Design and in vitro evaluation of a novel synergic intra-articular treatment for post-traumatic osteoarthritis

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Purpose: Post-traumatic (PT) OA, subsequent to joint injury, accounts for over 12% of the overall disease burden of OA. In the development of PTOA, the chondrocytes activity is progressively impaired with upregulation of proteolytic enzymes and downregulation of the most important growth factors involved in promoting chondrocyte anabolism. Based on the literature, we selected three promising target for PTOA treatment: (1) MMP3 is one of the metalloproteases mainly involved in collagen degradation, since several clinical studies reported an increase of synovial fluid MMP3 level in OA patients. In our previous work a Hyaluronic Acid (HA) partial hexadecylamide (HYADD®4) was selected as the strongest MMP inhibitor (MMPI) among a series of glycosaminoglycans. (2) TRPV4 channel is a Ca2+-permeable osmomechano-TRP channel, highly expressed in articular chondrocytes. It plays a pivotal role in chondrocyte mechanotransduction; if activated after a trauma, it is able to induce anabolic responses in vitro and chondroprotection in vivo. RN-1747, a small synthetic hydrophobic molecule was already reported as selective TRPV4 agonist. (3) mTOR (mammalian target of rapamycin) is a serine/threonine protein kinase that plays a vital role in cartilage development and homeostasis as well as contributing to the process of cartilage degeneration associated with OA. Rapamycin is an mTOR inhibitor already FDA-approved for the prevention of organ transplant rejection. The objective of these studies are: (1) to optimize the HYADD®4 chemical structures in order to increase the MMPI potency and selectivity; (2) to develop a stable formulation of the MMPI with an active molecule (RN-1747 or Rapamycin) in order to obtain a novel synergic intra-articular (i.a.) treatment for PTOA. Methods: A series of HA derivatives were synthesized, varying the polymer backbone and the hydrophobic side chain, chemically characterized by 1H-NMR and tested in vitro as MMP3 inhibitors. The inhibitor selected, FID-145 (a partial oleylamide of sulphated HA; patent pending), was screened in vitro against 10 different human MMPs and other targets. The efficacy of FID-145 was assessed in an in vitro model of inflammatory arthritis, where bovine cartilage biopsies were exposed to IL-1β/Oncostatin M (OSM) for 3 weeks; at the same time, cartilage explants were treated with FID-145. Collagen release in the surnatants was quantified and compared to controls. The formulation of FID-145 in PBS was characterized by TEM analysis and a Nile Red staining, where increasing concentrations of FID-145 were mixed with the same concentration of Nile Red and the fluorescence emission at 600 nm was measured. Micellar formulations of FID-145 with either RN-1747 or Rapamycin were obtained through mixing, sonication and sterilizing filtration. The loading degree in either RN-1747 or Rapamycin and the stability of both formulations were analyzed by HPLC-MS. The mTOR inhibition activity and the TRPV4 activation potency were assessed by two specific assays (K-Elisa mTOR Activity Kit, Merck, and Fluo 8 AM calcium assay, AAT Bioquest, in primary chondrocyte, respectively). Kinetics of RN-1747 release from FID-145 formulation was determined in vitro, in human synovial fluid, at 37°C. Results: Among the tested HA derivatives, FID-145 showed the highest inhibition potency against MMP3 and no activity against ADAMTS4 and elastase in vitro. In the cartilage explant model, a strong collagen release was detected in inflammatory conditions after 3 weeks; this tendency was reversed when FID-145 was added to the cartilage biopsies and collagen release decreased compared to control. FID-145 is an amphiphilic polymer and its ability to form micelles in water was confirmed by: 1) the TEM images showing the presence of spherical aggregates with diameter around 40 nm and 2) a Critical Micelle Concentration (CMC) of 0.2±0.1 mg/mL, measured with the Nile Red staining. RN-1747 and Rapamycin were successfully loaded at 0.15±0.05 mg/mL in 12 mg/mL FID-145 in PBS; both formulations proved to be stable, sterile and suitable for i.a. injection for OA treatment. The mTOR inhibition and the TRPV4 activation effect of the Rapamycin and TRPV4 formulations, respectively, was showed to be maintained in vitro. The controlled release of RN-1747 from the FID-145 micellar formulation showed that only about 50% of total loaded RN-1747 was released within 7 days. Conclusions: FID-145, a new amphiphilic HA derivative, was screened as a relatively potent and selective MMP3 inhibitor. When formulated in water, FID-145 yielded a stable micellar formulation easily loaded with hydrophobic, pharmacologically active molecules such as RN-1747 or Rapamycin, of promising relevance in the development of a novel synergic i.a. treatment for PT-OA.