use human instead of animal cell types as well as the ability to perform cellular uptake and bidirectional cell transport studies for evaluation of absorptive and secretory processes. The potential for automation to achieve higher throughput in the early drug discovery setting is an added attraction. Regardless of the cell type used, the utility of these models in transport studies is based on the correlation between permeability properties determined in these models and those obtained in vivo, such as fraction of dose absorbed (Fig. 3). To date, numerous laboratories have established a correlation between apparent permeability coefficients (P pf) from Caco-2 or MDCK cells and in vivo fraction absorbed of drugs in solution [58—62]. Construction of correlation plots for known compounds or reference markers then provides an opportunity for interpolation of fraction absorbed for NMEs for which in vivo fraction absorbed is unknown. 

Two biotin transporters have been described a multivitamin transporter present in many tissues and a biotin transporter identified in human lymphocytes. In 1997, Prasad and coworkers discovered a Na" "-coupled, saturable, structurally specific transporter present in human placental choriocarcinoma cells that can transport pantothenic acid, lipoic acid, and biotin. This sodium-dependent multivitamin transporter has been named SMVT and is widely expressed in human tissues. Studies by Said and coworkers using RNA interference specific for SMVT provide strong evidence that biotin uptake by Caco-2 and HepG2 cells occurs via SMVT thus, intestinal absorption and hepatic uptake are likely mediated by SMVT. The biotin transporter identified in lymphocytes is also Na coupled, saturable, and structurally specific. Studies by Zempleni and coworkers provide evidence in favor of monocarboxylate transporter-1 as the lymphocyte biotin transporter. 

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