Background/Purpose: The melanocortins (MCs) are endogenous peptides (including ACTH, α-MSH and γ-MSH), which bind 5 G protein-coupled receptors (MCRs 1 through 5) with varying affinity. They exert anti-inflammatory effects primarily via MCR3, and also MCR1 and possibly other MCRs. ACTH is unique among MCs in stimulating adrenal corticosteroid production via MCR2. It is rapidly effective and has been used to treat acute gout and several other inflammatory conditions, but can be associated with some corticosteroid-related toxicities. Our objective was to generate novel ACTH-containing MC fusion peptides that maximize anti-inflammatory activity in part through MCR3 while reducing corticosteroid induction.  

Methods: A series of peptides containing ACTH linked to other MCs were chemically synthesized, and selectivity for each MCR was evaluated using CHO cells transfected with individual MCRs. Anti-inflammatory activity was evaluated in the mouse air pouch model of acute gout. Peptides were administered via sc injection (doses: 3, 10, 30 nmoles [nm]) at 1 hr before (pre-Rx) or 1 hr after (post-Rx) urate crystal injection into the pouch. The pouch was lavaged 4 hrs after crystal injection, and neutrophils and other inflammatory mediators were quantified. Plasma corticosterone levels were measured at 60 min in healthy mice.  

Results: AQB-565, a 47aa peptide containing ACTH_1-24 and NDP-MSH (a more potent analog of α-MSH) fused with a 10aa linker, showed an 11-fold more potent agonist of MCR3 than ACTH (Table). It also showed ~20-fold greater activity on MCR4 and MCR5 relative to ACTH.

AQB-565 was more potent than ACTH (all data expressed as percent relative to control). Pre-Rx with AQB-565 significantly reduced neutrophil influx compared to ACTH at 3 nm (78±23% vs. 5±42%, p<.001) with no significant difference at 10 nm (92±7% vs. 82±18%) or 30 nm (92±4% vs. 89±11%). Pre-Rx with AQB-565 significantly reduced IL-1β levels compared to ACTH at 3 nm (39±21% vs. 10±19%, p<0.0001) and 10 nm (60±13% vs. 46±16%, p<.05) with no significant difference at 30 nm (76±7% vs. 61±9%). AQB-565 (3 nm) reduced accumulation of cytokines and chemokines in the air pouch exudate: TNFα (-55%), IL-6 (-84%), IL-17 (-60%), GM-CSF (-37%), MCP-1 (-46%), MIP-1β (-53%), and KC (-84%). Treatment with AQB-565 decreased mouse corticosterone levels by 40% (p=0.05) at similarly effective doses to ACTH. AQB-565 was also effective when given post-Rx with decreases of 85±15% and 46±26% of neutrophils and IL-1β levels, respectively.  

Conclusion: The fusion of the MCs NDP-MSH and ACTH creates a molecule (AQB-565) reflecting a strategy of targeted MCR modulation with greater anti-inflammatory effects in an in vivo model of acute gout and decreased adrenal steroidogenesis. This novel compound has promise as a potential therapeutic for acute gout and possibly multiple additional inflammatory conditions.