Mapracorat, a novel selective glucocorticoid receptor agonist, inhibits hyperosmolar-induced cytokine release and MAPK pathways in human corneal epithelial cells

Abstract

Purpose: Increasing evidence suggests that tear hyperosmolarity is a central mechanism causing ocular surface inflammation and damage in dry eye disease. Mapracorat (BOL-303242-X) is a novel glucocorticoid receptor agonist currently under clinical evaluation for use in the treatment of dry eye disease. This study assessed the anti-inflammatory effects of mapracorat in an in vitro osmotic stress model which mimics some of the pathophysiological changes seen in dry eye.

Methods: Human corneal epithelial cells were cultured in normal osmolar media (317 mOsM) or 440 mOsM hyperosmolar media for 24 h. Luminex technology was used to determine the effect of mapracorat on hyperosmolar-induced cytokine release. Effects of mapracorat on mitogen-activated protein kinase (MAPK) phosphorylation were determined by cell based ELISA. Effects of mapracorat on nuclear factor kappa B (NFkappaB) and activator protein-1 (AP-1) transcriptional activity were assessed by reporter gene assay. Dexamethasone was used as a control.

Results: Hyperosmolar conditions induced release of the pro-inflammatory cytokines interleukin-6 (IL-6), interleukin-8 (IL-8), and monocyte chemotactic protein-1 (MCP-1) from cultured human corneal epithelial cells, and altered the phosphorylation state of p38 and c-Jun N-terminal kinase (JNK) and transcriptional activity of NFkappaB and AP-1. Incubation of cells with mapracorat inhibited hyperosmolar-induced cytokine release with comparable activity and potency as dexamethasone. This inhibition was reversed by the glucocorticoid receptor antagonist mifepristone (RU-486). Increased phosphorylation of p38 and JNK caused by hyperosmolarity was inhibited by mapracorat. Mapracorat also significantly decreased the hyperosmolar-induced increase in NFkappaB and AP-1 transcriptional activity.

Conclusions: Mapracorat acts as a potent anti-inflammatory agent in corneal epithelial cells challenged with osmotic stress, with comparable activity to the traditional steroid dexamethasone. These in vitro data suggest that mapracorat may be efficacious in the treatment of dry eye disease.

Figure 1

Mapracorat demonstrates similar activity to dexamethasone in inhibiting hyperosmolar-induced cytokine release in T-HCEpiC. Cells were cultured in complete (HCGS containing) medium, followed by glucocorticoid-free medium for 48 h. Cells were then treated with 440 mOsm hyperosmotic basal media in the presence of dexamethasone or mapracorat for 24 h. IL-6 (A) and MCP-1 (B) release into the media was analyzed by Luminex. Light gray bar represents control (317 mOsm); dark gray bar represents hyperosmolarity (440 mOsm); open circles + dashed line represent mapracorat; closed circles + solid line represents dexamethasone. For A, lines are the result of a re-parameterized four-parameter logistic equation fit to the data; for B, lines are the linear interpolation between data points. Data are presented as mean±SEM, n=3. *Versus 440 mOsm hyperosmotic media; ^#versus dexamethasone at the identical dose; p<0.05.

Figure 2

Mapracorat demonstrates similar activity to dexamethasone in inhibiting hyperosmolar-induced cytokine release in P-HCEpiC. Cells were cultured in complete (HCGS containing) medium, followed by glucocorticoid-free medium for 48 h. Cells were then treated with 440 mOsm basal media in the presence of dexamethasone or mapracorat for 24 h. IL-8 (A) and MCP-1 (B) release into the media was analyzed by Luminex. Light gray bar represents control (317 mOsm); dark gray bar represents hyperosmolarity (440 mOsm); open circles + dashed line represent mapracorat; closed circles + solid line represents dexamethasone. For A, lines are the result of a re-parameterized four-parameter logistic equation fit to the data; for B, lines are the linear interpolation between data points. Data are presented as mean±SEM, n=3. *Versus 440 mOsm media; ^#versus dexamethasone at the identical dose; p<0.05.

Figure 3

Effect of RU-486 on mapracorat or dexamethasone inhibition of hyperosmolarity-induced cytokine release in T-HCEpiC. Cells were cultured in complete (HCGS containing) medium, followed by glucocorticoid-free medium for 48 h. Cells were treated with 440 mOsm hyperosmotic basal media + RU486 and/or mapracorat or dexamethasone for 24 h. Cytokine release into the media was analyzed by Luminex. A: IL-6 release; B: MCP-1 release. For both A and B, the white bar represents 440 mOsm + 100 nM mapracorat and the black bar represents 440 mOsm + 100 nM dexamethasone; open circles + dashed line represents 440 mOsm + mapracorat + RU-486; closed circles + solid line represents 440 mOsm + dexamethasone + RU-486, dark gray bar represents 440 mOsm alone. Data are presented as mean±SEM, n=3. *Versus respective 440 mOsm + mapracorat or 440 mOsm + dexamethasone; for IL-6, dark gray 440 mOsm bar denotes significantly different from 440 mOsm + dexamethasone and 440 mOsm + mapracorat; for MCP-1, dark gray 440 mOsm bar denotes significantly different from 440 mOsm + dexamethasone; p<0.05.

Figure 4

Effects of mapracorat and dexamethasone on hyperosmolarity-induced phosphorylation of p38 and JNK in T-HCEpiC. Cells were cultured in complete (HCGS containing) medium, followed by basal medium for 20 h. Cells were pre-treated with dexamethasone or mapracorat for 2 h. Cells were then treated with 440 mOsm hyperosmotic media + dexamethasone or mapracorat for 30 min. Phosphorylated p38 (A) and JNK (B) concentrations were determined by cell-based ELISA. Data are presented as mean±SEM, n=6. *Versus 440 mOsm media; p<0.05.

Figure 5

Effects of mapracorat and dexamethasone on hyperosmolarity-induced T-HCEpiC NFκB and AP-1 activity. Cells were transfected with an AP-1 or NFκB reporter gene (expressing firefly) and a constitutively expressing Renilla construct. Thirty hours after transfection, cells were treated with 440 media +/− mapracorat or dexamethasone. Cells were assayed for firefly luciferase followed by Renilla luciferase luminescence 48 h after transfection. Data are expressed as normalized firefly: Renilla luciferase ratio. A: NFκB transcriptional activity; B: AP-1 transcriptional activity. Data are mean±SEM, n=6. *Versus 440 mOsm media; p<0.05.