Water-soluble PDE4 inhibitors for the treatment of dry eye.

PDE4 inhibitors have the potential to alleviate the symptoms and underlying inflammation associated with dry eye. Disclosed herein is the development of a novel series of water-soluble PDE4 inhibitors. Our studies led to the discovery of coumarin 18, which is effective in a rabbit model of dry eye and a tear secretion test in rats.

Human conjunctival epithelial cell responses to platelet-activating factor (PAF): signal transduction and release of proinflammatory cytokines.

PURPOSE: The aims of the study were to characterize the signal transduction responses to platelet-activating factor (PAF) and to monitor the downstream effects of PAF on the production of proinflammatory cytokines in human conjunctival epithelial cells (HCECs). METHODS: The generation of inositol phosphates ([(3)H]IPs) from [(3)H]phosphoinositide (PI) hydrolysis and the mobilization of intracellular calcium ([Ca(2+)](i)) were evaluated using ion exchange chromatography and Fura-2 fluorescence techniques, respectively. The production of the cytokines (interleukin-6 [IL-6], interleukin-8 [IL-8], and granulocyte macrophage colony-stimulating factor [GM-CSF]) from PAF-stimulated HCECs was quantified using specific ELISA assays. Specific PAF antagonists were used to study the pharmacological aspects of PAF actions in HCECs. RESULTS: PAF (100 nM) maximally stimulated PI turnover in HCECs by 2.3+/-0.02 fold (n=21) above basal levels and with a potency (EC(50)) of 5.9+/-1.7 nM (n=4). PAF or its stabilized analog, methyl carbamyl (mc)PAF (EC(50)=0.8 nM), rapidly mobilized [Ca(2+)](i), which peaked within 30-60 s and remained elevated for 3 min. PAF (10 nM-1 microM) stimulated the release of the proinflammatory cytokines, IL-6, IL-8, and GM-CSF, 1.4-3.5 fold above basal levels. The effects of PAF (100 nM) on PI turnover and [Ca(2+)](i) were potently antagonized by the PAF antagonists, 1-o-hexadecyl-2-o-acetyl-sn-glycero-3-phospho (N,N,N-trimethyl) hexanolamine (IC(50)=0.69 microM; K(i)=38 nM), methyl 2-(phenylthio)ethyl-1,4-dihydro-2,4,6-trimethyl-pyridine-3,5-dicsrboxylate (PCA-42481; IC(50)=0.89 microM; K(i)=50 nM), rac-3-(N-octadecylcarbomoyl)-2-methoxy) propyl-(2-thiazolioethyl) phosphate (CV-3988; IC(50)=13 microM; K(i)=771 nM), and (+/-)-cis-3,5-dimethyl-2-(3-pyridyl)thiazolidin-4-one HCl (SM-10661; IC(50)=14 microM; K(i)=789 nM [n=3 for each antagonist]). PAF-induced production of IL-6, IL-8, and GM-CSF from HCECs was also blocked by these PAF antagonists (IC(50)=4.6- 8.6 microM). CONCLUSIONS: HCECs respond to PAF by generating IPs, mobilizing [Ca(2+)](i), and then secreting cytokines into the extracellular medium. These results suggest that HCECs may be key target cells for the PAF released from conjunctival mast cells following ocular allergic reactions. Therefore, HCECs in culture represent suitable in vitro models for the investigation of the role of PAF in human ocular allergic and inflammatory diseases and for the discovery of therapeutically useful PAF antagonists.

MUC16 expression in Sjogren's syndrome, KCS, and control subjects.

PURPOSE: To investigate the expression of MUC16 protein in tears and conjunctival cell membranes and MUC16 mRNA in conjunctival cells of Sjogren's syndrome (SS), keratoconjunctivitus sicca (KCS) and non-dry eyed (NDE) subjects. The relationship of tear flow and soluble MUC16 concentration was also measured. METHODS: Seventy-six subjects were recruited for this study: 25 SS (confirmed via American-European Consensus Criteria 2002), 25 KCS (confirmed by symptoms and Schirmer scores < or =10 mm) and 26 NDE. Tear flow was measured by the Schirmer test without anesthesia for 5 min. Tears were collected using an eye-wash technique. Protein and mRNA were isolated from conjunctival epithelial cells collected via impression cytology. Soluble and membrane bound MUC16 were quantified via western blotting and MUC16 mRNA was quantified by real time qPCR. RESULTS: The SS group demonstrated significantly higher concentrations of soluble MUC16 (7.28 [SS] +/- 3.97 versus 3.35 [KCS] +/- 4.54 [p=0.004] and versus 1.61 [NDE] +/- 1.22 [p<0.001]) and MUC16 mRNA (4.66 [SS] +/- 5.06 versus 1.84 [KCS] +/- 2.26 [p=0.01] and 1.52 [NDE] +/- 1.04 [p=0.003]) compared to both KCS and NDE groups, respectively. No differences in soluble MUC16 or MUC16 mRNA were found between the KCS and NDE groups. Membrane bound MUC16 was similar in all three groups. No significant correlation was found between mean Schirmer values and any measure of MUC16 expression. CONCLUSIONS: These results demonstrate that SS subjects display a significant increase in both soluble MUC16 and MUC16 mRNA concentrations compared to other forms of aqueous deficient dry eye and non dry-eyed individuals. There was no correlation between tear flow and soluble MUC16 concentration.

Preservation of tear film integrity and inhibition of corneal injury by dexamethasone in a rabbit model of lacrimal gland inflammation-induced dry eye.

PURPOSE: The aim of this study was to establish a clinically relevant short-term animal model of dry eye with utility in identifying compounds with potential therapeutic efficacy. METHODS: Rabbit lacrimal glands were injected with the T-cell mitogen Concanavalin A (Con A) and inflammation, tear function, and corneal epithelial cell integrity were subsequently assessed. The inflammatory response was characterized by quantifying biochemical markers of inflammation ex vivo and by confirming inflammatory cell influx by histology. Matrix metalloproteinase-9 (MMP-9) and proinflammatory cytokines IL-1beta, IL-8, and TGF-beta1 were quantified in tissue extracts. Tear function was monitored by measuring tear fluorescein clearance and tear breakup time (TBUT). Corneal epithelial cell integrity was determined by quantifying the uptake of methylene blue dye following the exposure of rabbits to a low-humidity environment. The anti-inflammatory corticosteroid, dexamethasone, was administered topically as indicated for each study. RESULTS: Histopathologic evaluation of lacrimal glands injected with Con A revealed a pronounced inflammatory process characterized by lymphocytic infiltration, multifocal necrosis, and fibroplasia. Elevated levels of MMP-9 and cytokines IL-1beta, IL-8, and TGF-beta1 were detected in the lacrimal gland and cornea. Inflammation of the rabbit lacrimal gland following an injection of Con A significantly reduced tear clearance and TBUT and increased susceptibility to desiccation-induced corneal damage. Dexamethasone was prophylactically and therapeutically effective in this inflammation model of dry eye, restoring tear function and inhibiting corneal injury following topical ocular application. CONCLUSIONS: Characteristics of this rabbit lacrimal gland inflammation model of dry eye are consistent with the current understanding of dry eye as a local ocular surface inflammatory response to abnormal tear volume and composition. These results suggest that this rabbit model of dry eye may be employed to assess the therapeutic efficacy of mechanistically diverse agents on clinically relevant signs of ocular surface disease. These methods were strategically developed to be applicable for advancing drug discovery for a broad spectrum of dry eye patients.

Preclinical and clinical antiallergic effect of olopatadine 0.2% solution 24 hours after topical ocular administration.

Pharmacologic studies examined the potential of a solution containing olopatadine to maintain and extend antiallergic efficacy after single topical ocular drop administration over 24 hours. Results of these preclinical experiments conducted in guinea pigs indicated that olopatadine 0.2% (wt/vol) solution was significantly effective 24 hours after dosing. This concentration of olopatadine provided significantly more efficacy than Patanol (olopatadine 0.1%) 24 hours after administration while being as effective as Patanol (olopatadine 0.1%) 5 minutes after administration. Results from a human conjunctival allergen challenge trial in sensitive subjects confirmed clinical efficacy of olopatadine 0.2% solution over 24 hours. When individuals were challenged with antigen at onset, 16 and 24 hours after drug administration onto the eye, significant reductions were observed in the scores for active drug as compared with placebo for pruritus (77, 77, and 61%), conjunctival redness (35, 28, and 20%), and chemosis (53, 41, and 31%), respectively. These data suggest that topically applied olopatadine 0.2% solution will be an effective once-a-day therapy for allergic conjunctivitis.

AL-2512, a novel corticosteroid: preclinical assessment of anti-inflammatory and ocular hypertensive effects.

The anti-inflammatory efficacy and ocular hypertensive effect of AL-2512 were characterized in rodent and feline models of ocular inflammation. Neutrophil influx into ocular tissue following topical ocular administration of test drugs was evaluated in models of endotoxin-induced uveitis. In rats, the anti-inflammatory efficacy of AL-2512 was compared with that of 0.1% dexamethasone. Test drug or vehicle was administered topically before subplantar injection of endotoxin. Neutrophil influx was assessed at 24 hours. Feline eyes, injected intravitreally with endotoxin, were treated topically with 0.1% AL-2512, 1.0% prednisolone acetate or vehicle at various timepoints before and after endotoxin injection. At 12 hours, protein concentration and leukocyte count in aqueous humor were determined. In the feline intraocular pressure (IOP) model, after baseline IOP values were established, AL-2512, dexamethasone, or vehicle was administered topically to both eyes of cats. IOP was measured daily before and during treatment. Topical ocular administration of AL-2512 inhibited endotoxin-induced leukocyte influx in rodent and feline models of uveitis. In rats, AL-2512 significantly inhibited neutrophil influx by 89%, compared with 93% by dexamethasone. In feline eyes, AL-2512 significantly (p < 0.05) inhibited leukocyte infiltration of aqueous humor by 59%, compared to 37% inhibition by prednisolone acetate. Intraocular pressure in cats treated for 32 days with AL-2512 or dexamethasone increased 6% and 18%, respectively. The ocular anti-inflammatory effect of AL-2512 was equivalent to dexamethasone and superior to prednisolone acetate in rat and feline models of ocular inflammation, respectively. This steroid provides anti-inflammatory efficacy equivalent to dexamethasone with a reduced risk of inducing ocular hypertension.

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.