Melanocortins are peptides endowed with anti-inflammatory and pro-resolving activities. Many of these effects are mediated by the Melanocortin receptor 1 (MC 1) as reported in several experimental settings. As such, MC 1 can be a viable target for the development of new therapies that mimic endogenous pro-resolving mediators. The aim of this study was to assess the immunopharmacology of a selective MC 1 agonist (PL8177) in vitro and in a mouse model of inflammatory arthritis.
PL8177 and the natural agonist αMSH were tested for activation of mouse and human Melanocortin receptors (MC 1,3,4,5), monitoring cAMP accumulation and ERK1/2 phosphorylation, using transiently transfected HEK293A cells. The anti-inflammatory and pro-resolving effects of PL8177 and αMSH were evaluated using mouse peritoneal Macrophages. Finally, a model of K/BxN serum transfer induced arthritis was used to determine the in vivo potential of PL8177.
PL8177 activates mouse and human MC 1 with apparent EC 50 values of 0.01 and 1.49 nM, respectively, using the cAMP accumulation assay. Similar profiles were observed for the induction of ERK phosphorylation (EC 50: 0.05 and 1.39 nM). PL8177 displays pro-resolving activity (enhanced Macrophage efferocytosis) and counteracts the inflammatory profile of zymosan-stimulated macrophages, reducing the release of IL-1β, IL-6, TNF-α and CCL-2. In the context of joint inflammation, PL8177 (3mg/kg i.p.) reduces clinical score, paw swelling and incidence of severe disease as well as the recruitment of immune cells into the arthritic joint.
These results demonstrate that the MC 1 agonism with PL8177 affords therapeutic effects in inflammatory conditions including arthritis.
Drugs targeting the Melanocortin system have emerged as promising therapeutics for several conditions including inflammation or obesity. Multiple candidates are under clinical development, and some have already reached approval. Here we present the characterization of a novel drug candidate, PL8177, selective for the Melanocortin 1 receptor (MC 1), demonstrating its selectivity profile on cAMP and ERK1/2 phosphorylation signaling pathways, of relevance as selective drugs will translate into lesser off-target effect. PL8177 also demonstrated, not only anti-inflammatory activity, but pro-resolving actions due to its ability to enhance efferocytosis (i.e. the phagocytosis of apoptotic cells), endowing this molecule with therapeutic advantages compared to classical anti-inflammatory drugs. Using a mouse model of inflammatory arthritis, the compound demonstrated in vivo efficacy by reducing clinical score, paw swelling and overall disease severity. Taken together, these results present Melanocortin-based therapies, and specifically targeting MC 1 receptor, as a promising strategy to manage chronic inflammatory diseases.
Definition of mechanisms and mediators of the resolution of inflammation can guide the development of therapies that mimic the way our own body terminates this response. Multiple pro-resolving molecular mediators have been discovered and are currently in translational studies and drug discovery programs for the treatment of pathologies with an inflammatory component. The Melanocortin (MC) system constitutes one of these endogenous pro-resolving pathways. All natural Melanocortin agonists derive from the same proopiomelanocortin (POMC) protein, further cleaved into ACTH and the smaller melanocyte stimulating hormones (α, β, γMSH). ACTH was the first agonist studied and shown to be effective for the treatment of patients affected by rheumatoid arthritis (RA). Melanocortins act on Melanocortin receptors (MC 1-5), G-protein coupled receptors (GPCRs) that regulate multiple functions, such as skin pigmentation (MC 1), steroidogenesis (MC 2), energy homeostasis (MC 3,4) or sebaceous gland function (MC 5). The high degree of similarity among these GPCRs makes challenging to achieve receptor selectivity. Although ACTH is the only MC agonist able to activate MC 2-dependent steroidogenesis, regulatory functions on the inflammatory response can be achieved through the other MCRs (MC 1, 3-5), independently of endogenous cortisol. Among them, MC 1 stands out due to its wide distribution among the immune system and its influence on the inflammatory response.
MC 1 activation reduces leucocyte recruitment and immune cell activation, Macrophage reactivity, promotes tolerogenic responses, and favors wound healing. The MC 1 selective small molecule BMS-470539 has shown therapeutic efficacy in various models of neuro-inflammation, ability to reduce leukocyte infiltration in a model of lung inflammation, improvement of membranous nephropathy and reduction of joint inflammation using the K/BxN serum induced transfer arthritis (STIA) model. In addition, the MC 1 selective peptide PL8177 can reduce experimental autoimmune uveitis and intestinal inflammation in a model of inflammatory bowel disease. Data obtained from the use of mice lacking a functional MC 1 receptor point to a major role for this receptor in regulating inflammation and maintaining homeostasis. For example, Mc1r-/- mice present with increased predisposition to vascular endothelial dysfunction, they develop more severe cartilage damage in experimental osteoarthritis as well as more severe intestinal damage in an experimental model of colitis.
Of relevance for joint diseases, the Melanocortin system is also functional in non-immune cells, such as fibroblasts, osteoclasts, osteoblasts and chondrocytes. In fact, Melanocortin peptides have been detected in the synovial fluid of rheumatoid arthritis, osteoarthritis and juvenile chronic arthritis and their levels negatively correlate with disease severity. Collectively, these reports highlight the relevance and therapeutic potential of MC 1 and the Melanocortin pathway for the control of joint inflammation and tissue repair.
Disorders of the musculoskeletal system lead to chronic pain and disability that affects 19% of European population. Current pharmacological strategies may stop disease progression but are rarely able to induce healing. We proposed that a fresh approach to the control of these diseases may be the development of agonists of endogenous protective mechanisms. However, among the major limitations of natural melanocortins is their lack of selectivity and an unfavorable pharmacokinetics. Therefore, the development of more selective and stable Melanocortin analogues may lead to novel therapeutics with improved translational potential for the treatment of joint inflammation as well as other conditions.
Herein, we evaluated the pharmacological profile of PL8177, a synthetic cyclic heptapeptide selective for MC 1. PL8177 has previously shown an interesting therapeutic potential in in vivo models of intestinal and ocular inflammation. In this study we investigated the signaling pathways engaged by PL8177 in human and mouse MC receptors and the post-receptor downstream functional outcomes using mouse primary peritoneal macrophages, known to express Melanocortin receptors. We also established the potential of PL8177 in a model of arthritis that recapitulates some features of active rheumatoid arthritis.
PL8177 (provided by Palatin Technologies Inc) and αMSH (Tocris, Bristol, UK) solutions were prepared at 1mM stocks in DMSO (for in vitro studies) or PBS (in vivo studies) and single-use aliquots were frozen at -20°C. All other chemicals were obtained from Sigma-Aldrich, Poole, UK, unless otherwise indicated.
B16-F1 melanocyte cell line naturally expressing MC 1 were maintained in RPMI containing 10% fetal calf serum (FCS), 2 mM L-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin and kept at 37°C with 5% CO 2. Confluent monolayers were washed with PBS, harvested by gentle scrapping and centrifuged at 600 x g for 10 min. Pellets were resuspended in harvesting buffer and incubated with radioligand [3 H]-PL8177 at a range of 1x10-6 M to 1x10-13 M for 90 min at room temperature. The endogenous Melanocortin agonist peptide αMSH was used as control. Binding was detected by scintillation counting and results are expressed as a percent of control specific binding and as a percent inhibition of control specific binding obtained in the presence of the test compound, PL8177. The inhibition constants (K i) were calculated using the Cheng Prusoff equation.
Vectors for human and mouse MC 1, MC 3, MC 4 and MC 5 and empty vector pCMV6 were originally purchased from Origene (Rockville, Maryland, USA) and in-house transformed into bacteria. Bacterial clones were grown overnight in 150ml LB medium supplemented with kanamycin (25µg/ml). Plasmid DNA isolation was performed using Zyppy™ Plasmid Maxiprep kit (Zymo Research; Irvine, California, USA).
HEK293A cells were maintained in DMEM containing 10% fetal calf serum (FCS), 2 mM L-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin and kept at 37°C with 5% CO 2. Cells were seeded in 96-well plates at 2x10 4 cells/well and transfected 24 hours later with 50ng of plasmid DNA (encoding for human and mouse MC 1, MC 3, MC 4 and MC 5, TrueORF cDNA clones (Origene) and Lipofectamine 2000 (Invitrogen; Waltham, Massachusetts, USA) according to manufacturer’s instructions. Cells were used 24 h later.
Twenty-four hours after transfection, cells were serum-starved for 3 hours to reduce and stabilize basal levels of cAMP. Melanocortin agonists were tested using 1/5 serial dilutions to generate concentration response curves starting at 10 or 0.4 µM. Vehicle was used as negative control, and forskolin (3 µM, Tocris Bioscience) as positive control. Compound solutions were prepared in serum-free DMEM containing 10 mM 3-isobutyl-1-methylxanthine (IBMX; Sigma), to inhibit phosphodiesterase activity. Cells were stimulated for 15 min with the respective compounds and lysed immediately with 0.1M HCl followed by scrapping and freezing at -80°C until the assay was performed. cAMP was quantified using the Cyclic AMP Select ELISA kit (Cayman Chemicals, CAY501040, Ann Arbor, Michigan, USA) according to manufacturer instructions. Optical Density (OD) measurements were converted to cAMP concentration using a standard curve and results were normalized subtracting background signal and calculated as % of forskolin. The endogenous agonist αMSH (10 µM) was used to define the 100% agonistic effect for each receptor subtype.
Twenty-four hours after transfection, cells were serum-starved for 3 hours to reduce and stabilize basal levels of phospho-ERK1/2. Concentration response curves were generated by 1/5 serial dilutions as above. Drug solutions w
