Caco-2 permeability studies

Caco-2 cells have been valuable in the estimation of drug absorption potential, transport mechanisms, and effect of permeation enhancers on transepithelial transport.35,39,53,67-69,78-81 Owing to the sensitivity of the cells and the limited solubility of new molecular entities, Caco-2 permeability studies are routinely done with relatively low concentration of compounds. One way to increase the solubility of these compounds is to use organic solvents. The low tolerability of Caco-2 cells to organic solvents limits the use of this approach in permeability studies. 

VolSurf was also successfully applied in the literature to predict absorption properties [156] from experimental drug permeability data of 55 compounds [165] in Caco-2 cells (human intestinal epithelial cell line derived from a colorectal carcinoma) and MDCK cell monolayers (Madin-Darby canine kidney). In this interesting case, it was shown that models including counterions for charged molecules clearly show significantly better quality and overall performance. The final model was also able to correctly predict, to a great extent, the relative ranking of molecules from another Caco-2 permeability study by Yazdanian et al. ]166]. 

The permeability of the drug substance can be determined by different approaches such as pharmacokinetic studies in humans (fraction absorbed or mass balance studies) or intestinal permeability studies (in vivo intestinal perfusion studies in humans or suitable animal models or in vitro permeation studies using excised intestinal tissue or epithelial cell culture monolayers like CaCo-2 cell line). In order to avoid misclassification of a drug subject to efflux transporters such as P-glycoprotein, functional expression of such proteins should be investigated. Low- and high-permeability model... 

Other cell lines used in permeability studies include the T84 human colonic adenocarcinoma colonic crypt cell model. This line has a reduced carrier expression, secrets mucus, and has very high resistance [31, 32], The IEC cell line is a rat fetal intestinal epithelium cell with higher permeabilities than Caco-2 cells [33], LLC PKi is a pig kidney epithelial cell line with low expression of efflux systems, but expression systems for transport proteins [32], 2/4/A1 cells are a conditionally immortalized rat fetal intestinal epithelium line with crypt cell-like morphology and temperature-sensitive differentiation [34], They form differentiated monolayers with tight junctions, increased brush border enzymes when grown on extracellular matrices with laminin. Transport of drugs with LP in 2/4/A1 monolayers was comparable to that in the human jejunum and up to 300 times faster than that in Caco-2 monolayers. In contrast, the permeability of HP drugs was comparable in both cell lines [34],... 

Although the potential to use Caco-2 cells to screen large numbers of formulations under carefully controlled experimental conditions appears attractive, the assumption that this model is suitable to evaluate drug formulations should not be made. In fact, the utility of Caco-2 cells in formulation evaluation is limited because these cells are sensitive to pharmaceutical excipients.112 In addition, the dilution of formulations into the simple buffer solutions used in permeability studies is likely to break the physical integrity of the... 

The setup of this membrane permeability study involves the use of culture inserts that contain Caco-2 cells grown as epithelial layers. A drug candidate is delivered to the apical side of the cell mono-layer (donor) and allowed to incubate for approximately 60 min. Samples from the apical and basolateral (recipient) side are collected for analysis by LC/MS/MS. Membrane permeability is expressed as the percentage of substrate transported across the monolayer from the apical to the basolateral side. 

The use of Caco-2 cell permeability studies has resulted in more accurate oral bioavailability predictions. Using the predicted hepatic clearance for compound X in humans (see above), estimating Fa by extrapolation from the Caco-2 cell Papp and assuming hepatic blood flow for humans (see, for example Rane et al., 1977) of 20 ml min 1 kg the human oral bioavailability of 69-98% is predicted for compound X. This compares well with the known oral bioavailability of this compound in rats and dogs (83 and 72%, respectively). 

R Wei, H Lee, LYT Li, JN Kryanos. Development of a high throughput analytical technique for compound permeability studies using Caco-2 Cells and HPLC/MS. Proceedings of the 47th American Society for Mass Spectrometry Conference, Dallas, TX, 1999. 

Caco-2 cells are currently used at all levels of pharmaceutical research and development. Automation technologies allow tissue culture labs to easily maintain a large number of Caco-2 cells as well as to perform numerous permeability studies without the introduction of many common human errors. Combinatorial chemistry provides vast arrays of compounds, and Caco-2 assays can be used to assess potential permeability and metabolic issues before much money is invested in the candidate.6... 

The Calu-3 human submucosal gland cell line forms polarized cell monolayers with tight junctions, produces mucus, and develops apical cilia when grown at an air interface. Transport studies can be performed after 10-14 days in culture, and it has been shown that Calu-3 cells express P-gp and actively transport amino acids, nucleosides, and dipeptide analogs organic anions, organic cations, polyamines, and efflux pump substrates are not actively transported.43,51,53-56 Because Calu-3 cells are not subject to the influence of multiple in vivo cell types, the expression of carrier proteins and enzymes may not reflect in vivo levels. Nevertheless, values obtained in Calu-3 permeability studies correlate well with those obtained from primary cultured rabbit tracheal epithelial cells and in vivo rat lung absorption studies.54 Mannitol permeation in Calu-3 cells is about 10 times less than that in vivo, but this is the same ratio difference between Caco-2 cells and in vivo intestinal epithelium.51... 

FIGURE 4 Plot of the success of simultaneous optimization of pK, and predicted absorption over time for an optimization project at Pharmacopoeia Laboratories. Predicted absorption classes (ABSCLS) are 0 (good), I (moderate, 2 (poor), and 3 (bad). Caco-2 cell permeability studies confirm the general trend of absorption predictions (results not shown). 

Laitinen, L. et al., N-in-one permeability studies of heterogeneous sets of compounds across Caco-2 cell monolayers, J. Pharm. Res., 20, 187, 2003. 

The parallel artificial membrane permeability assay (PAMPA) is a recent development in the area of artificial membranes that appears to offer considerable potential. Measuring the flux values (membrane permeation levels) of a range of test compounds by PAMPA and relating these values to the flux curves obtained in Caco-2 studies have shown good correlations, indicating that the PAMPA assay could be a good alternative to Caco-2 cells for the measurement of passively diffusing compounds. 

X 10 cm/s high permeability Papp Often, bi-directional permeability studies (apical to basolateral and basolateral to apical) are performed in Caco-2 cells to evaluate both passive diffusion and efflux potential. In this... 

Another relatively new lipophilicity scale proposed for use in ADME studies is based on MEKC [106]. A further variant is called BMC and uses mobile phases of Brij35 [polyoxyethylene(23)lauryl ether] [129]. Similarly, the retention factors of 16 P-blockers obtained with micellar chromatography with sodium dodecyl sulfate as micelle-forming agent correlates well with permeability coefficients in Caco-2 monolayers and apparent permeability coefficients in rat intestinal segments [130]. 

Calculated molecular descriptors including H-bond parameters were used for QSAR studies on different types of permeabiUty. For example, the new H-bond descriptor characterizing the total H-bond ability of a compound, was successfully appUed to model Caco-2 cell permeability of 17 drugs [30]. A similar study on human jejunal in vivo permeabiUty of 22 structurally diverse compounds is described in Ref. [62]. An exceUent one-parameter correlation of human red ceU basal permeabiUty (BP) was obtained using the H-bond donor strength [63] ... 

Yamashita et al. [82] also studied the effect of BSA on transport properties in Caco-2 assays. They observed that the permeability of highly lipophilic molecules could be rate limited by the process of desorption off the cell surface into the receiving solution, due to high membrane retention and very low water solubility. They recommended using serum proteins in the acceptor compartment when lipophilic molecules are assayed (which is a common circumstance in discovery settings). 

Measurements of Pe in fixed-pH solutions but at various different stirring speeds need to be made. The double-reciprocal analysis, HPe versus 1/v , for Caco-2 permeability measurements in the Transwell (Corning Costar) system produced a linear plot for x- 0.8 [514]. The intercept yields the membrane permeability for the particular pH value in the study the slope determines the k constant. From the analysis of testosterone transport, for the stirring speed of 25 rpm (planar rotating shaker), the thickness of each UWL (assuming symmetric geometry) was calculated to be 465 pm at 150 rpm, haq= 110 pm [514], Karlsson and Artursson [512] found x = 1.0 to best represent their stirring-based analysis of the UWL permeability. 

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