The Therapeutic Effects of Purified Cortrophin Gel on Experimental Autoimmune Uveitis

Abstract

Purpose: The melanocortin pathways are central in maintaining the normal anti-inflammatory microenvironment of the eye. A repository corticotropin injection (RCI) from ANI Pharmaceuticals that activates multiple melanocortin pathways was studied for its effects on experimental autoimmune uveitis (EAU), a murine model of human endogenous uveitis.

Methods: At the chronic phase of EAU, the mice received an injection of the RCI. Clinical scoring of the eyes was conducted every 3 to 4 days using fundus microscopy until the uveitis resolved. The eyes were collected, sectioned, and H&E stained for histological scoring. A T-cell reaction assay was performed using spleen cells to measure IFN-g, IL-17, and IL-10 following restimulation with retinal antigen. The effects of the RCI's active pharmaceutical ingredient (API) on LPS-stimulated macrophage production of IL-1β, IL-10, and TNF-α were assessed by ELISA.

Result: The RCI in a dose-dependent manner suppressed the clinical scores of EAU, showing no significant difference in scoring between 400 U/kg of RCI and a group of native α-MSH-treated mice. In parallel, the retinas of the treated mice had a significant decrease in the histology scores. A significant reduction in IFN-γ and IL-17 production was observed in spleen T-cells of the treated EAU mice, with no regulatory immunity to retinal antigens. The API significantly suppressed the production of IL-1β, IL-10, and TNF-α by stimulated macrophages.

Conclusions: RCI treatment effectively suppressed uveitis and protected the retina from inflammatory damage. These findings further illustrate the potential of melanocortin-based therapies for uveitis.

Keywords: Adrenocorticotropic hormone; experimental autoimmune uveitis; immunosuppression; melanocortin-based therapy; repository corticotropin gel.

Figure 1.

Effects of RCI treatment on EAU. Mice entering the chronic stage of EAU, with a sustained EAU score of 3, were injected with α-MSH (50 μg) or RCI at 0 (Gel Carrier), 40, or 400 U/kg. (A) The mouse eyes were continued to be scored until the α-MSH treated mice reached an average EAU score of 1. Each mouse eye was scored, and the maximum score was used per mouse. There was a 100% incidence of EAU. Presented are the mean ± Standard Error of the Mean (SEM) for each group of mice (N = 15 per group). There was a significant suppression in the scores of α-MSH and 400 Ukg RCI from 0 to 40 U/Kg. The EAU scores were significantly lower (***p ≤ 0.005) than the 0 U/kg gel-treated mice on Day 15 for 400 U/kg RCI and α-MSH-treated EAU mice. (B) The final EAU score on Day 42 when the eyes were collected for histology. The mean ± SEM for each mouse in each group shows a significant suppression in EAU scores between 0 U/kg treated mice and 400 U/kg and α-MSH-treated EAU mice. **p ≤ 0.01, ****p ≤ 0.001, ns = no statistical difference.

Figure 2.

Effects of RCI treatment on retinal structure. The eyes collected on day 40 were sectioned and H&E stained. Representative images of the stained sections from (A) Normal, (B) untreated EAU mice (0 U/kg), or mice treated with (C) 40 U/kg RCI, (D) 400 U/kg RCI, (E) native α-MSH peptide. The sections were scored. (F) The total histology core is provided for each mouse per group (N = 13 per group). Normal mouse eyes have scores equal to zero. Statistical differences (** p ≤ 0.01) exist between the EAU mice treated with 400 U/kg RCI or α-MSH and the untreated 0 U/kg gel. There were no statistical differences (ns) between 40 U/kg RCI and untreated 0 U/kg gel. (G) Scores of the six individual criteria per group showed significant discovery (*d ≤ 0.05, **d ≤ 0.01, ****d ≤ 0.001) of differences between the RCI-treated groups in retinal folds and vasculitis.

Figure 3.

Effects of RCI treatment on retinal antigen-specific spleen T cells. Spleen immune cells were collected 42 days after treatment and cultured with IRBPp, and 48 h later, the culture media was assayed by ELISA (A) IFN-γ, (B) IL-17, and (C) IL-10. There was significant (**p ≤ 0.01, ****p ≤ 0.001, N = 10) suppression in IFN-γ and IL-17 in the EAU mice treated with the RCI. While there was no significant (ns) difference in IL-10 between untreated (0 U/kg) and 40 U/kg RCI-treated EAU mice, there was significant (*p ≤ 0.05) suppression in IL-10 production by the spleen T cells of mice treated with 400 U/kg.

Figure 4.

Effects of API on LPS-stimulated macrophages. Cultured macrophages were treated with API (0, 2.5, 5, 10, 20, 40, and 80 U/ml) and then with LPS (1 μg/ml). After 48 h, the culture media was assayed by ELISA for TNF-α, IL-1β, and IL-10. The API significantly (**p ≤ 0.01, ****p ≤ 0.001, N = 4) suppressed the production of TNF-α in a dose-dependent manner and a monophasic response for IL-1β and IL-10 with higher concentrations of API suppressing cytokine production. ns = not statistically different.