Sustained intra-articular delivery of cyclin-dependent kinase 9 inhibitors protects against proteolytic activity in an ACL rupture rat model of PTOA

Abstract

Purpose: Pathogenesis of PTOA starts with a joint injury, which rapidly induces a cellular response that establishes a highly catabolic and inflammatory environment within the joint. This catabolic environment is itself a “secondary injury” that irreversibly degrades joint tissues and greatly accelerates PTOA. The initial step in creating the catabolic environment is the transcriptional activation of primary response genes (PRGs). PRGs are rapidly induced without de novo protein synthesis. Although the complete set of joint injury-induced PRGs is poorly defined, a well-characterized subset of PRGs is the inflammatory genes and lytic enzymes such as MMPs. In our ACL-rupture mouse PTOA model, we've shown that PRG activation is dependent on cyclin-dependent kinase 9 (Cdk9). We postulate that targeting Cdk9 might be more effective than targeting individual inflammatory pathways, cytokines, or matrix-degrading proteinases. Systemic administration of a small-molecule Cdk9 inhibitor (Flavopiridol) effectively prevented early molecular signatures of OA, including inflammatory cytokine and MMP production. However, because of the short in vivo half-life of flavopiridol, multiple injections were required. To address this limitation, we fabricated a sustained-release formulation of flavopiridol by encapsulating it in poly (lactic-co-glycolic acid) (PLGA) microparticles (MPs). Here we characterize these microparticles, and test whether intra-articular injection would reduce signs of joint degradation in an ACL-rupture rat model of PTOA. Methods: Fabrication of flavopiridol-PLGA MPs was performed using a single emulsion-solvent evaporation technique. Briefly, PLGA was dissolved in methylene chloride (5% w/v), flavopiridol added, and the solution added to a bulk volume of a polyvinyl alcohol in DiH2O whilst homogenizing (35,000 rpm for 2 min) to form an emulsion. MPs, thus formed, were stirred for 24 h to evaporate residual methylene chloride. MPs were washed, lyophilized and stored at −20°C. MP size distribution was measured by a Microtrac Nanotrac Dynamic Light Scattering Particle Analyzer, and confirmed by scanning electron microscopy. Flavopiridol release from the MPs was quantified over 42 days in PBS-Tween, by absorbance at 247 nm. To induce PTOA in rats (IACUC approved), 4 rats (Sprague Dawley) were anesthetized and a single mechanical overload applied to the knee joint to rupture the ACL. 5 mg of MPs were suspended in 50ul saline and administered into the intra-articular space using a 23-gauge needle, with 2 rats receiving flavopiridol-PLGA and 2 rats receiving blank-PLGA. To assess OA development and joint degradation, we performed longitudinal (up to 3 weeks) in vivo imaging of MMP activity using intra-articular injections of MMPSense750-FAST on an IVIS-200. Results: Flavopiridol was successfully encapsulated in PLGA microparticles at a mass concentration of ∼8 μg/mg PLGA. The average MP diameter was ∼6.8 μm. The in vitro release kinetics of flavopiridol from the PLGA MPs was rapid for the first 5 days, then linear with approximately 70% of the flavopiridol released by day 42. In the rat PTOA model, we observed a strong increase in the in vivo MMP activity 3 days after injury. However, intra-articular injection of flavopiridol-PLGA microparticles markedly reduced the in vivo MMP activity at all time points tested. Conclusions: Intra-articular delivery of a sustained release formulation of flavopiridol-PLGA microparticles provided long-term protection against injury-induced MMP activity in a rat PTOA model. These results indicate the therapeutic potential of sustained release formulations for locally targeting Cdk9 to effectively suppress injury-induced cellular response that leads to secondary joint tissue damage and potentiates PTOA pathogenesis.