Epithelial cell layer

The tissue distribution of P-glycoprotein yields important clues to its function. In most tissues it is localized to the apical (luminal) membrane of polarized epithelial cell layers. This location suggests that P-glycoprotein extrudes its substrates from the epithelial cells into the adjacent lumen. It is anticipated that P-glycopro-tein plays an important role as a protective mechanism against naturally occur-... 

In situ models are to evaluate absorption or membrane permeability under the physiologically relevant tissue condition. While the luminal environment can be modulated by the administered solution, the tissue condition is physiologically controlled. The estimated membrane permeability can be, in most cases, assumed to represent the transport across the epithelial cell layer at steady state or quasisteady state. However, one needs to be aware that the involvement of metabolic degradation, which may occur at the cellular surface or within the cytosol, can be a factor leading to biased estimates of membrane permeability and erroneous interpretation of the transport process. Particularly,... 

Ehrhardt C, Kneuer C, Bies C, Lehr C-M, Kim K-J, Bakowsky U (2005) Salbu-tamol is actively absorbed across human bronchial epithelial cell layers. Pulm Pharmacol Ther 18 165-170. 

Physiological Buffer Systems Recently, a lot of efforts have been made on how to increase the biorelevance of the Caco-2 model [63, 47, 64, 65,105], Historically, the media used for Caco-2 experiments were buffered at pH 7.4 on both sides of the monolayer. The pH in the cellular interstice and blood compartment is known to be 7.4. However, the pH in the upper GI tract under fasted conditions ranges from 5.0 to 6.5, with an acidic microclimate existing just above the epithelial cell layer estimated to be between 5.8 and 6.3 [90], The pH of the apical medium can have a critical effect on the transport of drugs, especially for drugs with a pKa close to 7, or when pH-dependent transporters are involved. 

Figure 9.1 Relationship between the transepithelial electrical resistance (TEER) value of the passage-cultured human nasal epithelial cell layer and permeability of 14C-mannitol (o, passage-2 A, passage-3 , passage-4) and budesonide ( , passage-2 , passage-3 , passage-4). (Data from Ref. [40]).

For AIC conditions, the apical surface of the epithelial cell layer is exposed to air after the nasal cells reached confluence on the Transwell insert, while the basolateral side is fed with culture fluid. Figure 9.3 shows TEER changes in epithelial cell layers cultured up to 20 days in LCC versus AIC methods [46], In AIC condition (initiated from day 3 after seeding), TEER peaked on day 5 and maintained above the TEER values observed for LCC counterparts. By contrast, TEER observed for LCC conditions peaked on day 2 and declined toward zero by day 15. These data indicate that human nasal epithelial cells at an air interface culture exhibit better electrophysiological characteristics than those cultured by the conventional liquid-covered conditions. 

Figure 9.3 Changes in transepithelial electrical resistance (TEER) of human nasal epithelial cell layers grown under LCC ( ) versus AIC (A) conditions. Each data point represents the mean SD of three determinations. (Data from Ref. [46]).

Because mucin and/or cilia systems of AIC cultured epithelial cells may work as a barrier for drug transport, lower Papp values are expected in cell layers cultured in AIC than in LCC methods. However, it was interesting to note that no significant differences in Rapp values were observed between the cell layers cultured with the two methods (Table 9.1). This is in contrast to solute permeabilities reported previously for cell layers cultured with LCC versus AIC methods [76, 80], For example, Yang et al. reported that Rapp of lipophilic solutes (e.g., various /3-blockers) across the primary cultured conjunctival epithelial cell layer are about threefold lower when cultured under AIC than LCC conditions, suggesting that the permeability of AIC cultured cell layers generally better reflects that of the excised tissue than LCC counterparts. Mathias et al. [76] also reported that the permeability of hydrophilic solutes across the primary rabbit tracheal epithelial cell layer cultured under AIC conditions was only half of that observed for cell layers cultured under... 

Table 9.1 Apparent permeability coefficients (Papp) of 14C-mannitol and budesonide across the passage-cultured human nasal epithelial cell layer grown under AIC or LCC conditions for 7 days.

Ehrhardt C, Kneuer C, Laue M, Schaefer UF, Kim KJ, Lehr CM (2003) 16HBE14o- human bronchial epithelial cell layers express P-glycoprotein, lung resistance-related protein, and caveolin-1. Pharm Res 20(4) 545-551. 

Chang JE, Basu SK, Lee VHL. Air-interface condition promotes the formation of tight comeal epithelial cell layers for drug transport studies. Pharm Res 17 670-676 (2000). 

Chang-Lin JE, Kim KJ, Lee VH. Characterization of active ion transport across primary rabbit corneal epithelial cell layers (RCrECL) cultured at an air-interface. Exp Eye Res 80 827-836 (2005). 

Figure 13.3 A flowchart illustrating various steps for the preparation and maintenance of primary rabbit conjunctival epithelial cell layers, cultured under liquid-covered and air-interfaced conditions (See also color insert).

Primary Culture Models of Conjunctival Epithelial Cell Layers... 

J. J. Yang, K. J. Kim, and V. H. Lee. Role of P-glycoprotein in restricting propranolol transport in cultured rabbit conjunctival epithelial cell layers. Pharm Res 17 533-538 (2000)... 

The successful application of in vitro models of intestinal drug absorption depends on the ability of the in vitro model to mimic the relevant characteristics of the in vivo biological barrier. Most compounds are absorbed by passive transcellular diffusion. To undergo tran-scellular transport a molecule must cross the lipid bilayer of the apical and basolateral cell membranes. In recent years, there has been a widespread acceptance of a technique, artificial membrane permeation assay (PAMPA), to estimate intestinal permeability.117118 The principle of the PAMPA is that, diffusion across a lipid layer, mimics transepithelial permeation. Experiments are conducted by applying a drug solution on top of a lipid layer covering a filter that separates top (donor) and bottom (receiver) chambers. The rate of drug appearance in the bottom wells should reflect the diffusion across the lipid layer, and by extrapolation, across the epithelial cell layer. 

No exposure-related clinical signs or lesions of systemic toxicity and no oncogenic responses were observed in rats exposed by inhalation at concentrations of 0, 15, 45, or 135ppm 6 hours/day, 5 days/week, for 24 consecutive months." Dose-related changes occurred in the anterior portion of the olfactory epithelium and consisted of atrophy of the neurogenic epithelial cells followed by progressive hyperplasia of the reserve cells and ultimately loss of the upper epithelial cell layer. Opacity and neovascularization of the cornea were also observed in methyl acrylate-exposed animals. 

The mucus layer also bathes the cilia of ciliated epithelial surface cells and provides a stimulus for ciliary motility (i.e., ciliary beating). The cilia consist of microtubules with a 9 + 2 configuration (nine pairs of peripheral microtubules and two central microtubules) that beat rhythmically to rapidly move mucus from the anterior to the posterior portion of the nasal cavity. To successfully cross the nasal permeability barrier, peptide drugs must penetrate the mucus layer and cross the epithelial cell layer, and do so in a limited time, because mucociliary clearance will limit the time of exposure of the peptide to the absorptive surface [19-21,27-29], Typically, drugs or inspired particles that are delivered nasally are removed via mucociliary clearance, with a clearance time of approximately 15 min in humans however, this transit time can vary from person to person and can be impacted by the addition of mucoadhesive agents to the formulation [30-37],... 

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