Background/Purpose: The inflammatory osteoarthritis (OA) phenotype is characterized by the presence of synovial membrane inflammation, pain and pathological changes in joint structure leading to rapid disease progression and debilitating chronic conditions. Efforts to develop effective OA therapies against a single target failed. Therefore, we designed a small molecule, CR10049, which preferentially inhibits the pro-inflammatory Src family kinases members, along with kinases that have a major role in OA development and maintenance such as members of the Ephrin receptor, Trk and the VEGFR family. The aim of this study was to characterize the pharmacological activity of CR10049 in vitro and in long-lasting small (rats) and large (rabbits) animal models of OA. Moreover, preliminary acute toxicology studies were employed to support the next development for human clinical trials.

Methods: A panel assay of 159 kinases was used to evaluate the binding affinity of CR10049. In vivo, OA was induced by injecting 2 mg of MIA into the knee of Sprague Dawley rats. On day 3 (D3) after MIA injection, animals received CR10049 (125 and 250 µg) or vehicle intraarticularly (IA, 30 µl). On D3 and D45 an additional control group received IA triamcinolone acetonide (TCA, 60 µg). Pain was blindly evaluated by electronic Von Frey testing and dynamic weight bearing for 91 days, at variance with the 28 days of the conventional MIA OA model. In another experiment, New Zealand rabbits that underwent ACL transection (ACLT), were administered IA with CR10049 (6 mg in 600 µl) or vehicle on D7, D20 and D40. At sacrifice (D120), explanted joints were processed for blinded histology evaluation. For toxicology studies in Wistar rats, CR10049 was injected intravenously (IV) at doses up to 60 mg/kg and IA up to 5 mg.

Results: CR10049 inhibited, in a nanomolar range (IC50 < 25 nM), the Src family member cSrc, Lck, Fyn, Fgr, Frk and Yes as well as other kinases including VEGFR1/2, FGFR1, EPHA3, EPHA5, TrkB and TrkC that have a major role in synovial inflammation and fibrosis, cartilage degradation, bone remodeling and pain status.In the long MIA rat paradigm, CR10049 significantly reduced weight-bearing imbalance for long period (Fig.1A) in a dose-dependent fashion; these effects were confirmed by mechanical allodynia and were significant up to D91. TCA exerted the known acute benefit that was rapidly lost and not replicated with a second injection (Fig.1A). In ACLT rabbits, CR10049 significantly preserved cartilage tidemark integrity and reduced GAG degradation, cartilage and cell damage, osteophytes formation and subchondral bone thickening for 3 months after the last IA injection (as summarized by total score in the fig.1B). In preliminary rat toxicology studies, CR10049 did not induce toxicological effects when injected systemically (IV) up to 60mg/kg and locally (IA) up to 5 mg, i.e. a 20x higher dose than the maximum effective dose.

Conclusion: We identified the first intra-articular drug, CR10049, with a long-acting effect on pain reduction and joint structure improvement in the experimental inflammatory OA setting. Its unique characteristics of selectively targeting kinases with a major role in OA, makes CR10049 a good candidate for the treatment of the OA inflammatory phenotype.

Figure 1. Representative data of the weight-bearing imbalance (A) and Mankin score (B) of CR10049-treated animals. Results are expressed as mean ± SEM (A) or SD (B) for n=11/group-n=6/naïve (A) or n=7/9 (B) animals and analyzed by two-way ANOVA with Dunnett (A) or Mann-Whitney t-test (B) comparisons. ** p < 0.01, ***p < 0.001, **** p < 0.0001 vs Vehicle.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/cr10049-the-first-intra-articular-src-family-kinase-inhibitor-as-a-long-acting-symptom-and-disease-modifying-drug-for-the-inflammatory-oa-phenotype/