Epithelia ocular epithelium

The purpose of this chapter is to present overviews of a selection of the major endothelial and epithelial barriers to drug delivery for which there are either primary culture or cell line systems that recapitulate the characteristics of the in vivo barrier. Our objective is to define some general characteristics of cell culture models and highlight the more commonly applied primary cell cultures and cell lines in use today. Specifically, we focus on cell culture models for the intestinal epithelium, blood-brain barrier, pulmonary and nasal epithelium, ocular epithelium, placental barrier, and renal epithelium. Renal epithelium was included here primarily because some cell lines derived from this tissue [e.g., Madin-Darby canine kidney cells (MDCK)] are often used as surrogates for other barriers by pharmaceutical scientists. We have arbitrarily chosen to exclude the skin and liver from the scope of this overview. However, it should be noted that hepatocyte cell culture models, for example, are becoming more widely available and have been the subject of recent reviews.1,2... 

The obtained results confirm earlier findings where vitamin A-deficient rats were used to prove the uptake of retinyl esters into lung, liver, kidney, and plasma after inhalation thereof (Biesalski, 1996). However, long-term topical administration of high vitamin A concentrations is a well-established therapy in atrophic rhinitis, rhinitis sicca, and metaplastic changes in the nasal or ocular epithelium (Deshpande et ah, 1997 Simm, 1980). The application leads to the normalization of mucous membranes and reappearance of a normal function with no side effects. 

Proteolysis. Proteolysis is the cleavage of amide bonds that comprise the backbone of proteins and peptides. The reaction can occur spontaneously in aqueous medium under acidic, neutral, or basic conditions. This process is accelerated by proteases, ubiquitous enzymes that catalyze peptide-bond hydrolysis at rates much higher than occur spontaneously. In humans, these enzymes only recognize sequences of L-amino acids but not d-amino acids. They are found in barrier tissues (nasal membranes, stomach and intestinal linings, vaginal and respiratory mucosa, ocular epithelium), blood, all internal solid organs, connective tissue, and fat. The same protease may be present in multiple sites in the body. 

But, unfortunately, this was not always enough, particularly for eyes bums, because of the ocular epithelium being extremely fragile, or in case of wide bums or bums due to specilic substances such as hydrofluoric acid. 

The carbomer polymeric gel base itself has been used successfully to treat moderate to severe cases of dry eye (keratoconjunctivitis sicca) [282]. The dry eye syndrome is usually characterized by deficiency of tear production and, therefore, requires frequent instillation of aqueous artificial tear eyedrops to keep the corneal epithelium moist. The gel base applied in a small amount provides a prolonged lubrication to the external ocular tissues, and some patients have reduced the frequency of dosing to control their symptoms to three times a day or fewer. 

Although the ocular absorption of peptide as well as nonpeptide drugs is poor [96,196-198], the ocular route is by far the least studied for the usefulness of penetration enhancers. This is in part due to the perceived sensitivity of ocular tissues to irritation and the fear of corneal and conjunctival damage caused by the enhancers. Whereas the rat nasal epithelium may tolerate up to 5% sodium glycocholate [199], ocular administration of sodium glycocholate at a concentration of 2% and beyond induces reddening of the eye and tear production in rabbits (Kompella and Lee, unpublished observation). 

Morimoto et al. [33] demonstrated that the ocular absorption of hydrophilic compounds over a wide range of molecular weights could be increased by 2 and 10 mM sodium taurocholate and sodium taurodeoxycholate in a dose-dependent manner. The compounds were glutathione (307 Da), 6-carboxyfluorescein (376 Da), FTTC-dextran (4 kDa), and insulin (5.7 kDa). Of the two bile salts, sodium taurodeoxycholate was more effective. At 10 mM, this bile salt increased the permeability of 6-carboxyfluorescein from 0.02% to 11%, glutathione from 0.08% to 6%, FITC-dextran from 0% to 0.07%, and insulin from 0.06% to 3.8%. Sodium taurocholate, on the other hand, increased the permeability to 0.13%, 0.38%, 0.0011%, and 0.14%, respectively. Taurodeoxycholate was more effective than taurocholate in the nasal epithelium as well [202], This difference in activities can possibly be attributed to their micelle-forming capability, which is higher for taurodeoxycholate, a dihydroxy bile salt [190],... 

RL Shih, VHL Lee. (1990). Rate limiting barrier to the penetration of ocular hypotensive beta-blockers across the corneal epithelium in the pigmented rabbit. J Ocular Pharmacol 6 329-336. 

JA Zadunaisky, B Spinowitz. (1977). Drugs affecting the transport and permeability of the corneal epithelium. In S Dikstein, ed. Drugs and Ocular Tissues. Basel Krager, pp 57-78. 

For in vitro toxicity studies and assessment of the barrier function, drug transport, cell physiology, and metabolism as well as the development of delivery systems, cell culture models provide powerful systems for scientific research. As the corneal epithelium is the main barrier for ocular penetration, various corneal epithelial cell cultures were established besides the corneal constructs that mimic the whole cornea and serve as reductionist models for the ocular barrier. In general, two types of cell culture models are available primary cell cultures and immortalized, continuous cell lines. 

Thus far, a wide array of useful cell culture models of the corneal epithelium has been established. Many of these cell culture models focus on toxicity testing and ocular irritation, but some cell layer models for drug permeation studies are also available. Indispensable for successful drug penetration testing is a cell layer that exhibits a tight epithelial barrier. This latter requirement of tight barrier properties disqualifies some of the models that were established as substitutes for the Draize test. At least two cell lines are available for pharmaceutical studies and some newer models may qualify as a useful tool, once they are characterized for their barrier properties. 

Toropainen E, Ranta VP, Talvitie A, Suhonen P, Urtti A. Culture model of human corneal epithelium for prediction of ocular absorption. Invest Ophthalmol Vis Sci 42 2942-2948 (2001). 

A variety of automatic voltage clamp devices with special modifications have been extensively utilized in electrophysiological studies of /sc in several ocular tissues including the amphibian corneal epithelium [42] and human fetal retinal pigment epithelium [43, 44], as well as non-ocular tissues like the rat tracheal epithelium [45], A strong temperature dependency and inhibitory effect of serosally instilled ouabain on the rabbit conjunctival /sc are characteristic of active ion transport driven by Na+/K+-ATPases in the conjunctiva [6, 7],... 

The ocular route is used mainly for the local treatment of eye pathologies. Absorption of drugs administered by conventional eyedrops can result in poor ocular bioavailabilities (2-10%). This is due to the limited area of absorption, the lipophilic character of the corneal epithelium, and a series of elimination factors that reduce the contact time of the medication with the comeal surface, such as drainage of instilled solutions, lacrimation, and tear turnover and tear evaporation [56]. 

The ocular membranes comprise the cornea (not vascularized) and the eonjuetiva (vascularized). The corneal epithelium consists of five or six layers of nonkeratinized squamous cells, and is considered to be the major pathway for ocular drug penetration [57]. 

Rats and mice exposed to 31, 63, or 12 5 ppm 6 hours/day for 16 days developed lesions in the nasal respiratory epithelium and/or olfactory epithelium, and the severity of the lesions generally increased with increasing exposure concentrations. Clinical findings included dyspnea, hypoactivity, and nasal and ocular discharge. At 250ppm all animals died within 4 days. In 2-year inhalation studies at doses of 2, 8, or 32 ppm rats and male mice had increased incidences of nonneoplastic lesions of the nose and increased severity of nephropathy female mice had increased incidences of nonneoplastic lesions of the nose and corneal degeneration. In addition, there was some evidence of carcinogenicity in male rats based on increased incidences of combined neoplasms of the nose and equivocal evidence... 

Dermal/Ocular Effects. Skin rashes have been infrequently reported in humans after inhalation exposure (Gordon 1944 McGuire 1932). No data were available on effects by oral exposure or dermal contact. Because the effects were sporadic, no firm conclusions can be made regarding the potential effects of carbon tetrachloride on the skin in humans. No reports are available on the effects of carbon tetrachloride on the eyes. In mice, selective localization of bound radioactivity was observed in the conjunctival epithelium after intravenous injection (Brittebo et al. 1990). However, in the absence of carbon tetrachloride-induced lesions in the conjunctiva, the significance of this metabolism and molecular binding is not clear. 

Sites of expression Ocular and extraocular photoreceptors Retinal pigment epithelium and Muller cells Ocular and extraocular photoreceptors... 

Glaucoma Timolol and other ocular P-blockers are used to treat glaucoma. Propranolol is effective in diminishing intraocular pressure in glaucoma. This occurs by decreasing the secretion of aqueous humor by ciliary epithelium. It neither affects the ability of eye to focus for near vision, nor changes pupil size. Used in chronic cases only. 

Ocular chemical bums are a significant problem [1] because they may destroy the entire comeal epithelium and extend into the fomices [2], More than 25,000 chemical products - oxidizers, reducing agents, corrosives, etc. - have the potential to cause chemical bums [3], Because serious eye bums can result in loss of sight or require comeal transplants, such chemical bums must be taken seriously. 

The epithelium, the most superficial cellular layer of the cornea of the eye, is chemically less resistant than the keratinized epidermis of the skin. However, during ocular accidents, we know that it takes a few seconds for the first lesions to appear. This delay is bound to multiple factors, winking reflex, protective and diluting effect of the lachrymal liquid, effect of sweeping of the palpebral movements. After a short period, a kinetic of diffusion will set up in a variable way according to the nature of the corrosive. 

The amyelinic nervous fibers are to be found in the corneal stroma and also in the superficial epithelium, which is a noticeable ocular specificity. The nervous endings are to be found unconnected between the cells of the superficial epithelium and react with any contact, with chemical aggressions, with drought... 

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