John M. Yanni

Nepafenac, a Unique Nonsteroidal Prodrug with Potential Utility in the Treatment of Trauma-Induced Ocular Inflammation: II. In Vitro Bioactivation and Permeation of External Ocular Barriers

Nepafenac, the amide analog of the NSAID amfenac, was examined in vitro for its bioactivation by ocular tissue components and its ability to permeate external ocular barriers. Rabbit tissues catalyzed a concentration-dependent conversion of nepafenac to amfenac. The order of specific hydrolytic activity is retina/choroid ≫ iris/ciliary body. Corneal tissue showed only minimal activity. Similarly, in human ocular cadaver tissue the specific activity of iris/ciliary body was greater than cornea. Continued perfusion of the corneal epithelium demonstrated a nearly six-fold greater permeation coefficient for nepafenac (kp = 727 × 10−6 min−1) than for diclofenac (kp = 127 × 10−6 min−1). Superior permeation of conjunctival and scleral tissue by nepafenac (kp = 128 × 10−6 min−1) compared to diclofenac (kp = 80 × 10−6 min−1) was also evident. Short term perfusion (5 min) of the corneal surface with 0.1% nepafenac resulted in sustained flux of drug across the cornea for 6 h. Under identical conditions only 3.3 μM of diclofenac accumulated on the corneal endothelial side compared to 16.7 μM nepafenac. The enhanced permeability of nepafenac, combined with rapid bioactivation to amfenac by the iris/ciliary body and retina/choroid, make it a target specific NSAID for inhibiting prostaglandin formation in the anterior and posterior segments of the eye.

Nepafenac, a Unique Nonsteroidal Prodrug with Potential Utility in the Treatment of Trauma-Induced Ocular Inflammation: I. Assessment of Anti-Inflammatory Efficacy

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 μM), nepafenac exhibited only weak COX-1 inhibitory activity (IC50 = 64.3 μM). However, amfenac was a potent inhibitor of both COX-1 (IC50 = 0.25 μM) and COX-2 activity (IC50 = 0.15 μM). 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. Diclofenacs inhibition of prostaglandin synthesis in the retina/choroid was minimal. Nepafenacs 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 nepafenacs topical ocular efficacy in the treatment of postoperative ocular pain, inflammation, and posterior segment edema.

Comparative effects of topical ocular anti-allergy drugs on human conjunctival mast cells.

BACKGROUND The concept of mast cell heterogeneity is well established. Recent data indicate that human conjunctival tissue mast cells and human connective tissue mast cells respond to various secretagogues in similar fashion. It is now recognized that different mast cell populations respond differently to anti-allergic drugs. OBJECTIVE The purpose of the study is to compare the effects of three new ocular anti-allergic drugs (nedocromil, olopatadine, and pemirolast) on mediator release from the target human conjunctival mast cell population with those of cromolyn sodium. The affinity of the compounds for the histamine H1 receptor was also compared. METHODS A monodispersed suspension of partially purified human conjunctival mast cells was prepared from cadaver conjunctival tissue. Mast cells (5 x 10(3)) were challenged with anti-human IgE in the presence or absence of test drugs, and histamine content of the cell supernatants was determined using a specific radioimmunoassay. H1 receptor binding activity was assessed using a radioligand binding assay. RESULTS Cromolyn and pemirolast (100 nM to 1 mM) failed to significantly inhibit histamine release from human conjunctival mast cells using exposure times of 1 and 15 minutes prior to challenge. Using identical nedocromil concentrations and exposure times, statistically significant (P < .05) inhibition (28%) of histamine release was observed at only the 100 microM concentration and 1-minute exposure time. In contrast, olopatadine inhibited histamine release in a concentration-dependent fashion (r = 0.891, n = 59, IC50 = 653 microM). Only olopatadine exhibited significant H1 receptor binding activity at relevant concentrations (Ki = 36 nM, n = 13). CONCLUSIONS These data indicate that olopatadine possesses anti-allergic activity in the appropriate targets for topical ocular anti-allergic drug therapy, human conjunctival mast cells. Coupled with the compounds antihistaminic activity, this suggests that olopatadine will have efficacy advantages in allergic conjunctivitis patients over the other drugs tested.

Secretion of proinflammatory cytokines by human conjunctival epithelial cells.

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.

Inflammation-Mediated Retinal Edema in the Rabbit Is Inhibited by Topical Nepafenac

The purpose of this study was to evaluate the ability of the nonsteroidal anti-inflammatory drug nepafenac to prevent development of mitogen-induced pan-retinal edema following topical ocular application in the rabbit. Anesthetized Dutch Belted rabbits were injected intravitreally (30 μg/ 20 μL) with the mitogen concanavalin A to induce posterior segment inflammation and thickening (edema) of the retina. The Heidelberg Retina Tomograph was used to generate edema maps using custom software. Blood-retinal barrier breakdown was assessed by determining the protein concentration in vitreous humor, whereas analysis of PGE2 in vitreous humor was performed by radioimmunoassay. Inhibition of concanavalin A-induced retinal edema was assessed 72 h after initiation of topical treatment with nepafenac (0.1–1.0%, w/v), dexamethasone (0.1%), Voltaren® (0.1%), or Acular® (0.5%). Concanavalin A elicited marked increases in vitreal protein and PGE2 synthesis at 72 h postinjection. Retinal thickness was also increased by 32%, concomitant with the inflammatory response. Topical application of 0.5% nepafenac produced 65% reduction in retinal edema which was correlated with 62% inhibition of blood-retinal barrier breakdown. In a subsequent study, 0.5% nepafenac significantly inhibited (46%) blood-retinal barrier breakdown concomitant with near total suppression of PGE2 synthesis (96%). Neither Voltaren nor Acular inhibited accumulation of these markers of inflammation in the vitreous when tested in parallel. This study demonstrates that nepafenac exhibits superior pharmacodynamic properties in the posterior segment following topical ocular dosing, suggesting a unique therapeutic potential for a variety of conditions associated with retinal edema.

Improved myeloperoxidase assay for quantitation of neutrophil influx in a rat model of endotoxin-induced uveitis

Previously described models of endotoxin-induced uveitis quantify neutrophil influx into the eye using biochemical or direct cell count methods that result in an underestimation of ocular leukocyte accumulation following the inflammatory stimulus. We have optimized the rat model of endotoxin-induced uveitis by first overcoming interference in the biochemical assay of myeloperoxidase due to endogenous ocular reductants and cellular constituents containing free thiol functional groups. This was accomplished by simultaneously 1) extensively diluting soluble, interfering substances and 2) blocking tissue sulfhydril functional groups during tissue homogenization. Uveitis was induced in rats by subplantar injection of endotoxin. Twenty-four hours later, eyes were enucleated, homogenized, fractionated, and myeloperoxidase activity of neutrophils sedimenting with the membranous pellet was extracted. Previously published extraction procedures yielded only 40% of total assayable myeloperoxidase activity. Optimal recovery of myeloperoxidase activity (>twofold increase) was achieved only with two sequential extractions using 50 mM phosphate buffer (pH 7.4) containing 10 mM N-ethylmaleimide, and subsequent solubilization of myeloperoxidase activity by extraction with 0.5% hexadecyltrimethylammonium bromide in 50 mM phosphate buffer (pH 6.0). This modified extraction procedure and optimized myeloperoxidase assay conditions (300 microM hydrogen peroxide and 1.5 mM o-dianisidine) were then used to enhance the uveitis model. Maximum ocular neutrophil accumulation was observed at endotoxin doses of 100-200 microg. Total ocular neutrophil infiltrations ranged from 250,000 to 800,000 cells/globe. This leukocyte influx was inhibited dose-dependently by topical ocular administration of dexamethasone, with half-maximal inhibition observed at a concentration of 0.01%, w/v. Further validated by the correlation of biochemical results with histological evaluation, the refined methodology described in this report has application in assessing the ophthalmic therapeutic potential of antiinflammatory agents.

Histamine-stimulated cytokine secretion from human conjunctival epithelial cells: inhibition by the histamine H1 antagonist emedastine.

The present studies demonstrate that histamine induces the secretion of IL-6, IL-8 and GM-CSF from human conjunctival epithelial cells in a dose- and time-dependent manner. The histamine antagonists emedastine (H1), ranitidine (H2) and thioperamide (H3) were evaluated for their ability to inhibit secretion of these cytokines. Emedastine potently inhibited histamine-induced IL-6, IL-8 and GM-CSF secretion with mean IC50 values of 2.23, 3.42 and 1.50 nM, respectively. Ranitidine and thioperamide failed to inhibit cytokine secretion over a wide dose range. These data suggest that mast cell derived histamine may stimulate inflammatory cytokine production in allergic conjunctivitis via activation of epithelial cell H1 receptors. The histamine H1 antagonist emedastine potently inhibits this response.

Interactions of olopatadine and selected antihistamines with model and natural membranes.

Objective: Olopatadine, an effective topical ocular human conjunctival mast cell stabilizer/antihistaminic antiallergic drug, was evaluated and compared to selected classical antihistamines for their interaction with model and natural membranes to ascertain potential functional consequences of such interactions. Methods: The model membranes examined consisted of the argon-buffer interface and monomolecular films of 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC) at the argon-buffer interface. Interactions with the model membranes were detected as changes in surface tension, i.e., surface pressure. Functional consequences of these interactions were assessed with natural membranes by 6-carboxyfluorescein leakage, hemoglobin release, lactate dehydrogenase release, and histamine release from appropriate cell types. Results: Measurements at the argon-buffer interface revealed intrinsic surface activity for all agents that ranged from highly surface-active to weakly surface-active in the order of: desloratadine > clemastine > azelastine = ketotifen > diphenhydramine > pyrilamine > emedastine > epinastine =olopatadine. This order of amphipathic behavior was confirmed for most of the compounds by estimates of their dissociation constants (K d,L ) determined from interactions with SOPC monolayers adjusted to a surface pressure approximating that of natural membranes. Epinastine was the only antihistamine that showed a disproportionately greater increase in surface activity toward SOPC in monolayer when compared to other antihistamines. Dissociation constants could not be established for olopatadine because of its low affinity for both the argon-buffer interface and the SOPC monolayer. Functional consequences of these interactions were assessed with natural membranes by 6-carboxyfluorescein leakage (erythrocyte ghosts), hemoglobin release (erythrocytes), lactate dehydrogenase release (conjunctival mast cells, corneal epithelial cells), and histamine release (conjunctival mast cells). Aside from olopatadine and emedastine, all antihistamines promoted a concentration- dependent leakage of hemoglobin from intact erythrocytes. The concentration of drug required to cause half-maximal hemoglobin release (H 50 ) from erythrocytes correlated linearly (r = 0.98) with the SOPC dissociation constants (K d,L ) estimated for the different antihistaminic agents interacting with SOPC monolayers.A similarly high correlation (r = 0.85) emerged from a plot with a slope approaching unity that related drug concentrations required for half-maximal hemoglobin leakage from erythrocytes to threshold doses of drug that caused histamine release from human conjunctival mast cells. Olopatadine was the only agent that did not promote membrane perturbation as monitored by either hemoglobin release from intact erythrocytes, LDH release from human conjunctival mast cells, or 6-carboxyfluorescein release from erythrocyte ghosts. Assessment of the lytic potential of marketed concentrations of ketotifen (0.025%), azelastine (0.05%), and epinastine (0.05%) revealed significant membrane perturbation of human conjunctival mast cells and, importantly, human corneal epithelial cells as indexed by LDH release. This was in contrast to marketed concentrations of olopatadine (0.1%) which maintained normal mast cell and corneal epithelial cell membrane function. Conclusions: Combined, these results support the notion that the disruption of natural cell membranes by surface-active antihistamines occurs not through a receptor-mediated process, but is the consequence of a direct interaction of these agents with the cell membrane. This is corroborated by surface pressure-concentration isotherms for adsorption of five different antihistaminic agents to SOPC monolayers where 50% lysis occurred at a surface pressure of 42.9 ± 1.1mN/m. Olopatadine appears to be unique among the agents tested by demonstrating low intrinsic surface activity, thus limiting its interaction with natural membranes. At concentrations of about half-maximal compound solubility (i.e., 5.0mM or a 0.19% drug solution), olopatadine generated SOPC monolayer surface pressures (i.e., 39.82 ± 0.10 mN/m) that were below those that promoted membrane perturbation and onset of hemoglobin leakage. Olopatadine’s restricted interaction with membrane phospholipids limits the degree of membrane perturbation and release of intracellular constituents, including histamine, LDH,and hemoglobin, which is believed to contribute to olopatadine’s topical ocular comfort and patient acceptance.

Effect of Lodoxamide on in vitro and in vivo Conjunctival Immediate Hypersensitivity Responses in Rats

The antiallergic compound, lodoxamide, was evaluated for its abilities to attenuate a local allergic reaction in rat conjunctiva in vivo and to inhibit rat conjunctival mast cell mediator release in vitro. Topically applied lodoxamide (0.01, 0.10 and 1.0%, w/v) dose-dependently reduced the allergic response (23, 43 and 72%, respectively) in vivo. Lodoxamide was more effective than cromolyn sodium, N-acetyl aspartyl glutamic acid (Naaxia) and levocabastine, and 25 (7-200) times more potent than nedocromil sodium in direct comparisons. Addition of lodoxamide (10 micrograms/ml) to sensitized conjunctival tissue in vitro immediately prior to antigen challenge significantly reduced the amount of histamine released by the tissue. These data suggest that lodoxamides in vivo anti-allergic activity in the conjunctiva is associated with its ability to prevent allergic mediator release from mast cells contained in this same tissue.

Corneal Protection by the Ocular Mucin Secretagogue 15(S)-HETE in a Rabbit Model of Desiccation-induced Corneal Defect

The mucin secretagogue 15(S)-HETE was found to stimulate glycoprotein secretion in human ocular tissue at submicromolar concentrations in the present studies. Therefore, the ability of topically applied 15(S)-HETE to preserve corneal integrity was investigated in a rabbit model of desiccation-induced corneal defect. Desiccation-induced corneal injury was elicited in anesthetized rabbits by maintaining one eye open with a speculum. Corneal staining and corneal thickness changes were determined immediately following desiccation. 15(S)-HETE dose-dependently reduced corneal damage (ED50 = 120 nM) during a two-hour desiccation. Corneal staining was unchanged relative to control using a 1 microM dose of 15(S)-HETE. Through four hours of desiccation, 15(S)-HETE (500 nM) decreased corneal staining by 71% and completely prevented corneal thinning. 15(S)-HETE (1 microM) was significantly more efficacious than an artificial tear product over the 4-hour desiccation period. There was no evidence of tachyphylaxis following repeated topical ocular dosing of 15(S)-HETE. These studies demonstrate that 15(S)-HETE stimulates ocular mucin secretion in vitro and effectively protects the cornea in a rabbit model of desiccation-induced injury. The results suggest that the ocular mucin secretagogue 15(S)-HETE may have therapeutic utility in dry eye patients, alleviating corneal injury and restoring corneal integrity.