Identifying the Skeletal Benefits of SFX-01

Lead Research Organisation: Royal Veterinary College
Department Name: Comparative Biomedical Sciences CBS

Abstract

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.

Publications

10 25 50

Studentship Projects

Project Reference Relationship Related To Start End Student Name
BB/M009513/1 01/10/2015 31/03/2024
1906049 Studentship BB/M009513/1 01/10/2017 30/03/2022 Polymnia Louka
BB/R506011/1 01/10/2017 30/09/2021
1906049 Studentship BB/R506011/1 01/10/2017 30/03/2022 Polymnia Louka
 
Description Sulforaphane exerts both anti-oxidant and anti-inflammatory effects, but is unstable. A chemically complexed stable form (sulforaphane/alpha-cyclodextrin, SFX-01) has previously been found to exert beneficial bone effects in a mouse (STR/Ort) model of spontaneous osteoarthritis (OA) and inhibition of osteoclasts derived from C57/BL6. In terms of in vitro work, SFX-01 has been shown to be more beneficial and influential in differentiation phase to exert predominantly lasting restriction upon osteoclast resorptive function. We thereby, hypothesise that SFX-01 inhibits osteoclast fusion and thereby associated genes will be examined. In addition, preliminary work has shown that SFX-01 treated osteoclasts exhibit fewer nuclei and smaller in size. In addition, we have explored whether in vitro osteoclastogenic potential and sensitivity to SFX-01 is related to mass in different mouse strains to find that osteoclasts derived from mice with higher bone mass exhibit lower in vitro osteoclastogenic potential as well as a correspondingly reduced sensitivity to inhibition by SFX-01. It is tempting to speculate that the bone marrow origin of osteoclasts imparts conserved and inherent differences in osteoclast behaviour that are matched to bone mass and to sensitivity to SFX-01. Finally, NRF2 and NF-kB activators inhibited osteoclastogenesis and resorption while the activators did not show any significant changes. Thereby, further work is required in order to examine whether the pharmacological activation of NRF2 influence SFX-01 mediation inhibition of osteoclastogenesis/resorption.
However, in vitro osteoblastogenic potential of SFX-01 has shown no significant changes suggesting that SFX-01 has anti-resorptive activities rather than formation.
In addition, in order to examine whether SFX-01 influence the disease development in a spontaneous model of mouse OA, male STR/Ort mice at 13-14 weeks of age, were assigned randomly to vehicle(water) and treated in drinking water of 100mg/kg SFX-01 for 5 months. Gait was recorded using treadmill based DigiGait imaging system every two weeks till the end of experiment. Tibial from vehicle and SFX-01 were scanned using micro-CT scanner. Slices were reconstructed and then 2D/3D analysis has been performed. Results so far, indicate that SFX-01 treatment changes metaphyseal trabecular bone by leading to significant enhancement in bone area and trabecular thickness. In terms of the cortical bone, it has been shown that SFX-01 treatment also alters cortical bone by enhancing bone area and indices of tibial strength. Interestingly, tibial length has been increased in treated groups, making it tempting to further examine the endochondral ossification involvement.

In addition to the above, further in vitro assays have shown the early stage exposure of SFX-01 in sufficient for lasting inhibitin of osteoclastogenesis resorption and just 24hr exposure is enough for this effect suggesting the rapid effect of SFX-01 on osteoclasts. In terms of the mechanisms, NRF2 activator in osteoclast cultures indicated that it ihnibited osteoclastogenesis and that SFX-01 increases the inhibition achieved by activation of NRF2. Regarding the NF-KB, activation of NF-KB increased the inhibition of osteoclastogenesis achieved by SFX-01. Finally preliminary data also suggest that NRF2 activation is more profound in early stage of osteoclastogenesis suggesting that the effect of NRF2 and SFX-01 is more effective in the differentiation stage.
Exploitation Route According to Arthritis Research UK nearly 30% of the patients live with disability, and much higher percent account for cases of work-related ill-health (42%). This also means that OA patients have also high out of pocket expenditures including the direct costs for drugs, medications and indirect costs related with their productivity at work, or early retirement. This also costs to NHS almost 5 million and the no cure-cost will continue to increase each year.
If we manage to further prove the effectiveness of SFX-01 in pre-clinical models, it could finally enter human clinical trials for treatment of arthritic joints which could a major advance in the socio-economic burden we currently have with OA.
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology
 
Description Osteoarthritis (OA) is common cause of gait impairment as well as physical disability. Aging and obesity are the most common contributions as one to five people over the age of 45 years old seem to develop the condition. In total 9 million people in the UK suffer from OA and this is not only a major barrier to their mobility but also affects their day-to day function. OA causes health but also huge economic burden in humans and in animals. According to Arthritis Research UK nearly 30% of the patients live with disability, and much higher percent account for cases of work-related ill-health (42%). This also means that OA patients have also high out of pocket expenditures including the direct costs for drugs, medications and indirect costs related with their productivity at work, or early retirement. This also costs to NHS almost 5 million and the no cure-cost will continue to increase each year. Despite the fact that plentiful studies have been performed to improve our understanding of OA, the effective treatment options remain limited. Most of the drugs provide pain relief and indeed improve function and quality of life but none of them have proven disease modifying activity, and they have also side effects. Joint replacement is considered as the treatment of last resort for people with OA. However, many patients are not satisfied with the final outcome and most of them need to revise the surgery due to problems such as implant wearing and loosening. OA now been accepted as a serious disease by the FDA and it thereby requires development of new disease-modifying drugs which effectively target OA changes in cartilage and bone. Our project focuses on a stable sulforaphane based drug in that could be the key for OA. Sulforaphane is release by eating cruciferous and more particularly broccoli. Several studies have shown that sulforaphane which is released by broccoli has anti-inflammatory and anti-cancer properties. However, all those studies rely on the use of sulforaphane in the form of a frozen broccoli extract. Thereby what holds sulforaphane back from the clinical development is the fact that it is highly unstable. Evgen has incorporated it into a stable form medication called Sulforadex (SFX-01) which seems to correspond to eating arounf 2.5kg of broccoli in a day and phase II clinical trials are already in place for metastatic breast cancer and haemorrhage. Novel results have recently showed improved bone architecture and greater symmetry in gait and movement on osteoarthritic mice model that have been treated with sfx-01 compared to untreated mice. Thereby, this strongly encourages us that the stable form of sulforaphane could be a promising drug for the treatment of osteoarthritis that could effectively target OA. If we manage to further prove the effectiveness of SFX-01 in pre-clinical models, it could finally enter human clinical trials for treatment of arthritic joints which could a major advance in the socio-economic burden we currently have with OA.
Sector Healthcare
Impact Types Cultural,Societal,Economic