FID-134: a hyaluronan-bisphosphonate macromolecular drug delivery system for intra-articular treatment of osteoarthritis with potential combined cartilage and subchondral bone targeting

Purpose: Osteoarthritis (OA) is a degenerative disease of the joint leading to chronic pain and disability, and consequently resulting in a major socioeconomic health burden. OA, which has long been believed to be a cartilage disease, is now considered a whole-joint disorder that affects various anatomical structures, including subchondral bone; moreover, periarticular bone abnormalities seem also to be involved in the disease initiation and progression. Hyaluronic Acid (HA), a physiological component of synovial fluid and extracellular matrix, is commonly used as intra-articular viscosupplementation therapy for its viscoelastic and lubricant features. In addition, exogenous HA has been proven to stimulate endogenous HA synthesis, chondrocyte metabolism and synthesis of cartilage matrix components and to inhibit chondrodegenerative enzymes, as well as the inflammatory process. Bisphosphonates (BPs) are anti-resorptive agents that inhibit the recruitment and maturation of osteoclast precursors and the activity of mature osteoclasts in the bone. Although it is still not clear if oral administration of BPs is beneficial in OA therapy, pre-clinical evidences are reported in the literature showing that intra-articular BPs could have an impact in slowing down or reversing OA progression. The combination of the biological and mechanical role of HA as viscosupplement and the antiresorptive effect of Alendronate (ALD) could be an interesting strategy for OA treatment. The present study describes the synthesis and characterization of FID-134, a new chemical derivative of HA, conjugated with ALD by means of a covalent bond, cleavable in physiological condition. The activity of the substance, acting as a macromolecular drug delivery system, was tested in an in vitro model of inflammatory OA. Methods: FID-134 was synthesized starting from 500 kDa HA: the chemical structure and functionalization degree with ALD were investigated by 1H NMR and ICP-OES, respectively. Kinetics of ALD release from FID-134 was determined in TRIS buffer at 37°C and compared to a simple mixture of HA and ALD; a 10 kDa cut-off membrane was employed to mimic the synovial membrane. A 20mg/mL FID-134 formulation was investigated for viscoelastic properties, in absence and presence of Ca2+ ions, and compared to HA and HA+ALD mixture formulated at the same concentration. The cytotoxicity of FID-134 was tested on Saos-2 osteoblasts (ATCC HTB-85) and on primary bovine chondrocytes at 24h, 3 and 7 days and compared to free ALD. The efficacy of FID134 was assessed in an in vitro model of inflammatory arthritis, where bovine cartilage biopsies were exposed to IL-1β/OSM (10ng/mL) for 3 weeks; at the same time, cartilage explants were treated with FID-134. Collagen release in the surnatants was quantified and compared to controls. Results: The structure of FID-134 was confirmed by 1H NMR, with diagnostic ALD signals resonating at about 3.0 and 1.7 ppm respectively, and the molar functionalization degree of 20% mol/mol was determined by ICP-OES. Compared to HA+ALD mixture, ALD release assay demonstrated the slow release of the bisphosphonate from FID-134, showing that only about 50% of total bound ALD was released from FID-134 within 7 days. In presence of Ca2+ ions, viscoelastic properties of FID-134 dramatically improved with respect to HA and HA+ALD formulations, which remained unaffected. The cytotoxicity of ALD was evident at the concentration of 100 μM on Saos-2 and primary bovine chondrocytes after 3 days, while no cytotoxicity was observed at 7 days with FID-134. In the cartilage explant model, a strong collagen release was detected in inflammatory conditions after 3 weeks; this tendency was reversed when FID-134 was added to the cartilage biopsies, with collagen release halved compared to control. Conclusions: The synthesized HA-ALD adduct was demonstrated to act as a drug delivery system and ALD was slowly released during time in a controlled manner. FID-134 opens the door for a new approach for OA treatment, as it combines viscosupplementation and biological effect due to HA presence with the pharmacological activity of BPs, targeting both cartilage and subchondral bone. In vitro results showed reduced cytotoxicity for FID-134, compared to free ALD, towards the main cell populations in the joint: chondrocytes and osteoblasts. Moreover, the beneficial effect of FID-134 against inflammatory conditions in cartilage were demonstrated on an in vitro model of inflammatory OA, through the reduction of collagen degradation. These findings suggest that FID-134 could be beneficial in both cartilage degradation and restoration of subchondral bone function, as previously reported in the literature for HA and ALD. Finally, local administration and controlled BP release would likely overcome the drawbacks of ALD oral administration, such as aspecificity and long-term toxic side effects.