Anti-inflammatory properties of SM04690, a small molecule inhibitor of the Wnt pathway as a potential treatment for knee osteoarthritis V. Deshmukh 1, T. Seo 1, C. Swearingen 1, Y. Yazici 1 1 Samumed, LLC, San Diego, CA, USA
Disclosures Shareholders and employees of Samumed, LLC: Vishal Deshmukh Tim Seo Christopher Swearingen Yusuf Yazici
The Wnt pathway, osteoarthritis (OA), and inflammation Increased Wnt signaling drives bone formation, cartilage breakdown, and inflammation 1-4 Wnt pathway mutations (e.g., FrzB, DOT1L) are associated with OA 5,6 Wnt proteins are over-expressed in OA joints 7,8 1. Hamerman D. (1993) N Engl J Med. 2. Yuasa, t et al. (2008) Lab Invest. 3. Ma B and Hottiger, MO (2016) Frontiers Immun 4. Sokolove J and Lepus CM. (2013) Ther Adv Musculoskelet Dis. 5. Blom AB, et al. (2009) Arthritis Rheum. 6. Monteagudo S, et al. (2017) Nat Commun. 7. Rudnicki JA and Brown AM. (1997) Dev Biol. 8. Thomas RS, et al. (2011) Arthritis Res Ther. Figure adaptation: Bush and Beier. (2013) Nature Medicine. 667-9.
SM04690: A Wnt pathway inhibitor for knee OA SM04690 is a small molecule, intra-articular (IA), Wnt pathway inhibitor in development for treatment of knee OA 1,2 In previous preclinical studies, SM04690: inhibited inflammation 1 decreased cartilage degradation 1 regenerated cartilage 1 demonstrated sustained local exposure and no observable systemic toxicity 1,2 In previous phase 1 and phase 2a clinical studies, a single IA SM04690 injection appeared well-tolerated and showed potential for improving symptoms and maintaining joint space width in knee OA subjects 3 The current studies evaluated SM04690 effects in an inflammatory model of OA. 1. Deshmukh V, et al. (2017) OAC. 2. Yazici Y, et al. (2017) OAC. 3. Yazici Y, et al. (2017) Arthritis Rheum.
SM04690 exhibited broad anti-inflammatory properties PBMCs or T and B cells or HDF or ECs Superantigen or LPS or IgM SM04690 or cyclosporin A or prednisolone or vehicle In vitro anti-inflammatory activity of SM04690 was measured on the DiscoverX BioMAP platform using an empirical scale (0-5), where 0=weak activity and 5=highly potent activity. SM04690 demonstrated comparable or better activity than prednisolone and cyclosporin A across several anti-inflammatory assays. Highly potent Abbreviations: PBMC, peripheral blood mononuclear cells; HDF, human dermal fibroblasts; EC, endothelial cells; LPS, lipopolysaccharide Weakly active
SM04690 anti-inflammatory activity - In vitro
C y to k in e le v e ls (p g /m l) Decreased inflammation: SM04690 suppressed inflammatory cytokines R e la tiv e E x p re s s io n R e la tiv e E x p re s s io n Cellular assay: Synovial fibroblasts were stimulated with IL-1β to induce cytokine production, then treated with SM04690 Cytokine production was quantified by ELISA and qrt-pcr Dose dependent inhibition of IL-1β, IL-6, IL-8, and TNF-α production was demonstrated Synovial fibroblasts 6 0 0 4 0 0 T N F - IL - 6 2 0 IL -1 4 IL -8 1 5 3 2 0 0 1 0 5 ** ** 2 1 ** *** 0 0 1 2 3 4 L o g C o n c. (n M ) 0 Unstimulated 0 IL-1β (100 ng/ml) IL-6 EC 50 = 24 nm; TNF-α EC 50 = 35 nm IL-1β + SM04690 (100 nm) IL-1β + SM04690 (30 nm) n=3 replicates, Mean ± SEM, **p<0.01, ***p<0.001
N o rm a liz e d fo ld c h a n g e Decreased inflammation: SM04690 suppressed inflammatory cytokines N o rm a liz e d fo ld c h a n g e Cellular assays: Synovial fibroblasts were stimulated with LPS and peripheral blood mononuclear cells (PBMCs) were stimulated with super antigen (sag) SM04690 inhibited pro-inflammatory cytokine secretion compared to vehicle 1.5 Synovial Fibroblasts stimulated with LPS 1.5 PBMCs stimulated with sag 1.0 0.5 0.0 * * * * * * * * * * * * * * * * IL - 1 IL - 2 IL - 5 IL - 6 IL - 8 T N F IF N 1.0 0.5 ** *** *** *** 0.0 P ro lif e ra tio n I L -1 I L -1 I L -2 I L -6 I L -8 T N F - I L -1 7 A *** *** *** *** DMSO SM04690 (30 nm) DMSO SM04690 (110 nm) SM04690 (330 nm) n=3 replicates, Mean ± SEM, *p<0.05, **p<0.01, ***p<0.001.
SM04690 inhibited LPS-stimulated inflammation in human monocytes via NFκB Cellular assay: Human monocytes were stimulated with LPS and treated with SM04690 for 4hrs Levels of proteins were measured by Western blot SM04690 specifically inhibited NFκB phosphorylation in vitro and had no effects on other pathways
SM04690 anti-inflammatory activity - In vivo
Inflammatory model of rat OA: Monosodium Iodoacetate (MIA) injection OA induction Day 0 Day 3 Day 11 Day 28 MIA injection Rat MIA model: Inflammation within 2 hours and cartilage degeneration within 1-2 weeks Monosodium iodoacetate (MIA) intra-articular (IA) injection on Day 0 SM04690 IA injection on Day 3 (0.3 μg) Joint histology performed on Day 11 for histology and Day 28 for joint health
S y n o v ia l m e m b ra n e th ic k n e s s (u m ) SM04690 attenuated acute inflammation in the rat MIA knee OA model compared to vehicle H&E staining after a single IA injection of SM04690 decreased inflammatory infiltrates, decreased hypercellularity, and improved structural integrity, compared to vehicle treatment at Day 11 Synovial membrane thickness was significantly decreased in SM04690 joints compared to vehicle at Day 11 Sham H&E Staining Synovial membrane thickness 1 5 0 0 1 0 0 0 * * MIA + Vehicle 5 0 0 MIA + SM04690 (0.3µg) 0 S h a m M IA + V e h ic le M IA + S M 0 4 6 9 0 (0.3 g ) n=30 sections, Mean ± SEM, **p<0.01, one-way ANOVA
SM04690 attenuated acute inflammation and protected cartilage in the rat MIA knee OA model A single IA injection of SM04690 decreased inflammatory cytokines and matrix metalloproteinases (MMPs), compared to vehicle treatment at Day 11 Cytokine gene expression Protease gene expression 3 IL 1-3 T N F - 4 IL -6 1.5 M M P 1 1.5 M M P 3 1.5 M M P 1 3 1.5 A D A M T S 5 R e la tiv e E x p r e s s io n 2 1 * R e l a t i v e E x p r e s s i o n 2 1 * R e la tiv e E x p r e s s io n 3 2 1 * R e la tiv e E x p re s s io n 1.0 0.5 * R e la tiv e E x p re s s io n 1.0 0.5 * R e la tiv e E x p re s s io n 1.0 0.5 * * R e la tiv e E x p re s s io n 1.0 0.5 * * 0 0 0 0.0 0.0 0.0 0.0 Sham MIA + Vehicle MIA + SM04690 (0.3 µg) MIA + Vehicle MIA + SM04690 (0.3 µg) n=10 rats/group, Mean ± SEM, *p<0.05, one-way ANOVA n=8 rats/group, Mean ± SEM, *p<0.05, **p<0.01, t-test
SM04690 attenuated pain in the rat MIA knee OA model A single IA injection of SM04690 decreased pain (measured by Von Frey) and improved gait (measured as weight distribution), compared to vehicle treatment n=10, estimated treatment effect ± 95% CI, *p<0.05, **p<0.01, ***p<0.001, generalized estimating equation regression
O A R S I S c o re SM04690 protected cartilage in the rat MIA knee OA model A single IA injection of SM04690 improved Safranin O staining and OARSI scores compared to vehicle at Day 28 Sham * 6 Safranin O staining 4 800 μm 2 MIA + Vehicle MIA + SM04690 (0.3µg) 0 S h a m M IA + V e h ic le M IA + S M 0 4 6 9 0 (0.3 g ) 800 μm 800 μm n= 10, Mean ± SD, *p<0.05, Mann-Whitney U test
Conclusions From in vitro models: SM04690 demonstrated potent anti-inflammatory effects across a broad range of cytokines These effects appeared to be mediated via NFĸB In the MIA rat knee OA model, SM04690, compared to vehicle: Attenuated inflammation and structural damage to the knee Improved pain in treated rats Protected cartilage from catabolic breakdown (Model limitations included exaggerated inflammatory and degenerative responses compared to human knee OA.) Further studies elucidating the role of SM04690 in inflammatory pathways are on-going A human Phase 2b clinical trial is in progress
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