Caco carrier-mediated transport

Hilgendorf, C. Spahn-Langguth,H. Regardh,C. G. Lipka, E. Amidon,G. L. Langguth, P., Caco-2 vs Caco-2/HT29-MTX co-cultured cell lines Permeabilities via diffusion, inside- and outside-directed carrier-mediated transport, J. Pharm. Sci. 89, 63-75 (2000). 

The rat intestinal cell line IEC-18 has been evaluated as a model to study small intestinal epithelial permeability. This cell line forms very leaky monolayers with TER of 50 n cm2 and permeability to mannitol of 8 x 10-6 cm s 1. The IEC-18 model was proposed to be a better model than the Caco-2 monolayers for evaluating the small intestinal paracellular permeation of hydrophilic molecules. However, the leakier paracellular pathway is related to the poor differentiation level of the cells and an undeveloped paracellular barrier lacking peri-junctional actin-belt. In addition, due to the poor differentiation the cells have minute expression of transporters and are therefore not useful for studies of carrier-mediated transport [82, 84]... 

Several attempts have been made to estimate the dose required in humans in relation to a drug s potency, and to put this into the context of solubility and permeability for an optimal oral drug [2, 3]. A relatively simple example of this is where a 1.0 mg kg-1 dose is required in humans, then 52 pg mL"1 solubility is needed if the permeability is intermediate (20-80%) [3]. This solubility corresponds approximately to 100 pM of a compound with a MW of 400 g mol-1. Most screening activities for permeability determinations in, e.g., Caco-2, are made at a concentration of 10 pM or lower due to solubility restrictions. The first implication of this is that the required potency for these compounds needs to correspond to a dose of <0.1 mg kg-1 in humans if the drug should be considered orally active. Another implication would be the influence of carrier-mediated transport (uptake or efflux), which is more evident at low concentrations. This could result in low permeability coefficients for compounds interacting with efflux transporters at the intestinal membrane and which could either be saturated or of no clinical relevance at higher concentrations or doses. 

Molecules with a large molecular weight or size are confined to the transcellular route and its requirements related to the hydrophobicity of the molecule. The transcellular pathway has been evaluated for many years and is thought to be the main route of absorption of many drugs, both with respect to carrier-mediated transport and passive diffusion. The most well-known requirement for the passive part of this route is hydrophobicity, and a relationship between permeability coefficients across cell monolayers such as the Caco-2 versus log P and log D 7.4 or 6.5 have been established [102, 117]. However, this relationship appears to be nonlinear and reaches a plateau at around log P of 2, while higher lipophilicities result in reduced permeability [102, 117, 118]. Because of this, much more attention has recently been paid towards molecular descriptors other than lipophilicity [86, 119-125] (see section 5.5.6.). The relative contribution between the para-cellular and transcellular components has also been evaluated using Caco-2 cells, and for a variety of compounds with different charges [110, 112] and sizes [112] (see Section 5.4.5). 

Hidalgo, I. J., Li, J., Carrier-mediated transport and efflux mechanisms in Caco-2 cells, Adv. Drug Delivery Rev. 1996, 22, 53-66. 

Behrens I, Kissel T (2003) Do cell culture conditions influence the carrier-mediated transport of peptides in Caco-2 cell monolayers Eur J Pharm Sci 19 433-442. 

In addition to screening molecules for intestinal absorption, Caco-2 cells have also been used to study mechanisms of drug transport. For many compounds, intestinal permeation involves a transporter to either aid or limit transepithelial transport. The value of Caco-2 cells in this type of studies is due to the fact that these cells express various membrane transporters relevant to drug absorption.1719-23,28,30 However, when interpreting results of studies that involve carrier-mediated transport, discretion, and scaling factors may be required because of the difference in expression level of transporters between in vitro and in vivo systems.12 Another important consideration in carrier-mediated transport studies is that some transport systems in Caco-2 cells may achieve maximal expression level at different days in culture.17,21,38,74 Thus, validation of Caco-2 cells for mechanistic studies should include the identification of the time for optimal expression of transporters as well as the qualitative evaluation of the transporters to establish that they are representative of the native intestinal transporters. 

Takanaga, H., Tamai, I., and Tsuji, A., pH-dependent and carrier-mediated transport of salicylic acid across Caco-2 cells, /. Pharm. Pharmacol., 46, 567,1994. 

Prognosis of a compounds permeability should be made stressing limitations of the model. There is no bioavailability prognosis from in vitro data - a cellular assay can provide only permeability potential through a biological membrane. The membrane, in most cases CACO-2 cells, is very similar to what we observe in vivo in the small intestine and resembles many characteristics to in vivo enterocytes. CACO-2 cells can be used for prediction of different pathways across intestinal cells. Best correlation occurs for passive transcellular route of diffusion. Passive paracellular pathway is less permeable in CACO-2 and correlations are rather qualitative than quantitative for that pathway. CACO-2 cells are an accepted model for identification of compounds with permeability problems, for ranking of compounds and selection of best compounds within a series. Carrier-mediated transport can be studied as well using careful characterization of transporters in the cell batch or clone as a prerequisite for transporter studies. 

Hidalgo IJ and LiJ. Carrier-mediated Transport and Efflux Mechanisms in Caco-2 Cells. Adv Drug Del Rev 1996 22 53-66. 

Significant differences were revealed when comparing overall gene expression profiles and permeability data obtained from Caco-2 cells with data derived from human enterocytes (GeneChip analysis) these were consistent with observed differences in carrier-mediated transport. It has been concluded that in vivo/in vitro (Caco-2)... 

When applying any of these models it is crucial to understand the main transport mechanisms as well as the metabolic route and characterization of the activity of the transporter/enzyme involved. It is well recognized that the activities of carrier-mediated processes in Caco-2 cells are considerably lower than in vivo [20, 42, 48] therefore, it is crucial to extrapolate in vitro cell culture data to the in vivo situation with great care [18, 20, 42, 48], This is especially important when carrier-mediated processes are involved, as evidenced by a recent report which showed significant differences in gene expression levels for transporters, channels and metabolizing enzymes in Caco-2 cells than in human duodenum [48], If an animal model is used, then potential species differences must also be considered [18, 20, 45],... 

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. 

Figure 9.14 The effective permeabilities (Pefr, different clinically relevant concentrations to mean SD) of cimetidine in human jejunum at investigate saturation in any carrier-mediated two clinically relevant luminal concentrations. transport across the intestinal epithelium. No The rate and extent of intestinal absorption of difference in Pefr values between the two cimetidine have been widely discussed, and Fa concentrations was noted, and, togetherwith the for this drug has been estimated at around observation that human permeability in vivo is 75% [90, 91], It has been reported that cimetidine similar to permeability in the Caco-2 model (with is a substrate for both P-gp and/or organic cation low expression of carrier proteins), this suggests transporters (OCNT1 and OCNT2) [82, 92], We that passive diffusion is the dominant determined the human jejunal in vivo Pe - at two mechanism even for cimetidine [82],...

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