Pharmacological profiling of novel melanocortin peptides targeting the hMC1, hMC3 and hMC5 receptors in 2-D and 3-D models of activated chondrocytes

Musculoskeletal diseases such as Osteoarthritis (OA) affects over 8.75 million people aged 45 and over in the UK alone, resulting in pain, joint stiffness and lack of mobility of the affected joint. The pathology of OA is mediated by a complex inflammatory response predominantly driven by elevated levels of interleukin-1beta (IL-1b), in addition to tumour necrosis factor-alpha (TNF-a). Endotoxins such as lipopolysaccharide (LPS), or Nitric Oxide donors such as S-nitroso-Nacetylpenicillamine (SNAP) has also been reported to play a role in the inflammatory response seen in this degenerative joint disease. Current treatment options include pain management and maintenance of mobility by utilising symptom relivers such as non-steroidal anti-inflammatory drugs or disease-modifying antirheumatic drugs and joint replacement surgery. These are not feasible long-term treatment solutions for OA. Therefore, the melanocortin system, comprised of melanocortin peptides could provide a novel treatment option for OA.

Melanocortin peptides are endogenous pro-opiomelanocortin-derived peptides that display potent anti-inflammatory effects via the activation of the 7-transmembrane Gprotein coupled melanocortin receptors. To date, five human melanocortin (hMC) receptors have been identified (hMC1 - hMC5), whereby, hMC1, hMC3 and hMC5 receptor subtypes display potent anti-inflammatory effects modulating inflammatory pathways found in arthritic pathologies. The receptor subtype involved in the modulation of inflammatory pathways in OA is unclear. Therefore, molecular investigations via RT-PCR/qPCR was initially carried out to ensure that the C-20/A4 cell-line was sufficient to use. Melanocortin receptor expression and regulation of key inflammatory biomarkers in both monolayer 2-D and 3-D micromass constructs of the C-20/A4 chondrocytic cell-line was determined. Cell signalling assays such as ELISA, Caspase 3/7 assay, and cell viability assays was used to evaluate the effect of the melanocortin peptides in IL-1b activated chondrocytes, and Western blotting was carried out to evaluate the induction of HO-1.

The expression and functional activity of hMC1, hMC3 and hMC5 receptors were identified and confirmed in monolayer (2-D) and 3-D micromass C-20/A4 cells. 2-D and 3-D chondrocytes were activated using either IL-1 b, TNF-a, LPS or SNAP to allow evaluation of the effect of selective melanocortin peptides. IL-1 b (100 pg/mL) activation of chondrocytes caused ~40% and ~38% cell death in 2-D and 3-D cultures respectively. This trend of cell death was observed with the use of TNF-a (60 pg/mL), LPS (0.1 μg/mL) and SNAP (1.0 mM) at 6 hours. The effect of the selective hMC1 receptor agonist BMS-470539 dihydrochloride, the hMC1 and hMC3 receptor agonist [DTrp8]-g-MSH, and the novel selective hMC3 receptor agonists PG-990 and PG-992 in activated chondrocytes was evaluated initially in 2-D and then in 3-D cultures. It was determined that 3-D micromass C-20/A4 chondrocytes activated by IL-1b (100 pg/mL) and pre-treated with the novel selective hMC3 receptor agonist PG-990 (10.0 μg/mL) for 6h displayed chondroprotection (determined by the cell viability studies), and significantly inhibited pro-inflammatory mediator (IL-6, IL-8, MMP-1, MMP-3 and MMP-13) release determined by ELISA. The selective hMC3 receptor agonist PG-990 also displayed protective effects on glycosaminoglycan content and induced HO-1 expression. Whereby, the selective hMC1 receptor agonist BMS-470539 dihydrochloride (10.0 μg/mL) did not display a significant effect. This data possibly suggests that targeting the hMC3 receptor could provide an alternative treatment option for OA in the future.