Caco-2 cell monolayer system

Table 11 Delineation of Permeability Coefficients of Steroids in Caco-2 Cell Monolayer System... 

The Madin-Darby canine kidney (MDCK) cell model is another commonly used cell monolayer system for the assessment of human intestinal absorption. MDCK cell lines offer a couple of key advantages over Caco-2 cells, including the ability to reach full differentiation in 3-7 days (as opposed to 21 days for Caco-2) as well as the ability to be co-transfected with Pgp, the primary intestinal efflux transporter. Good correlation has been observed between MDCK permeability and human absorption. " The primary disadvantage of the MDCK ceU line is that it is derived from canine, rather than human, cells and, as such, expression of transporters and metaboUzing enzymes is not identical. 

Figure 8 Appearance kinetics of radiolabeled solutes that diffuse across Caco-2 cell monolayers via the paracellular pathway. The Transwell system consisted of a donor and receiver solution at pH 7.4. Stirring by planar rotation up to 100 rpm had no effect. The insert with filter, cell monolayer, and donor were transferred to a new receiver chamber at time intervals to maintain sink conditions.

Figure 18 Linear fluxes of hydrocortisone across Caco-2 cell monolayers in the Transwell system into a receiver sink as a function of stirring (rotary platform shaker) rate at 25 °C.

Figure 20 Correlation of appearance kinetics of steroid permeants across Caco-2 cell monolayers in the Trans well system with log partition coefficients (n-octanol/water) and stirring dependency. 

Figure 21 Linearized double reciprocal plot of the effective permeability coefficients and corresponding stirring rates to determine the power dependency of the stirring rate and mass transfer resistances for the aqueous boundary layers and the Caco-2 cell monolayer in the Transwell system. 

Table 12 Effective Permeability Coefficients and Thicknesses of the Aqueous Boundary Layer of the Caco-2 Cell Monolayer/ Transwell System as a Function of Stirring by Planar Rotating Shaker3...

Using the side-by-side diffusion cell system, Hidalgo et al. (1992) quantified the transflux of testosterone in Caco-2 monolayers at 37°C as a function of the flow rate of the 02/C02 gas mixture (Table 13). They concluded that the kinetics were ABL-controlled and proceeded to calculate the ABL thickness on each side of the diffusion chambers using... 

Figure 22 Correlation between the log permeability coefficient for a series of peptides across a Caco-2 cell monolayer in the Transwell system and A log PC, which is defined as log PC(n-octanol/water) — log PC (isooctane/water). [Redrawn from Burton et al. (1992) with permission from the publisher.]...

Figure 25 Cumulative fraction of the initial donor concentration of [1-blockers that diffused across Caco-2 cell monolayers as a function of donor pH. Transwell systems were used, and stirring was done using a rotary platform shaker. (A), pH 7.4 (B), pH 6.5.

Figure 29 Intrinsic permeability of the monoester of PNU-82,899 using Caco-2 cell monolayers. A Transwell system was used with receiver sink conditions at 25°C. The initial donor concentration was 199 iM, and donor and receiver solutions were at pH 7.4.

The identification and characterization of cell culture systems (e.g., Caco-2-cells) that mimic in vivo biological barriers (e.g., intestinal mucosa) have afforded pharmaceutical scientists the opportunity to rapidly and efficiently assess the permeability of drugs through these barriers in vitro. The results generated from these types of in vitro studies are generally expressed as effective permeability coefficients (Pe). If Pe is properly corrected to account for the barrier effects of the filter (PF) and the aqueous boundary layer (PAbl) as previously described in Section II.C, the results provide the permeability coefficient for the cell monolayer... 

Fig. 37), suggesting that desolvation of the polar bonds in the molecule is a major determinant of permeability. Consistent with this, good correlations were found between the permeabilities of these peptides and their partition coefficients between heptane-ethylene glycol (r2 = 0.87) or the differences in partition coefficients between n-octanol-buffer and isooctane-buffer (r2 = 0.82) both these buffer systems provide experimental estimates of hydrogen-bonding potential. These results are qualitatively identical with those described earlier for the permeability of these peptides across Caco-2 cell monolayers. 

Figure 38 Correlations between appearance permeability coefficients for a related series of peptides measured in mesenteric blood draining perfused rat ileal segments and Caco-2 cell monolayers in the Transwell system. See Table 14 for identification of the peptides. The Pe for the rat ileum was not corrected for the aqueous boundary layer and blood flow effects. [Redrawn from Kim et al. (1993) with permission from the publisher.]...

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. 

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. 

There are several cell monolayer models that are frequently used for the evaluation of drug permeability and absorption potential (Table 18.1). For a more detailed discussion, please refer to Chap. 8. Caco-2 cells (adenocarcinoma cells derived from colon) are the most extensively characterized and frequently used of the available cell lines [5-9], A unique feature of Caco-2 cells is that they undergo spontaneous enterocyte differentiation in cell culture. Unlike intestinal enterocytes, Caco-2 cells are immortalized and replicate rapidly into confluent monolayers. When the cells reach confluency during culture on a semi-porous membrane, they start to polarize and form tight junctions, creating an ideal system for permeability and transport studies. During the past decade, use of... 

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... 

---