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.

Characterization of the in vitro anti-inflammatory activity of AL-5898 and related benzopyranyl esters and amides.

Selected ester- (AL-5898 and AL-8417) and amide-linked benzopyran analogues (AL-7538 and AL-12615) were evaluated in vitro for their ability to inhibit key enzymes/processes of the inflammatory response. AL-7538 and AL-12615 exhibited weak intrinsic cyclooxygenase inhibitory activity (IC50 = 13 microM, 37 microM). In contrast, 5-HETE and LTB4 synthesis in A(23187)-stimulated neutrophils was effectively inhibited by both ester and amide analogs (IC50 = 2-3 microM). While there was some indication for differing sensitivities among benzopyran esters and amides in the suppression of cytokine synthesis in stimulated U-937 cells, there appeared to be no great discrimination when assessing their effect on U-937 cell adhesion to IL-1beta activated HMVEC-L cells. Inhibition of cell adhesion was concentration-dependent, with IC50 values ranging between 18 microM and 30 microM for AL-5898. Concentration-dependent inhibition of inflammatory cytokine production (i.e., IL-1beta, TNF-alpha, GM-CSF and IL-6) was also apparent in LPS-stimulated, cultured PBMC as well as in PMA/A(23187) activated U-937 cells monitoring the synthesis of IL-1beta, IL-8, TNF-alpha, and MCP-1. Notably, the hydrolysis products of the benzopyranyl ester, AL-5692 and (S)-6-methoxy-alpha-methyl-2-naphthaleneacetic acid, were devoid of pharmacological activity when assessed for inhibition of monocyte adhesion or IL-1beta synthesis. Collectively, our data demonstrate the unique in vitro polypharmacology of a novel series of benzopyran analogs that suppress pivotal enzymes and processes in the inflammatory response.