Paracellular Uptake

Bypassing intestinal transmembrane transporters mainly by a paracellular absorption would avoid or limit exposure of the substrate to these efflux pumps. Improved paracellular uptake can be achieved by using fatty acids, calcium chelators such as EDTA, papain, bromelain, surfactants, chitosans, polyacrylic acid or thiolated polymers. 

Among hydrophilic polymers, PAA-based systems turned out to be of particular interest showing the capability of opening epithelial tight junctions, which are mainly responsible for limited paracellular uptake of hydrophilic macromolecules. Calcium-binding ability of these polymers... 

For cellular models, a more compUcated form of the above equation is needed, to factor in paracellular, facilitated uptake and effiux transport, etc. [22].)... 

PAMPA is typically used to make a prediction of the passive, transcellular absorption of a compound. Compounds which may be absorbed by a paracellular mechanism or may be substrates for active transport (uptake or efflux) are usually better assessed in a cell based system. A combination of assays can be applied to gain a greater understanding of the permeability and transport properties of a compound. 

Solute uptake can also be evaluated in isolated cell suspensions, cell mono-layers, and enterocyte membrane vesicles. In these preparations, uptake is normalized by enzyme activity and/or protein concentration. While the isolation of cells in suspension preparations is an experimentally easy procedure, disruption of cell monolayers causes dedifferentiation and mucosal-to-serosal polarity is lost. While cell monolayers from culture have become a popular drug absorption screening tool, differences in drug metabolism and carrier-mediated absorption [70], export, and paracellular transport may be cell-type- and condition-depen-dent. 

Culture protocols have been published which describes an accelerated differentiation process where monolayers are ready to be used after 3-7 days of culture [90-92]. One of these systems, the so-called BD BioCoat Intestinal Epithelium Differentiation Environment, is commercially available through BD Bioscience. This system is described to produce monolayers of a quality that are comparable with the typical Caco-2 cells with respect to permeability for drugs transported transcellularly. The paracellular barrier function is however low, as indicated by high mannitol permeability and low TER. The functional capacity for active uptake and efflux is not as thoroughly characterized as for the standard Caco-2 mono-layers. 

Figure 4.6 Likely mechanisms by which macromolecules cross cellular barriers in order to reach the bloodstream from (in this case) the lung. Transcytosis entails direct uptake of the macromolecule at one surface via endocytosis, travel of the endosome vesicle across the cell, with subsequent release on the opposite cell face via exocytosis. Paracellular transport entails the passage of the macromolecules through leaky tight junctions found between some cells...

Bourdet DL, Pollack GM, Thakker DR (2006) Intestinal absorptive transport of the hydrophilic cation ranitidine A kinetic modeling approach to elucidate the role of uptake and efflux transporters and paracellular vs. transcellular transport in Caco-2 cells. Pharm Res 23 1178-87. 

Major transport pathways in Caco-2 monolayers. A Passive transcellular B Passive paracellular C Transporter-mediated apical uptake D Transporter-mediated apical efflux E Transporter-mediated basolateral efflux F Transporter-mediated basolateral uptake. 

Cell culture models are routinely used to assess permeability of new potential drug candidates. The simplicity and higher throughput of these models makes them a useful alternative to in vivo studies. These models are used to predict absorption in vivo, rank order compounds and examine absorption mechanism. Transcellular, paracellular, active uptake and efflux mechanisms can be studied with these models. 

The availability of human Pgfr data is more limited than Fa% data and is biased towards highly absorbed compounds. Therefore, the use of in vivo P ff data is more limited for understanding incompletely absorbed compounds which may be subject to paracellular transport or active uptake and efflux mechanisms. 

The use of in vitro models for prediction of compounds that are predominantly absorbed passively by the transcellular route is generally good with these models. Predicting compounds which are absorbed paracellularly or via active uptake or efflux mechanisms is more difficult. There is a lack of understanding of expression levels of transporters in the gut, which makes in vivo predictions difficult. 

The most efficient rectal absorption enhancers, which have been studied, include surfactants, bile acids, sodium salicylate (NaSA), medium-chain glycerides (MCG), NaCIO, enamine derivatives, EDTA, and others [45 17]. Transport from the rectal epithelium primarily involves two routes, i.e., the paracellular route and the transcellular route. The paracellular transport mechanism implies that drugs diffuse through a space between epithelial cells. On the other hand, an uptake mechanism which depends on lipophilicity involves a typical transcellular transport route, and active transport for amino acids, carrier-mediated transport for (3-lactam antibiotics and dipeptides, and endocytosis are also involved in the transcellular transport system, but these transporters are unlikely to express in rectum (Figure 8.7). Table 8.3 summarizes the typical absorption enhancers in rectal routes. 

Permeation of mAbs across the cells or tissues is accomplished by transcellular or paracellular transport, involving the processes of diffusion, convection, and cellular uptake. Due to their physico-chemical properties, the extent of passive diffusion of classical mAbs across cell membranes in transcellular transport is minimal. Convection as the transport of molecules within a fluid movement is the major means of paracellular passage. The driving forces of the moving fluid containing mAbs from (1) the blood to the interstitial space of tissue or (2) the interstitial space to the blood via the lymphatic system, are gradients in hydrostatic pressure and/or osmotic pressure. In addition, the size and nature of the paracellular pores determine the rate and extent of paracellular transport. The pores of the lymphatic system are larger than those in the vascular endothelium. Convection is also affected by tortuosity, which is a measure of hindrance posed to the diffusion process, and defined as the additional distance a molecule must travel in a particular human fluid (i. e., in vivo) compared to an aqueous solution (i. e., in vitro). 

Generally, low molecular mass permeation enhancers can be divided into transcellular and paracellular permeation enhancers. On the one hand the potential of permeation enhancers to open the paracellular route of uptake can be determined by the reduction in the transepithelial electrical resistance (TEER) (enhancement potential = EP). On the other hand the potential of permeation enhancers to open the transcellular route of uptake can be determined by the lactate dehydrogenase (LDH) assay (LDH potential = LP). The parameter K = (EP—LP)/EP represents the relative contribution of the paracellular pathway. Consequently, a K value of 0 means predominantly transcellular and a K value of 1 means predominantly paracellular. Based on this classification system Whitehead and Mitragotri classified over 50 low molecular mass permeation enhancers showing that most of them are paracellular and only a few of them are transcellular permeation enhancers (2008). 

In case of systemic delivery of nucleic acid drugs via the oral route only comparatively small therapeutic agents such as oligonucleotides seem to reach the systemic circulation in significant quantities via the paracellular route of uptake. 

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