AP214, a new αMSH analogue, possesses anti-inflammatory activities

Trinidad Montero-Melendez1, Hetal B Patel1, Michael Seed1, Thomas Jonassen2, Mauro Perretti1. 1William Harvey Research Institute, Barts and The London SMD, Queen Mary University of London, London, United Kingdom, 2Action Pharma A/S, Copenhagen, Denmark.

Activation of melanocortin receptors (MCR) constitutes a new strategy for the modulation of inflammatory processes, capitalising on the biological activities of melanocortin peptides such as α-melanocyte-stimulating hormone (MSH). Targeting of MC1R or MC3R has revealed a viable approach that elicits anti-inflammatory activity in several disease models, including LPS-induced lung inflammation, gouty arthritis, or peritonitis. Research in this area is focused on the development of new ligands that modulate the activity at specific melanocortin receptors. In this work we have studied the anti-inflammatory properties of the new peptide AP214, a MSH analogue with increased binding affinity to MCR (1).

Acute peritonitis was induced with 1 mg i.p. zymosan A given i.p. to male C57Bl6 mice (WT), MC1R mutant (recessive yellow e/e colony) or MC3R null mice (∼25 g body weight). The recruitment of Gr1+ cells was assessed at 4 h, using flow cytometry. AP214 was injected at doses of 400 and 800μg/kg i.p. just before zymosan. In vitro, peritoneal macrophages collected from WT, MC1R mutant and MC3R null mice were stimulated with 25 μg/ml zymosan, and cytokine release in the supernatant measured by ELISA at 6 h.

Zymosan recruited 117±14, 122.5±26 and 102±14 x106 Gr1+ cells per cavity in WT, MC1R mutant and MC3R null mice, respectively (n = 6). AP214 peptide displayed inhibitory activity at the 400-μg/kg dose in both WT (35% of inhibition) and MC1R-/- mice (33.8%); however, this effect was lost in MC3R null mice. In vitro, AP214 (1-1000 nM) affected zymosan-induced cytokine release, with 128, 25 and 19% of inhibition on IL-1β, TNF-α and IL-6 levels, respectively, as calculated for the 300 nM concentration. These effects were conserved in macrophages from MC1R mutant mice. An uneven effect was observed in MC3R null macrophages, so that AP214 no longer affected IL-1β secretion, whereas it fully retained its inhibitory properties on TNF-α and IL-6 levels.

In summary, we report here that the melanocortin AP214 possesses in vitro and in vivo anti-inflammatory properties that could be exploited as a new anti-inflammatory strategy. Use of receptor mutant (inactive MC1R) or null (MC3R) mice and cells indicated a major role for the latter receptor in bringing about the inhibitory properties of AP214. However, the involvement of other receptors (possibly MC5R) can not yet been ruled out from mediation of some of the actions promoted by AP214.

(1) K Doi et al. Kidney International (2008) 73,1266-1274.