Paracellular route

Anderberg, E. K. Lindmark, T. Artursson, P., Sodium caprate ehcits dilations in human intestinal tight junctions and enhances drug absorption by the paracellular route, Pharm. Res. 10, 857-864 (1993). 

The intent of this chapter is to establish a comprehensive framework in which the physicochemical properties of permeant molecules, hydrodynamic factors, and mass transport barrier properties of the transcellular and paracellular routes comprising the cell monolayer and the microporous filter support are quantitatively and mechanistically interrelated. We specifically define and quantify the biophysical properties of the paracellular route with the aid of selective hydrophilic permeants that vary in molecular size and charge (neutral, cationic, anionic, and zwitterionic). Further, the quantitative interrelationships of pH, pKa, partition... 

While both paracellular and passive transcellular pathways are available to a solute, the relative contribution of each to the observed transport will depend on the properties of the solute and the membrane in question. Generally, polar membrane-impermeant molecules diffuse through the paracellular route, which is dominated by tight junctions (Section III.A). Exceptions include molecules that are actively transported across one or both membrane domains of a polarized cell (Fig. 2). The tight junction provides a rate-limiting barrier for many ions, small molecules, and macromolecules depending on the shape, size, and charge of the solute and the selectivity and dimensions of the pathway. 

It is assumed that the convective flow of water across the ABL, cell mono-layer, and filter owing to pressure gradients is negligible and that the cell mono-layer is uniformly confluent. When these conditions are not met, Katz and Schaeffer (1991) and Schaeffer et al. (1992) point out that mass transfer resistances of the ABL and filter [as described in Eq. (21)] cannot be used simply without exaggerating the permeability of the cell monolayer, particularly the paracellular route. An additional diffusion cell design was described by Imanidis et al. (1996). 

III. PARACELLULAR TRANSPORT KINETICS A. Morphology of the Paracellular Route... 

The paracellular route is the pathway of diffusion of a permeant between cells growing in a monolayer. The ability of solutes to move through this space is... 

The plot of permeability coefficient versus molecular radius in Figure 10 shows the interdependence of molecular size and electric charge. The permeability of the solutes decreases with increasing size. The protonated amines permeate the pores faster than neutral solutes of comparable size, and the anions of weak acids permeate the pores at a slower rate. The transport behavior of the ionic permeants is consistent with a net negatively charged paracellular route. These results are phenomenologically identical to those found in the transport kinetics of... 

Table 7 Permeability Coefficients of the Paracellular Route of Unperturbed and Cytochalasin D-Perturbed MDCK Cell Monolayers at 25°C... 

To estimate the relative importance of the tight junction and the lateral space composing the paracellular route, let us consider the permeability of mannitol across Caco-2 and MDCK cell monolayers. The results taken from earlier examples are presented below ... 

Figure 16 Correlation of effective pore radius of the paracellular route of rat alveolar epithelial cell monolayers with transepithelial electrical resistance and time in culture. Pore radii were calculated from the data shown in Figure 14.

In Section III, emphasis was placed on flux kinetics across the cultured monolayer-filter support system where the passage of hydrophilic molecular species differing in molecular size and charge by the paracellular route was transmonolayer-controlled. In this situation, the mass transport barriers of the ABLs on the donor and receiver sides of the Transwell inserts were inconsequential, as evidenced by the lack of stirring effects on the flux kinetics. In this present section, the objective is to give quantitative insights into the permeability of the ABL as a function of hydrodynamic conditions imposed by stirring. The objective is accomplished with selected corticosteroid permeants which have been useful in rat intestinal absorption studies to demonstrate the interplay of membrane and ABL diffusional kinetics (Ho et al., 1977 Komiya et al., 1980). 

The perturbation of monolayers with agents (e.g., disodium ethylenediamine tetraacetate, Ca+2-free medium, sodium citrate, cytochalasin D) to open tight junctions and the effect on the transmonolayer flux of permeants are addressed in this section. It has been observed that permeants taking predominantly the trans-cellular route are not affected by perturbants of the paracellular route, compared to extracellular or relatively hydrophilic permeants (Artursson and Magnusson, 1990). Let us put these general observations into a quantitative intepretation in the light of the transmonolayer kinetic studies of steroids in this section and of paracellular permeants in Section III. There are three cases to consider (1) ABL-controlled permeants, (2) monolayer-controlled permeants transported principally by the transcellular route, and (3) monolayer-controlled permeants for which the paracellular route dominates. 

If the paracellular route is perturbed to the extent that the pore radius R is changed from 12 A to 500 A, then F(5/500) 1.0 and Pparacell = 6.1 x 10 6 cm/sec. Although this represents a 12-fold gain from 5 X 10 7 cm/sec, the transcellular route taken by testosterone is yet the dominant pathway of the monolayer. In making the final calculations, we can conclude that the change in the observed / , will be imperceptible. 

Figure 24 Schematic model of passive diffusion of molecular species of a weak base through the transcellular and paracellular routes of a cell monolayer cultured on a filter support.

Eparaceii = permeability of charged species (cationic or anionic) for the paracellular route [Eqs. (45) and (46)]... 

Upon examination of the columns of Pe and PM values for each drug in Table 16, one discovers that the influence of PF is minimal. Therefore, Pe = PM, i.e., permeation of the drugs across the ABL/cell monolayer/filter barriers is governed by the cell monolayer. The remaining questions are (1) To what extent is the transmonolayer diffusional process of uncharged and cationic species gated by the transcellular and paracellular routes and (2) What are the governing factors ... 

Knowing the fraction of nondissociated solutes at pH 7.4 and 6.5, one obtains the permeabilities of the various molecular species across the parallel trans-cellular and paracellular routes of the cell monolayer. Hence, restating Eq. (83) as... 

Figure 27 Relative contributions of the transcellular and paracellular routes for diffusion of the neutral and charged molecular species of (3-blockers across Caco-2 cell mono-layers. APL, alprenolol ATL, atenolol PDL, pindolol PPL, propranolol.

Here, Pe takes into account the aqueous boundary layers (/W), transcellular diffusion with metabolism, the paracellular pathway (Pparaceu), and the filter support (PF). It is assumed that drug diffusing through the paracellular route escapes metabolism and contributes insignificantly to the appearance of intact drug in the receiver. 

The permeability coefficient of the diffusional-bioconversion pathway can be delineated with the aid of Eq. (Ill) once the permeability coefficients of the ABL, filter support, and paracellular routes are known (Table 18). It is seen that 98% of the diester molecules passing through the cell monolayer take the intracellular route. 

Figure 6 Epithelial penetration routes for topically applied drugs. The transcellular route (1) is preferred by lipophilic drugs, while the paracellular route (2) is preferred by hydrophilic drugs.

The permselectivity of the corneal and conjunctival paracellular routes was investigated by Huang et al. [159] in an attempt to show that nutrients can be extracted from the blood by the conjunctiva. Neither the blood vessels supplying the conjunctiva nor its basement membrane are rate-limiting to the transport of horseradish peroxidase. This 40 kDa tracer is restricted underneath the conjuncti-... 

Several of the postulated roles for nematode-secreted AChEs assume that they gain access to the intestinal mucosa. Several possibilities exist for transport of parasite AChE across the epithelial cell barrier, such as (i) utilization of existing pathways for receptor-mediated transcytosis (ii) a paracellular route facilitated by parasite-secreted proteases as observed for a bacterial elastase (Azghani et al., 1993) and (iii) increased paracellular permeability resulting from inflammatory events in the mucosa. We consider the latter suggestion most likely, as this has been duplicated by ex vivo perfusion with rat mast cell protease II (Scudamore et al., 1995). Moreover, cholinergic stimulation attenuates epithelial barrier properties to macromolecules in rat ileal crypts (Phillips et al., 1987). 

Below 0 Intestinal and CNS permeability problems. Susceptible to renal clearance. If MW < 300, may be absorbed by the slower paracellular route. 

For the evaluation of a possible relationship between the molecular structure of a potential candidate and its transport abilities to cross the epithelial membrane of the gut, the mechanism or route of transport must be known [1,4]. This is due to the structural requirements for the transcellular route being different from the paracellular route. During the lead optimization phase - when many mechanistically based studies are performed - the cell culture-based models can also be used with great confidence. 

Fig. 15.2. Physicochemical molecular descriptors affect the transport route utilised across the intestinal epithelium. To passively diffuse through the membrane (1), the compound (here illustrated with testosterone) should preferably be small, with a molecular weight <500 Da, as well as uncharged and fairly lipophilic. However, compounds that are too lipophilic can stick to the membrane and will not pass through the cells. The paracellular route (2), here exemplified with mannitol, is mainly utilised by smaller (Mw < 200 Da)...

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