Probing the Role of Melanocortin Type 1 Receptor Agonists in Diverse Immunological Diseases

Abstract

Background: The melanocortin α-melanocyte stimulating hormone (α-MSH), an endogenous peptide with high affinity for the melanocortin 1 receptor (MC1r), has demonstrated prevention and reversal of intestinal and ocular inflammation in animal models. Preclinical studies were performed to determine whether two MC1r receptor agonists, PL-8177 and PL-8331, exhibit actions and efficacy similar to α-MSH in preventing and reversing intestinal and ocular inflammation. Methods: Both PL-8177 and PL-8331 were assessed in a Eurofins LeadProfilingScreen selectivity panel including 72 in vitro assays. PL-8177 and PL-8331 were evaluated in an in vitro assay using human whole blood stimulated by lipopolysaccharide to determine inhibition of tumor necrosis factor alpha (TNF-α); for comparison, adrenocorticotropic hormone (ACTH) and α-MSH were used as positive controls. PL-8177, dosed at 0.5, 1.5, and 5.0 μg, was assessed in a cannulated rat model of dinitrobenzene sulfonic acid (DNBS)-induced bowel inflammation versus vehicle and oral sulfasalazine. PL-8177 was also dosed at 0.3 mg/kg/mouse injected intraperitoneally versus untreated controls and α-MSH treatment in mice with experimental autoimmune uveitis (EAU). PL-8331 at 3 doses, 3 times daily, was evaluated in a murine model of scopolamine-induced dry eye disease (SiccaSystem^TM model), versus twice-daily Restasis^® and Xiidra^®. Results: Both PL-8177 and PL-8331 demonstrated no significant activity at the 1 μm concentration in any of the 72 in vitro assays. PL-8177 and PL-8331 inhibited lipopolysaccharide-induced TNF-α to a similar degree as ACTH and α-MSH. In the DNBS rat model of bowel inflammation, PL-8177 was significantly superior to untreated controls at all 3 doses (P < 0.05) in reducing bowel inflammation parameters, with effects similar to sulfasalazine. In the murine EAU model, PL-8177 significantly reduced retinal inflammation scores versus untreated controls (P = 0.0001) over 3-5 weeks, and to a similar degree as α-MSH. In the murine scopolamine-induced model of dry eye disease, PL-8331 reduced corneal fluorescein staining scores at all doses, significantly (P = 0.02) for the highest dose (1 × 10^-5 mg⋅mL^-1), and similarly to Restasis^®; Xiidra^® demonstrated no effect. Conclusion: The MC1r receptor agonists PL-8177 and PL-8331 exhibited actions similar to those of α-MSH in preventing and reversing intestinal and ocular inflammation in preclinical disease models.

Keywords: PL-8177; PL-8331; alpha-melanocyte stimulating hormone; dry eye disease; experimental autoimmune uveitis; inflammatory bowel disease; melanocortin; melanocortin 1 receptor.

FIGURE 1

Inhibition of lipopolysaccharide-induced TNF-α inhibition in human whole blood. α-MSH, alpha-melanocortin stimulating hormone; ACTH, adrenocorticotropic hormone; TNF-α, tumor necrosis factor alpha.

FIGURE 2

Effects of PL-8177 and sulfasalazine on colon weight and inflammation scores in rats with DNBS-induced bowel inflammation. ^∗P < 0.05; DNBS, dinitrobenzene sulfonic acid; IC, intracolonic; PO, per oral.

FIGURE 3

Effect of PL-8177 treatment versus controls on inflammation scores of mice with induced experimental uveitis. ^∗P = 0.0001 by analysis of variance. α-MSH, alpha-melanocortin stimulating hormone; EAU, experimental autoimmune uveitis.

FIGURE 4

(A) Histology of retinas from healthy mice (Top), untreated EAU mice (Lower Left), and EAU mice treated with PL-8177 0.3 mg/kg/mouse (Lower Right). In contrast with the healthy retinas, the retinas from the untreated mice showed: cellular infiltration; uneven nuclear layers with folding; loss of the outer limiting membrane; loss of the intervening plexiform layer between the inner and outer nuclear layers in some places; a thinner photoreceptor layer, suggesting photoreceptor dropout; and signs of vasculitis in the central retinal vessels. (B) At higher magnification (40×), disruption of the retinal pigment epithelial monolayer and presence of immune cells in the photoreceptor layer were detectable. In contrast to the untreated retinas (Lower Left), EAU retinas treated with PL-8177 (Lower Right) retained the even layers of the retina with little evidence of photoreceptor loss [similar thickness of photoreceptor layer as in healthy eyes (Top)], and some of the outer limiting membrane. The retinal pigment epithelial monolayer was intact and there was a clear outer plexiform layer between the inner and outer nuclear layers. One detectable difference between the healthy retinas (Top) and the PL-8177-treated EAU retinas (Lower Right) is that photoreceptor nuclei were not lined up in the latter as they were in the healthy eyes.

FIGURE 5

Corneal fluorescein staining in mice with chronic dry eye (SiccaSystem^TM model). Data are presented as median ± interquartile range; data werecompared by paired non-parametric Wilcoxon test. ^∗Significant difference; see P-values under x-axis.