Caco active transport

Ho NFH, PS Burton, RA Conradi, CL Barsuhn. (1995). A biophysical model of passive and polarized active transport processes in Caco-2 cells Approaches to uncoupling apical and basolateral membrane events in the intact cell. J Pharm Sci 84 21-27. 

Some laboratories have found an alternative to the short-term cultures by using cell lines other than Caco-2 cells. The most popular of these is Madin-Darby canine kidney (MDCK) cells, an epithelial cell line from the dog kidney. MDCK cells have been suggested to perform as well as Caco-2 cells in studies of passive drug permeability [56]. These cells have also been used to optimise the conditions for studies of low-solubility drugs [53]. However, as noted previously, the active transport processes of this cell line can be quite different to those of Caco-2 cells [28-30], Another cell line that only requires short-term culture is 2/4/A1, which is a conditionally immortalised rat intestinal epithelial cell line [86]. The 2/4/A1 cell line is discussed in Section 4.3.2.2 below. 

In conclusion, there are several drawbacks to the use of Caco-2 cells in studies of active drug transport. Despite these drawbacks, we note that a recent comprehensive study comparing various P-glycoprotein drug efflux assays in drug discovery came to the conclusion that the Caco-2 transport assay is the method of choice, since it displays a biased responsiveness towards compounds with low or moderate permeability - in other words, towards compounds whose intestinal permeability is most likely to be significantly affected by drug efflux mechanisms [101]. 

Several active transport systems that are normally found in the small intestinal enterocytes have been characterized in the Caco-2 cell model [13]. These include transport systems for glucose [32, 33], amino acids [34-37], dipeptides [38-40], vitamins [41], and bile acids [42, 43]. 

The expression of the active transport systems is time-dependent and may vary with nutritional conditions [53, 54]. The culturing conditions, e.g., the passaging process, can dramatically alter the biological characteristics and transport properties of Caco-2 cell monolayers [55-58]. 

Very few published data exist on the evaluation of automated systems, though one report has been made of an automated absorption assay using Caco-2 cells cultured on both sides of polycarbonate membranes [93], The concept of culturing cells on the lower sides of the membranes was investigated as a means of improving the opportunity to study transport in the secretory basolateral to apical direction. However, this approach resulted in increased variability and impaired active transport properties of the cell monolayers, and was therefore not recommended. 

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... 

A very simple technique is to use a radiolabeled ligand (usually a well-known substrate) of the specific transporter of interest. A recent suggestion for functional quantitation of the apparent affinities (fQ values) to P-gp using Caco-2 cells and the substrate taxol has been published [143], The method can be described simply as (1) determination of b —> a transport of3 H -taxol in normal, untreated Caco-2 cells and (2) determination of b —> a transport of 3H-taxol in the presence of verapamil (0.2 mM). The difference between these two components represents the active transport via P-gp. The two concentrations of the test compound are chosen as approximately 0.25 x K, and 4.0 x K, and for the inhibition of taxol transport, and in the study of Gao et al. [143], 16 pM and 250 pM of the test compound were used... 

The use of Caco-2 cell monolayers has gained in popularity as an in vivo human absorption surrogate moreover, the monolayers are generally accepted as a primary absorption screening tool by several pharmaceutical companies [10]. However, Caco-2 cell permeability measurements exhibit certain limitations due to the mechanisms involved. Both passive and active pathways exist active transport tends to increase the absorption across the cells and, since Caco-2 cells overexpress the P-glycoprotein (P-gp) efflux pump, the absorption of some compounds across these cells may be underestimated. 

The applicability range of any model should be limited to molecules having a similar mechanism of transport. Therefore, we have selected from the literature only those compounds with well-characterized Caco-2 cell permeability and excluded compounds with a high efflux ratio. Known P-gp substrates and actively transported compounds were also excluded from the list. 

Cefadroxyl and cefaclor are beta-lactam antibiotics which show high affinity for the PepTl carrier system, whereas the other two beta-lactams, cephalotin and cef-metazole, are not recognized by PepTl protein and are not actively transported in the intestine. However, as the VolSurf Caco-2 model predicts that all the beta-lactams are nonpenetrating compounds, it is very probable that, as they rely only the diffusion mechanism, cefadroxyl and cefaclor will not cross the cell monolayer. 

Neuhoff S, Ungell AL, Zamora I, Artursson P (2005) pH-Dependent passive and active transport of acidic drugs across Caco-2 cell monolayers. Eur J Pharm Sci 25 211-220. 

Cell monolayers grown on permeable culture inserts form confluent mono-layers with barrier properties and can be used for drug absorption experiments. The most well-known cell line for the in vitro determination of intestinal drug permeability is the human colon adenocarcinoma Caco-2 [20, 21], The utility of the Caco-2 cell line is due to its spontaneous differentiation to enterocytes under conventional cell culture conditions upon reaching confluency on a porous membrane to resemble the intestinal epithelium. This cell model displays small intestinal carriers, brush borders, villous cell model, tight junctions, and high resistance [22], Caco-2 cells express active transport systems, brush border enzymes, and phase I and II enzymes [22-24], Permeability models... 

Ranaldi, G., Islam, K., and Sambuy, Y., D-cycloserine uses an active transport mechanism in the human intestinal cell line Caco-2, Antimicrob. Agent Chemother., 38, 1239, 1994. 

Another in vitro method for permeability screening was parallel artificial membrane permeation assay (PAMPA) initially reported by Kansy. In a PAMPA permeability screen, the Caco-2 cell mono-layer membrane is replaced by an artificially generated membrane. Versions of different artificial membranes that lack active transporter systems and pores have been developed to mimic the in vivo transcellular intestinal epithelial cell barrier. Therefore, the PAMPA screen only measures the intrinsic... 

Mizuma found that some DKPs are actively transported in Caco-2 cell monolayers by PEPTl. The... 

One of the main in vitro permeability assays used in the pharmaceutical industry has been for many years the Caco-2 monolayer. Therefore, most of the in silica models developed to predict permeability were based on Caco-2 data. Hou and Johnson produced a couple of reviews that comprehensibly summarizes the recent efforts using Caco-2 permeability data [92, 94]. All those models are designed to predict the influx or apparent permeability of drugs in the same direction as intestinal absorption occurs, that is, from the apical to the basal side of the cell line, regardless of the extent of active transport involved in the permeation process. 

L-a-methyldopa a substrate for the amino acid transporter. In Caco-2 cells, the active transport of this dmg by the amino acid transporter was seven times higher than transport by passive diffusion. Its absorption may be further increased by upregulating the amino acid transporter, as has been observed in the 20-70% stimulation of carrier-mediated amino acid transport by treatment of 0.2 mg/kg growth hormone. 

Soldner A, Benet L, Mutschler E, Christians U (2000) Active transport of the angiotensine II antagonist losartan and its main metabolite EXP 3174 across MDCK-MDR1 and CACO-2 cell monolayers. Br J Pharmacol 129 1235-1243... 

Nevertheless, several uptake transporters have been studied successfully in CACO-2 cells as possible drug delivery systems. CACO-2 cells are a suitable model for uptake studies given that they are characterized for the studied transporter (Anderle et al. 2003). Whereas permeability values for actively transported compounds may differ substantially from in vivo situation a ranking of compounds for higher or lower permeability seems to be possible. 

---