The production of cytokines by human conjunctival epithelial cells following stimulation was investigated. Primary cultures of human conjunctival epithelial cells were characterized by morphology and keratin expression. Cultured epithelial cells were treated with varying concentrations of lipopolysaccharide, interleukin (IL)-1 beta, calcium ionophore A23187, or phorbol myristate acetate, and cytokine secretion was determined over specified intervals. Culture supernatants and cell lysates were analyzed by ELISA for IL-1 beta, IL-3, IL-4, IL-5, IL-6, IL-8, IL-11, IL-1 receptor antagonist (IL-1ra), tumor necrosis factor-alpha (TNF-alpha), and granulocyte-macrophage colony stimulating factor (GM-CSF). With the exception of IL-1ra, unstimulated conjunctival epithelial cells produced cytokines at relatively low or undetectable levels. IL-1ra was detected in both culture supernatants and cell lysates under basal conditions. In response to stimuli, conjunctival epithelial cells secreted the proinflammatory cytokines TNF-alpha, IL-6, IL-8, and GM-CSF in a dose- and time-dependent fashion. After stimulation, the intracellular levels of IL-1ra increased in these cells but the supernatant-associated levels remained unchanged. None of the other cytokines evaluated (IL-1 beta, IL-3, IL-4, IL-5, and IL-11) were detected in supernatants or lysates of resting or stimulated cells. These findings suggest that conjunctival epithelial cells may contribute to the pathogenesis of human ocular diseases by production of proinflammatory cytokines. Further evaluation of these cells as targets of therapy is warranted.

Olopatadine (AL-4943A; KW-4679) [(z)-11-[3-(dimethylamino)propylidene]-6, 11-dihydrodibenz[b,e]oxepine-2 acetic acid hydrochloride] is an anti-allergic agent which inhibits mast cell mediator release and possesses histamine H1 receptor antagonist activity. Studies were conducted to characterize the in vitro and in vivo pharmacological profile of this drug relevant to its topical ocular use. AL-4943A inhibits histamine release in a concentration-dependent fashion (IC50 = 559 microM) from human conjunctival mast cell preparations in vitro. Histamine release was not stimulated by AL-4943A at concentrations as high as 10 mM. In contrast, ketotifen stimulated histamine release at concentrations slightly higher than effective inhibitory concentrations. AL-4943A did not display any in vitro cyclooxygenase or 5-lipoxygenase inhibition. Topical ocular application of AL-4943A effectively inhibits antigen- and histamine-stimulated conjunctivitis in guinea pigs. Passive anaphylaxis in guinea pig conjunctiva was attenuated by AL-4943A applied 30 min prior to intravenous or topical ocular antigen challenge (ED50 values 0.0067% and 0.0170%, w/v, respectively). Antihistaminic activity in vivo was demonstrated using a model of histamine-induced vascular permeability in guinea pig conjunctiva. AL-4943A applied topically from 5 min to 24 hrs prior to histamine challenge effectively and concentration-dependently inhibited the vascular permeability response, indicating the compound has an acceptable onset and a long duration of action. Drug concentrations 5-fold greater than those effective against histamine-stimulated conjunctival responses failed to inhibit vascular permeability responses induced with either serotonin or Platelet-Activating-Factor. These data indicate that the anti-histaminic effect observed with AL-4943A is specific. These anti-allergic/antihistaminic activities of AL-4943A observed in preclinical model systems have been confirmed in clinical trials in allergic patients.

To evaluate the effects of topical ocular drugs with histamine H 1 -antagonist activity on histaminestimulated phosphatidylinositol turnover and interleukin (IL) 6 and IL-8 secretion from human conjunctival epithelial cells.Methods: Primary human conjunctival epithelial cell cultures were stimulated with histamine in the presence or absence of test drugs. Phosphatidylinositol turnover was quantified by ion exchange chromatography and cytokine contentofsupernatantsbyenzyme-linkedimmunosorbentassay.Results: Antazoline hydrochloride, emedastine difumarate, levocabastine hydrochloride, olopatadine hydrochloride, and pheniramine maleate attenuated histaminestimulated phosphatidylinositol turnover and IL-6 and IL-8 secretion. Emedastine was the most potent in ligand binding, phosphatidylinositol turnover, and IL-6 secretion, with dissociation constant and 50% inhibitory concentrations of 1-3 nmol/L. Olopatadine, antazoline, and pheniramine exhibited similar H 1 -binding affinities (32-39 nmol/L). However, olopatadine was approximately 10-fold more potent as an inhibitor of cytokine secretion (50% inhibitory concentration, 1.7-5.5 nmol/L) than predicted from binding data, while antazoline and pheniramine were far less potent (20-to 140-fold) in functional assays. Levocabastine (dissociation constant, 52.6 nmol/L) exhibited greater functional activity (50% inhibitory concentration, 8-25 nmol/L) than either antazoline or pheniramine.Conclusions: Histamine-stimulated phosphatidylinositol turnover and cytokine secretion by human conjunctival epithelial cells are attenuated by compounds with H 1 -antagonist activity. However, antihistaminic potency alone does not predict anti-inflammatory potential. Olopatadine, emedastine, and levocabastine were notably more potent than pheniramine and antazoline.Clinical Relevance: Selected topical ocular drugs with antihistaminic activity may offer therapeutic advantages to patients with allergic conjunctivitis by inhibiting proinflammatory cytokine secretion from human conjunctival epithelial cells.

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Nepafenac, the amide analog of 2-amino-3-benzoylbenzeneacetic acid (amfenac), was examined in preclinical models for its potential utility as a topical ocular anti-inflammatory agent. Diclofenac was selected as the reference compound. In contrast to diclofenac (IC50 = 0.12 microM), nepafenac exhibited only weak COX-1 inhibitory activity (IC50 = 64.3 microM). However, amfenac was a potent inhibitor of both COX-1 (IC50 = 0.25 microM) and COX-2 activity (IC50 = 0.15 microM). Ex vivo, a single topical ocular dose of nepafenac (0.1%) inhibited prostaglandin synthesis in the iris/ciliary body (85-95%) and the retina/choroid (55%). These levels of inhibition were sustained for 6 h in the iris/ciliary body and 4 h in the retina/choroid. Diclofenac (0.1%) suppressed iris/ciliary body prostaglandin synthesis (100%) for only 20 min, with 75% recovery observed within 6 h following topical dosing. Diclofenac's inhibition of prostaglandin synthesis in the retina/choroid was minimal. Nepafenac's inhibitory efficacy and longer duration of action was confirmed in a trauma-induced rabbit model of acute ocular inflammation monitoring protein or PGE2 accumulation in aqueous humor. Results warrant further assessment of nepafenac's topical ocular efficacy in the treatment of postoperative ocular pain, inflammation, and posterior segment edema.