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Objective: NSAIDs are recommended for the first-line treatment for osteoarthritis pain but not for long-term use because of the increased risk of severe adverse events, such as gastrointestinal ulcers and heart attacks. To fulfill the unmet long-term use needs of these patients, a new therapeutic approach, which combines locally active drugs and sustained-release formulations, is proposed in this project. There are two major objectives of this research: one is to develop a locally active COX-2 (LA-COX-2) inhibitor that can be inactivated quickly upon enter into systemic circulation; the other is to establish a sustained release system to prolong the residence time of antedrug in the joint. Methods: The LA-COX-2 inhibitors were designed based on the scaffold of celecoxib by introducing an ester bond. Their inhibitory activity against COX-2 and COX-1 was determined in recombinant human enzyme or cell-based assays. Their stability was measured in different biofluids. Pharmacokinetic studies of lead compounds were conducted in male SD rats intravenously to estimate their systemic exposure. After optimization, 4-31 and 7a1 were selected for efficacy evaluations. Two formulations, crystalline suspension, and PLGA microparticles were prepared through wet milling and emulsion solvent evaporation methods respectively for 4-31 and 7a1. The long-term pharmacokinetic and pharmacodynamic studies of 4-31 and 7a1 were conducted in the monosodium iodoacetate(MIA)-induced osteoarthritis rat model. Results: More than forty compounds containing an ester bond were designed, synthesized, purified, and tested. According to the in vitro activity against COX-2 and solubility, three lead compounds were selected for pharmacokinetic study and pilot efficacy study. The AUCs of lead compounds 5a1-1, 5a2-4, and 8h6 were 321, 0.046, and 29.3 hrnM respectively. They were much lower than that of celecoxib (39092 hrnM) reflecting the lower systemic exposure of LA-COX-2 inhibitors. However, lead compounds failed to reduce the MIA-induced joint swelling and OA pain that may be attributed to the hydrolysis of lead compounds in rat joints. After optimization, we design and synthesized a new compound, named 4-31, which was more stable than lead compounds. Two sustained-release formulations, crystalline suspension, and PLGA microparticles were developed for 7a1 and 4-31 with slight variations in particle size distribution (7a1 CS: 9.44 μm, 4-31 CS: 5.53 μm, 7a1 MP: 14.49 μm, and 4-31 MP: 18.82 μm), zeta potential (7a1 CS: -29.83 mV, 4-31 CS: -27.67 mV, 7a1 MP: -8.3 mV, and 4-31 MP: -7.2 mV), and loading capacity (7a1 MP: 17% and 4-31 MP: 23.1%) among three batches. The long-term PK study of 7a1 and 4-31 demonstrated that crystalline suspension and PLGA microparticles can maintain the drug in the joint for at least one month. In efficacy studies, intra-articular injection of 1 mg 7a1 and 4-31 crystalline suspension or 0.33 mg 7a1 and 4-31 microparticles significantly reduced the joint swelling and the weight distribution changes and walking gait changes caused by osteoarthritis pain in the rat model. However, most of them only showed positive effects in the early stage but not the late stage of the MIA-induced osteoarthritis rat model. Among them, an intra-articular injection of 0.33 mg 4-31 MP was the most effective treatment and achieve statistical significance throughout the study period. Conclusion: We have successfully developed LA-COX-2 inhibitors which were effective to inhibit COX-2 activity and sensitive to the esterases. Compared to celecoxib, the extremely low blood drug concentration and AUC of LA-COX-2 inhibitors in the intravenous PK study indicate their minimal systemic exposure. The long-term PK study of the sustained-release formulation of 4-31 and 7a1 demonstrated that crystalline suspensions and PLGA microparticles can sustainably release LA-COX-2 inhibitors in the joint for at least one month. A single intra-articular injection of LA-COX-2 inhibitors crystalline suspensions or microparticles can significantly reduce joint swelling and improve the abnormal weight distribution and walking gait caused by MIA-induced osteoarthritis pain.



Locally Active Drugs, Osteoarthritis, COX-2 Inhibitors, Pain, Systemic Toxicity