HU308-mediated cannabinoid signaling mitigates age and trauma-related osteoarthritis

Purpose: Osteoarthritis (OA) is characterized by progressive, irreversible erosion of articular cartilage that may be evoked during progressive age and after traumatic insult. This study aims to assess the attributes of HU308- a selective cannabinoid receptor type 2 (CB2) agonist, to serve as a potential Disease modified OA drug (DMOAD). We further attempted to elucidate the mechanism, by which HU308 may contribute to preserving joint cartilage.

Methods: Wild type (wt) mice (female, 3 month-old, C57BL6/J) were subjected to post-traumatic OA induction using Destabilization of the Medial Meniscus (DMM) surgical procedure. The mice were Intra-articularly (IA) injected with HU308 (120 μM, 10 μL, twice a week) from 4-8 weeks post-surgery. After sacrifice, the joints were assessed for the presence of osteophyte and articular cartilage histopathology, following Safranin O/ Fast Green staining. Moreover, pain thresholds were assessed using a Pressure Applicator Measurement (PAM device) at baseline (i.e. prior to procedure), 4 weeks (i.e. prior to first injection) and at the 8-week experimental endpoint. Next, we assessed the effect of systemic HU308 administration (intraperitoneal-IP, 3mg/kg in 100μL; twice a week for 4 weeks) on aged wild type (wt; Sirt1^flox) mice (6, 16 and 21 months of age).To decipher the mechanism of action rendered by HU308, we similarly assessed joint histopathology of 20-month-old wt vs Cb2r nulls, which are genetically ablated for CB2 receptor. Human chondrocytes were stimulated with 10-200 μM HU308 and assessed via immunoblotting, immunofluorescence (IF) and quantitative PCR analysis, for targeted circuits affected by HU308. Furthermore, we monitored if Sirt1 is a downstream mediator of HU308 by IP administration of HU308 to 16-month-old wt (Sirt1^fl/fl) and cartilage-specific Sirt1 knockout mice, which were generated by inducible aggrecan-dependent Cre-recombinase driver, crossed with Sirt1^fl/fl strain (i.e. ATC^cre Sirt1^fl/fl).

Results: Intra-articularly administered HU308 attenuated cartilage damage, osteophyte appearance and pain sensitivity following DMM procedure. While old (16 and 21 month) mice exhibit severe OA and reduced pain thresholds vs. younger (6 months) mice, IP administration of HU308 to 16-month mice not only improved pain sensitivity, but also maintained cartilage integrity. Interestingly, 21-month mice exhibited higher pain thresholds following HU308 administration vs vehicle, however OA severity remained unaffected, indicating a certain timeframe for effective HU308 treatment. Mechanistically, Cb2r null mice showed increased age-induced severity of OA as compared to age-matched wt mice, indicating that Cb2r receptor mediated signaling is required for maintaining cartilage homeostasis during aging. Assessing human chondrocyte treated with HU308 (10-200nM), display a dose-dependent increase in aggrecan and SIRT1 transcription levels. Interestingly, aged-mice bearing cartilage specific Sirt1 ablation (i.e. 16 months; ATC^cre Sirt1^fl/fl) exhibited similar OA severity following systemic IP administration of HU308 vs vehicle, indicating that HU308 might partially require Sirt1 to bestow its joint protective effects.

Conclusions: Collectively, the results show that HU308 reduced trauma and age-induced OA severity and pain sensitivity, in a mechanism that may involve CB2 signaling, and partial involvement of Sirt1 as an intracellular mediator coaxing aggrecan mRNA expression. While these effects of HU308 require further in depth mechanistic evaluation, these results support that HU308 bestows a significant therapeutic effect in preventing joint pain and maintaining joint health and integrity.