Identifying the Skeletal Benefits of SFX-01

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Our project fully aligns to BBSRC's 'bioscience for health' priority. It focuses on bioscience that will identify new extensions to 'healthspan' to relieve medical intervention requirements and the world-wide osteoarthritis (OA) healthcare burden. It merges newly identified RVC insights in signalling during the natural deterioration of healthy joints with age with Evgen Pharma's compounds that target this pathway. Our project is clearly and acutely motivated to use bioscience to underpin new effective therapy for mutual benefit. Sulforaphane (Sulf, unstable) potently induces Nrf2 (nuclear erythroid related factor 2) to upregulate protective enzymes and inhibit proinflammatory signalling. We thus examined if SFX-01 (stable Sulf) modulates the natural OA that develops in STR/Ort mice, to find indeed that it severely limited symptoms (accustomed limp fails to develop) and increased bone mass. We hypothesise: that Nrf2-mediated blockade of NF-kB by SFX-01 promotes osteogenesis, limits bone resorption and restricts endochondral cartilage-to-bone conversion (EO) to slow OA. We will address 3 independent aims focused (annually) on SFX-01 targeting of osteoclasts, osteoblasts and chondrocytes in STR/Ort and CBA (control mice to define any reliance on Nrf2-mediated NF-kB inhibition, and seek confirmation in SFX-01-treated STR/Ort and CBA (ongoing), and Nrf2 KO mice (collaboration).Aim 1 (yr 1): Does SFX-01 regulate osteoclast (OC) formation/resorption?OC-forming marrow mononuclear cells from 8wk STR/Ort (pre-OA onset) and CBA control (non-prone) bones will be cultured and non-adherent cells suspended on dentine discs (24hr in medium +M-CSF/RANKL); attached OC precursors cultured for 8d with/without Sulf or SFX-01 (0-1mM; pH 7.0 for final 2d to activate resorption). Discs reacted for tartrate-resistant acid phosphatase (TRAP) activity will be used to asses OC number and resorbed area (activity/OC). Studies at optimal Sulf/SFX-01 levels will evaluate NF-kB activation in presence/absence of Nrf2-selective inhibitor (trigonelline) or activator (RA839) and corroboration sought in joints from Str/ort and CBA mice treated with SFX-01 and, if necessary, confirmed in Nrf2 KO and human OCs. Aim 2 (yr 2): Does SFX-01 regulate osteoblast (OB) proliferation/differentiation?Primary OBs from STR/Ort and CBA will be cultured with/without Sulf or SFX-01 (0-1mM); cell no./viability measured by crystal violet and proliferation by PCNA/BrdU labelling; OB differentiation by alkaline phosphatase (per protein; BCA) and by histochemistry; transcript/ protein levels for markers of distinct stages of OB differentiation measured by qPCR and immunoblotting; mineralisation quantified by Alizarin Red. As above, studies will corroborate Nrf2-mediated NF-kB roles using Nrf2 inhibitor/activator, Nrf2 KO OBs and SFX-01-treated STR/Ort/CBA joints and human primary osteoblasts (normal/OA shoulders, ongoing).Aim 3 (yr 3): Does SFX-01 regulate EO? Embryo (E15) metatarsals from STR/Ort and CBA mice will be cultured with/without Sulf or SFX-01 (0-1mM). Total/mineralisation zone length, growth plate organisation (histology) and mineral density and bone mass/architecture (microCT) assessed for 10d. Transcript/protein levels for Wnt signalling markers, proliferation, cell death, EO and NF-kB activation will be measured to define mechanisms of SFX-01 action. Corroboration sought in Nrf2 KO mice and in SFX-01-treated Str/ort and CBA joints.