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A Small Molecule Inhibitor of the Wnt Pathway (SM04690) As a Potential Disease Modifying Agent for the Treatment of Osteoarthritis of the Knee
Vishal Deshmukh, PhD, Haide Hu, PhD, Charlene Barroga, PhD, Carine Bossard, PhD, Sunil KC, PhD, Luis Dellamary, BS, Joshua Stewart, BS, Kevin Chiu, BS, Maureen Ibanez, MS, Melinda Pedraza, BS, Tim Seo, MS, Long Do, PhD, Shawn Cho, MS, Joseph Cahiwat, BS, Betty Tam, PhD, Jeyanesh R.S. Tambiah, MBChB, John Hood, PhD, Nancy E. Lane, MD, Yusuf Yazici, MD
PII: S1063-4584(17)31167-6
DOI: 10.1016/j.joca.2017.08.015
Reference: YJOCA 4078
To appear in: Osteoarthritis and Cartilage
Received Date: Revised Date: Accepted Date:
2 January 2017 18 July 2017 30 August 2017
Osteoarthritis
Please cite this article as: Deshmukh V, Hu H, Barroga C, Bossard C, KC S, Dellamary L, Stewart J, Chiu K, Ibanez M, Pedraza M, Seo T, Do L, Cho S, Cahiwat J, Tam B, Tambiah JRS, Hood J, Lane NE, Yazici Y, A Small Molecule Inhibitor of the Wnt Pathway (SM04690) As a Potential Disease Modifying Agent for the Treatment of Osteoarthritis of the Knee, Osteoarthritis and Cartilage (2017), doi: 10.1016/ j.joca.2017.08.015.
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1 Title Page
2 A Small Molecule Inhibitor of the Wnt Pathway (SM04690) As a Potential Disease
3 Modifying Agent for the Treatment of Osteoarthritis of the Knee
5 Running title: Wnt pathway inhibitor as potential DMOAD for OA
7 Authors: Vishal Deshmukh1, Haide Hu1, Charlene Barroga1, Carine Bossard1, Sunil
8 KC1, Luis Dellamary1, Joshua Stewart1, Kevin Chiu1, Maureen Ibanez1, Melinda
9 Pedraza1, Tim Seo1, Long Do1, Shawn Cho1, Joseph Cahiwat1, Betty Tam1, Jeyanesh
10 R.S. Tambiah1, John Hood1, Nancy E. Lane2 , Yusuf Yazici1 +
11 +corresponding author
13 Affiliations: 1 Samumed, LLC, San Diego, CA, USA
14 2 University of California, Davis, CA, USA
16 Author Emails: Vishal Deshmukh, PhD (vishal@samumed.com), Haide Hu, PhD
17 (huyong21@gmail.com), Charlene Barroga, PhD (charlene@samumed.com), Sunil KC,
18 PhD (sunil@samumed.com), Luis Dellamary, BS (luis@samumed.com), Joshua
19 Stewart, BS (josh@samumed.com), Kevin Chiu, BS (kevin@samumed.com), Carine
20 Bossard, PhD (carine@samumed.com), Maureen Ibanez, MS
21 (maureen@samumed.com), Melinda Pedraza, BS (melinda@samumed.com), Tim Seo,
22 MS (tim@samumed.com), Long Do, PhD (long@samumed.com), Shawn Cho, MS
23 (shawn@samumed.com), Joseph Cahiwat, BS (joec@samumed.com), Betty Tam, PhD
24 (betty@samumed.com), Jeymi Tambiah, MBChB (jeymi@samumed.com), John Hood,
25 PhD (john@impactbiosciences.com), Nancy E. Lane, MD (nelane@ucdavis.edu), Yusuf
26 Yazici, MD (yusuf@samumed.com)
28 + Main corresponding author: Yusuf Yazici, MD, Samumed LLC. 9381 Judicial Drive,
29 Suite 160, San Diego, CA 92121; Tel: 858-926-2962; yusuf@samumed.com
46 Abstract
48 Objectives
50 Osteoarthritis (OA) is a degenerative disease characterized by loss of cartilage and
51 increased subchondral bone within synovial joints. Wnt signaling affects the
52 pathogenesis of OA as this pathway modulates both the differentiation of osteoblasts
53 and chondrocytes, and production of catabolic proteases. A novel small molecule Wnt
54 pathway inhibitor, SM04690, was evaluated in a series of in vitro and in vivo animal
55 studies to determine its effects on chondrogenesis, cartilage protection and synovial-
56 lined joint pathology.
58 Design
60 A high-throughput screen was performed using a cell-based reporter assay for Wnt
61 pathway activity to develop a small molecule designated SM04690. Its properties were
62 evaluated in bone marrow derived human mesenchymal stem cells (hMSCs) to assess
63 chondrocyte differentiation and effects on cartilage catabolism by immunocytochemistry
64 and gene expression, and glycosaminoglycan breakdown. In vivo effects of SM04690
65 on Wnt signaling, cartilage regeneration and protection were measured using
66 biochemical and histopathological techniques in a rodent acute cruciate ligament tear
67 and partial medial meniscectomy (ACLT+pMMx) OA model.
69 Results
71 SM04690 induced hMSC differentiation into mature, functional chondrocytes and
72 decreased cartilage catabolic marker levels compared to vehicle. A single SM04690
73 intra-articular (IA) injection was efficacious in a rodent OA model, with increased
74 cartilage thickness, evidence for cartilage regeneration, and protection from cartilage
75 catabolism observed, resulting in significantly improved Osteoarthritis Research Society
76 International (OARSI) histology scores and biomarkers, compared to vehicle.
78 Conclusions
80 SM04690 induced chondrogenesis and appeared to inhibit joint destruction in a rat OA
81 model, and is a candidate for a potential disease modifying therapy for OA.
83 Keywords: Osteoarthritis, Wnt signaling, chondrocyte, ACLT, DMOAD, small molecule
85 INTRODUCTION
87 Osteoarthritis affects an estimated 27 million adults in the U.S. and is a leading cause of
88 disability worldwide1. The disease is characterized by breakdown of articular cartilage
89 and growth of subchondral bone causing pain, decreased mobility, limitation of function
90 and failure of synovial joints2. The only current therapeutic options for OA are
91 symptomatic pain management and surgical intervention3, 4 There is therefore an
92 unmet need for disease modifying OA drugs (DMOADs). Mesenchymal stem cells
93 (MSCs) in synovium and subchondral bone are capable of differentiation into cartilage
94 forming chondrocytes, bone forming osteoblasts, or adipocytes5. Compared to healthy
95 synovium, synovium of subjects with meniscal injury and early OA is enriched in stem
96 cells6-8, suggesting the failure to regenerate articular cartilage may not be due to
97 insufficient stem cell supply, but rather their inappropriate differentiation when
98 attempting to restore healthy cartilage homeostasis.
100 Wnt proteins interact with stem and differentiated cells to orchestrate organogenesis,
101 cell differentiation, morphogenesis and tissue remodeling. In adults, the role of the Wnt
102 pathway in tissue formation is extended to homeostatic control through the tightly
103 regulated differentiation of resident stem cells to replenish and repair adult tissues9, 10
104 Activation of Wnt signaling is initiated by Wnt proteins binding to their cognate
105 receptors, and is modulated by endogenous antagonists such as Dickkopf, Wnt-
106 inhibitory signaling protein, secreted Frizzled related protein and Cereberus. In the
107 canonical Wnt/p-catenin-signaling pathway, binding of Wnt proteins to cell surface
108 receptors leads to stabilized p-catenin levels by inhibition of p-catenin phosphorylation
109 and proteasome-mediated degradation. Stabilized p-catenin translocates to the nucleus
110 and interacts with the T-cell factor/lymphoid enhancer factor (TCF/LEF) family of
111 transcription factors to activate Wnt target genes9, 10 Wnt signaling is subject to
112 complex modulation at multiple levels, resulting in signaling acting more as rheostats,
113 than binary switches in most tissues. Modulating this pathway is an attractive
114 therapeutic approach for regenerative medicine, however success has been limited due
115 to lack of potent, safe therapeutic agents11.
117 Recently, increased understanding of cartilage growth mechanisms during OA
118 development and aging have identified new molecular mechanisms and targets.
119 Amongst these, Wnt signaling has been shown to play a critical role in OA
120 pathogenesis, particularly cartilage and subchondral bone remodeling12. A single
121 nucleotide polymorphism analysis demonstrated an association of both proximal femur
122 bone shape and risk of hip OA with an Arg324Gly substitution mutation in FrzB, a Wnt
123 antagonist, implicating the Wnt pathway as a potential regulator of the disease13, 14 At a
124 molecular level, the balance of Wnt signaling maintains osteoblast and chondrocyte
125 lineage fates and their homeostasis. Increased mechanotransductive force increases
126 Wnt signaling in the joint, which results in osteoblast formation and release of proteases
127 that remodel articular cartilage into an osteoconductive matrix, while decreased Wnt
128 signaling stimulates chondrogenesis12. Wnt signaling has also been implicated in the
129 induction of protease production, especially matrix metalloproteinases (MMP1, MMP-3
130 and MMP-13) by chondrocytes and synovial tissue in response to mechanical stress
131 and pro-inflammatory cytokines15. In the context of a joint with abnormal loading, this
132 has the potential to lead to a progressive disease such as OA, with Wnt playing a
133 central role in its pathogenesis.
135 In this study, SM04690, a small molecule Wnt pathway inhibitor being developed as a
136 potential DMOAD, was evaluated for its effects on chondrocyte differentiation, cartilage
137 regeneration and protection, and prevention of joint destruction in a preclinical model of
138 knee OA.
140 METHODS
142 Cell culture and differentiation
143 SW480 cells (ATCC) cultured in Dulbecco's modified Eagle's medium (DMEM,
144 ThermoFisher, Carlsbad, CA) with 10% fetal bovine serum (FBS) were transduced with
145 the TCF/LEF-luciferase lentivirus (Qiagen, Germantown, MD) and stable clones were
146 selected using puromycin. Primary bone marrow derived hMSCs (Lonza Inc., Basel,
147 Switzerland) were grown in MSCGM™ mesenchymal stem cell growth medium (Lonza),
148 and used between passages 2 and 6.
150 High-throughput screening
151 A chemical library was screened in a cellular TCF/LEF reporter-based assay using
152 SW480 cells. Compounds were transferred into screening plates using ECHO 550
153 (Labcyte, San Jose, CA). SW480 cells were plated (3000/well) in DMEM with 1% FBS.
154 After 48 hrs, BrightGlo (Promega, Madison, WI) luminescence was measured using
155 Envision (Perkin Elmer, Waltham, MA). For chondrogenic differentiation, hMSCs were
156 plated (20,000/well) in incomplete chondrocyte differentiation medium (iCDM; Lonza)
157 and treated with SM04690 or compounds- FH535, IWR-1, ICG001, iCRT14, KY02111,
158 and, CX-4945 (Sigma-Aldrich) or Transforming Growth Factor p3 (TGFp3; 20ng/ml,
159 Peprotech Inc., Rocky Hill, NJ). On Day 5, cells were fixed, stained with 1 pg/mL
160 Rhodamine B and imaged using Cellomics CellInsight (ThermoFisher).
162 Chondrocyte Differentiation
163 For chondrocyte differentiation, hMSCs were plated in either 48-well plates
164 (40,000/well) or dispensed into 15ml conical tubes (150,000/tube) in iCDM and treated
165 with SM04690 in DMSO or TGFp3 (20ng/ml). Cells were incubated for 21 days, with
166 media changes every 5 days, fixed and immunostained with specific antibodies or with
167 Alcian Blue (1% in acetic acid, pH 2.5) or Safranin O (0.1% aqueous solution). Cells
168 were imaged using EVOS FL Microscope (ThermoFisher). Gene expression was
169 measured by quantitative real time PCR (qRT-PCR) using SYBR Green based, gene
170 specific primers.
171 Primary calvarial cells were isolated from mouse E13.5 embryos using collagenase
172 (Sigma-Aldrich, St. Louis, MI) and plated in poly-L-lysine coated plates. After 5 days
173 cells were treated with DMSO or SM04690. On Day 21, cells were fixed and stained
174 with Alcian Blue.
176 Cartilage Matrix Degradation- Glycosaminoglycan (GAG) and Nitric Oxide (NO)
177 measurement
178 hMSCs were differentiated into chondrocytes using TGFp3 for 21 days followed by
179 treatment with either TNFa (20ng/ml) + Oncostatin M (10ng/ml) or IL1 p (10ng/ml) and
180 SM04690 for 72hrs. Chondrocytes were digested with papain (Sigma). GAG content
181 was measured using the dimethylmethylene blue (DMMB) kit (Chondrex, Redmond,
182 WA) and absorbance at 535nm was measured using Cytation 3 (Biotek, Winooski, VT).
183 NO was measured using Greiss reagent kit (Promega).
185 Pharmacokinetics
186 Following single IA SM04690 injection into Sprague-Dawley (SD) rats (12 weeks old,
187 male), knee joints were collected, flash frozen and stored at -70°C. Bone and cartilage
188 samples were isolated from tibias, homogenized and SM04690 extracted with acidified
189 organic solvent. Extracts were analyzed using Phenomenex PFP column and HPLC
190 gradient method in tandem to a triple quadrupole mass spectrometer (API Triple Quad
191 3000) with a Turboionspray source for detection in positive ion mode.
193 Surgery-induced OA model
194 All animal studies were performed in accordance with approved Samumed, LLC Animal
195 Committee protocols. Male SD rats were housed at Samumed LLC, and provided food
196 and water ad libitum. At 10 weeks postnatal age, 24 rats were subjected to severing of
197 the anterior cruciate, medial collateral and medial meniscotibial ligaments
198 (ACLT+pMMx). One-week post-surgery, all rats were randomized and given IA
199 SM04690 (0.3pg in 50pl) or vehicle (n=12 rats/group). Thirteen weeks post-surgery,
200 knee joints were isolated, fixed in 10% formalin, decalcified and embedded in paraffin
201 blocks. Frontal sections (5 pm thick, 3 sections to ensure redundancy) from different
202 levels, each 100 pm apart, were obtained and stained with Safranin O/Fast Green. At
203 least 12 sections were obtained from each rat and imaged using a light microscope
204 (EVOS FL, Life Technologies).
206 OARSI Scoring, Cartilage Protection and Regeneration
207 Histological evaluation was performed by 2 blinded observers. Images were scored
208 based on the OARSI cartilage histopathology scoring system by evaluating damage
209 based on grade (depth of progression into cartilage) and stage (extent of joint
210 involvement)16. Briefly, femurs and tibias were evaluated separately, and each assigned
211 a score for cartilage damage grade on a scale of 0 to 6 (0 - intact surface; 6 - extensive
212 deformation) and cartilage damage stage on a scale from 0 to 4 (0 - normal joint; 4 -
213 >50% involvement). Total score was the product of Grade x Stage (0 representing
214 normal joint; 24 representing severe OA)16. Twelve sections were scored per rat and no
215 rats were excluded. Following scoring, the study was unblinded and the 4 sections from
216 each rat with lowest scores (representing least cartilage damage) were excluded from
217 further analysis. Mean OARSI score for each rat was generated by averaging the
218 scores from the 2 blinded observers. Additionally, histological evaluation was repeated
219 by 4 independent blinded observers based on a modified objective quantitative
220 histomorphometrical OARSI scoring system17. Safranin O staining intensity and
221 cartilage thickness were measured using ImageJ18.
222 Quantification of articular chondrocytes was performed using immunohistochemistry for
223 doublecortin (Dcx), followed by imaging (Keyence, confocal mode), and analysis using
224 Cellomics (ThermoFisher). Levels of circulating biomarkers were measured by ELISA
225 on weeks 3, 4, 6.
226 In a second study, 24 rats (male, 10-weeks old) were subjected to the ACLT+pMMx
227 procedure. One-week post-surgery all rats were randomized and given an IA injection of
228 SM04690 (0.3pg) or vehicle (n=12 rats/group). On week 5 after surgery, cartilage was
229 isolated from the rats for gene expression measurement of chondrocyte markers and
230 proteases.
232 Statistical analysis
233 Statistical analysis was performed using Prism 7 (Graphpad Inc, La Jolla, CA). EC5o
234 values were obtained using sigmoidal dose-response curve-fitting. For those outcomes
235 in which an underlying normal distribution was assumed, an independent, two-tailed t-
236 test was utilized for two group comparisons, and for more than two groups one-way
237 ANOVA was utilized. OARSI scores, for which the normal distribution was not assumed,
238 were evaluated using Mann-Whitney U test. Data were represented as Mean ± 95%
239 confidence interval (CI) or Mean ± standard deviation (SD) as noted in the figure
240 legends, and significance values listed. Significance was set at p<0.05. *p<0.05,
241 **p<0.01, ***p<0.001
243 RESULTS
245 Wnt Pathway Modulation- Cellular High-Throughput Screening Identified SM04690 as a
246 Potent and Selective Inhibitor of Wnt Signaling.
248 Small-molecule inhibitors of Wnt signaling were identified using a high-throughput
249 TCF/LEF-reporter assay in SW480 colon cancer cells bearing a mutation in the APC
250 protein19, which leads to constitutively active canonical Wnt signaling. Hits were
251 counter-screened against SW480 cells expressing a SV40-driven luciferase reporter, to
252 eliminate compounds that acted non-specifically. Based on the elucidated structure
253 activity relationship from these compounds, iterative medicinal chemistry efforts
254 generated SM04690 (EC50= 19.5 nM) which did not affect the SV40 luciferase reporter
255 (Figure 1a). The activity of SM04690 was then compared with known Wnt pathway
256 inhibitors such as FH53520, IWR-121, ICG00121, iCRT1422, KY0211123, and, CX-494524.
257 SM04690 was found to be ~150-500-fold more potent than the other compounds
258 (Supplementary Figure 1a, b). Inhibition of Wnt signaling was confirmed with SM04690
259 treatment in SW480 cells by qRT-PCR, and Western blot for p-catenin and its target
260 proteins (Figure 1b, c). Bone-marrow-derived hMSCs (CD29+, CD44+ CD166+ CD105+
261 CD45-), treated with SM04690, showed a dose-dependent decrease in the expression
262 of Wnt pathway genes (ASCL1, LEF1, TCF7L2, TCF7, C-MYC and AXIN2), as
263 measured by qRT-PCR (Figure 1d) and Western Blot (Figure 1e). SM04690 also
264 inhibited the expression of AXIN2, TCF7 and LEF1 in hMSCs (Supplementary Figure
265 1c) and AXIN2 and LGR5 in IEC6 (intestinal stem cells sensitive to Wnt activation;
266 Supplementary Figure 1d) when the Wnt pathway was selectively activated using either
267 Wnt3a or a GSK3p inhibitor, CHIR-99021. The inhibition of Wnt target genes by
268 SM04690 (30nM) was comparable to the activity of CX-494524 (10|jM) and better than
269 KY02111 (10|M)23. SM04690 had minimal effects on the non-canonical Wnt pathway
270 and the BMP pathway (Supplementary Figures 2, 3). Changes in the expression of
271 genes in these pathways (e.g., decreased expression of DAAM1, LEF1, RHOA, WNT4,
272 BMP4 and increased expression of DKK1, NLK1, WNT16, MAPK13, SMAD7) correlated
7 25 28
273 with Wnt pathway inhibition and the induction of chondrogenic differentiation7
275 Cartilage Regeneration- Wnt Pathway Inhibitor SM04690 Induced Chondrocyte
276 Differentiation in vitro.
278 Chondrogenesis occurs as a result of MSC and progenitor cell differentiation. To assess
279 SM04690 effects on early chondrogenesis, hMSCs were treated for 5 days, and
280 Rhodamine B stained chondrogenic nodules29 were measured as an indicator of early
281 cell condensation phenotype associated with chondrogenesis induction (Figure 2a, b).
282 SM04690 promoted aggregation of hMSCs in a dose-dependent manner (EC5o = 10nM,
283 Supplementary Figure 4a), increasing Rhodamine B stained colonies >40-fold
284 (p<0.0001), compared to DMSO treated control. TGFp330, was used as a positive
285 control. hMSCs treated with SM04690 for 3 days demonstrated significantly increased
286 (p=0.0006) induction of Sox931 by immunocytochemistry; Figure 2c, d) and Western blot
287 (Supplementary Figure 4b), compared to DMSO, indicating an early chondrocyte
288 lineage phenotype.
290 hMSCs treated with SM04690 in high density three dimensional, 21-day pellet culture
291 conditions efficiently differentiated into mature chondrocytes, as evidenced by larger
292 pellets with increased Safranin O staining relative to DMSO treated control (Figure 2e).
293 Additionally, hMSCs treated with SM04690 for 21 days under monolayer conditions
294 demonstrated chondrocyte differentiation, with the presence of mature chondrocyte
295 specific proteins and cartilage matrix components (TIMP1, Type II collagen, aggrecan,
296 Alcian Blue, Safranin O, Rhodamine B, Toluidine Blue and CD44)30-32 as compared to
297 DMSO treated cells (Figure 2f). Treatment with SM04690 also induced significant
298 increases in the sulfated GAG content of differentiated chondrocytes32 (Figure 2g)
299 compared to DMSO control (p=0.023).
301 Following SM04690 treatment, qRT-PCR analysis of differentiated cells further
302 confirmed increased expression of chondrocyte associated genes, including SOX9,
303 cartilage oligomeric matrix protein (COMP), aggrecan, COL2A1, TGFp1, tissue inhibitor
304 of metalloproteinase 1 (TIMP1), CD44, and Type 10 collagen (COL10A1) (Figure 3a),
305 as compared to DMSO treatment, with expression levels greater than those induced by
306 CX-4945 (10pM; p<0.05 for all genes except COL2A1) and KY02111 (10pM; p<0.05)
307 (Supplementary Figure 5a). SM04690 treatment decreased expression of genes
308 associated with osteoblast differentiation (BGLAP [osteocalcin], ALPL [Alkaline
309 phosphatase], BMP4, RUNX2)29, 33 and tendon or ligament differentiation (COL1A1)
310 (p<0.01, Figure 3b) with levels comparable to CX-4945 (10|M) and KY02111 (10|M)
311 treatment (Supplementary Figure 5b). While increased C0L10A1 expression was
312 observed in vitro, a corresponding increase was not observed in the in vivo model
313 treated with SM04690 (Supplementary Figure 9a), suggesting increased C0L10A1
314 expression may be a cell culture system result and not indicative of hypertrophic
315 differentiation34 induced by SM04690 treatment.
317 Further, the ability of SM04690 to induce differentiation of mouse progenitor cells into
318 chondrocytes was evaluated. SM04690 treatment for 21 days induced chondrocyte
319 differentiation in ATDC5 cells (mouse chondrogenic cell line35), as measured by
320 Safranin O and Alcian Blue staining (Figure 3c). Additionally, primary calvarial cells
321 isolated from E13.5 mouse embryos36 and cultured in the presence of SM04690
322 (100nM) for 21 days showed a marked increase in the number and size of Alcian Blue
323 stained chondrocytes, increasing the area of field covered with chondrocytes to >30%
324 compared to <3% in the DMSO treated control (Figure 3d, e).
326 Since SM04690 inhibited Wnt signaling, several commercially available Wnt inhibitors
327 were tested in the early chondrogenic aggregation and chondrocyte differentiation
328 assays. In line with their activities in the TCF/LEF reporter assay (Supplementary Figure
329 1a, b), several Wnt pathway inhibitors were ~50-500 fold less potent than SM04690
330 (Supplementary Figure 6a, b). SM04690 also induced ~3-10-fold more Alcian Blue and
331 Safranin O stained chondrocyte colonies, at ~200-fold lower EC50 as compared to CX-
332 4945, KY02111 and ICG-001 (Supplementary Figure 6c-h).
334 Cartilage Protection- Wnt Pathway Inhibitor SM04690 Protected Chondrocytes from
335 Catabolic Breakdown in vitro.
337 In addition to regeneration of cartilage, long-term disease modification in OA requires
338 inhibited degradation of newly formed and existing cartilage. Therefore, the effects of
339 SM04690 on chondrocytes and hMSCs and cartilage catabolism under
340 pathophysiological OA-like conditions were evaluated. Chondrocytes and hMSCs were
341 treated either with a combination of TNF-a (20ng/ml) and oncostatin M (10ng/ml), or
342 IL1p (10ng/ml) to mimic cytokine-induced cartilage degeneration during OA progression.
343 When treated with these cytokines, cultured chondrocytes and hMSCs upregulated the
344 expression of MMP-1, MMP-3, MMP-13 and IHH37 as measured by qRT-PCR (Figure
345 4a, b, Supplementary Figure 7a-d), increased extracellular secreted GAG29, as
346 measured by DMMB reaction (Figure 4c), and released increased amounts of NO29
347 (Figure 4d), as measured by the Greiss reaction, compared to untreated chondrocytes.
348 Treatment with SM04690 inhibited cytokine-induced expression of matrix degrading
349 enzymes (MMP-1, MMP-3, MMP-13, IHH) (Figure 4a, b, Supplementary Figure 7a-d),
350 with potency similar or improved (p<0.05) compared to CX-4945 (10 pM) and KY02111
351 (10 pM). Consistent with this effect, SM04690 treatment also significantly decreased
352 breakdown and release of GAGs (Figure 4c) and NO (Figure 4d) compared to controls
353 (p<0.05).
355 Pharmacokinetics- IA SM04690 Demonstrated Sustained Local and Minimal Systemic
356 Exposure
358 Pharmacokinetic evaluation of a single IA SM04690 injection in rats demonstrated knee
359 joint residence time >180 days (Supplementary Figure 8a). Systemic exposure of drug
360 was below quantifiable plasma levels (lower limit of quantification= 10nM) and no
361 obvious adverse effects (weight loss, significant swelling, signs of pain or distress) were
362 observed in the treated rats.
364 Cartilage Regeneration- IA SM04690 Promoted Cartilage Growth and Improved Joint
365 Health in a Rat Model of Knee Osteoarthritis.
367 The ACLT+pMMx rat OA model causes severe joint destabilization, leading to OA-like
368 disease with cartilage degradation within 1-2 weeks16, 38, 39 The efficacy of SM04690
369 was evaluated in the ACLT+pMMx rat knee OA model, following a single IA injection of
370 SM04690 (0.3pg) or vehicle control. Histological examination of the joints 12 weeks post
371 vehicle injection (13 weeks post-surgery) showed loss of smooth cartilage articular
372 surface and decreased intercondylar space between the femur and tibia in these rats
373 (Figure 5a). In contrast, SM04690 treated animals showed increased articular cartilage
374 and smooth cartilage surface as indicated by significantly increased Safranin O-Fast
375 Green staining intensity (p<0.0001; Supplementary Figure 8c) and cartilage thickness
376 measurements (p=0.0042; Supplementary Figure 8d). Blinded OARSI histopathology
377 analysis16 of the most severely affected joint areas, revealed a significant decrease
378 (p=0.008) in treatment group scores (Figure 5b) compared to vehicle controls.
379 Additionally, blinded scoring performed using a modified objective quantitative
380 histomorphometrical OARSI scoring method17 also showed a significant decrease
381 (p=0.0005) in the OARSI score with SM04690 treatment (Supplementary Figure 8b),
382 indicating improvement in joint morphology.
384 Serum was evaluated during the 12-week post- treatment period for N-terminal
385 propeptide of collagen IIA (PIIANP), a biomarker of cartilage matrix synthesis40. Levels
386 of PIIANP in serum were significantly increased (p=0.0186) at 3 weeks post-treatment in
387 rats treated with SM04690 as compared to vehicle control and remained slightly
388 elevated even at 5 weeks post-treatment (p>0.05; Figure 5c).
390 Additionally, the onset of chondrogenic differentiation in the SM04690 treated animals
391 was confirmed by increased gene expression of Col2a1, aggrecan and COMP in the
392 cartilage as compared to vehicle treatment (Figure 5d). However, no significant changes
393 were observed in the expression levels of Col10a, a chondrocyte hypertrophy marker,
394 (Supplementary Figure 9a) after 4 weeks from treatment with SM04690, as compared to
395 vehicle. Further, quantification of chondrocytes in the superficial zone of the articular
396 cartilage at 12- weeks post injection showed a significant increase (p=0.001) in the
397 number of Doublecortin (Dcx) positive chondrocytes41 in SM04690 treated rats
398 compared to vehicle (Figure 5e, f).
400 Cartilage Protection- Single IA Injection of SM04690 Decreased Cartilage Breakdown in
401 a Rat Model of Knee Osteoarthritis.
403 Local regulation of matrix degradation markers has previously been demonstrated by a
404 significant increase in mRNA levels of ADAMTS4 and MMP-13 in animal models of
405 OA38, 42 MMP-1, MMP-3, MMP-13 and ADAMTS5 were highly expressed in the articular
406 cartilage of ACL-transected, vehicle-treated animals. In SM04690 treated rats,
407 significant (p<0.05) reductions of protease levels (approximately 2-3-fold) were
408 observed at 4 weeks post injection as compared to vehicle-treated animals (Figure 5g).
409 Further, circulating COMP levels, which correlate with OA disease severity43, were
410 slightly reduced in SM04690-treated rats as compared to control rats at 2 weeks post-
411 treatment, and significantly reduced (p=0.0064) at 5 weeks post-treatment (Figure 5h).
413 Wnt Pathway Modulation- SM04690 Inhibited the Wnt Pathway in a Rat Model of Knee
414 Osteoarthritis.
416 The expression of Wnt pathway genes in the cartilage from ACLT+pMMx rats was
417 evaluated using a qRT-PCR panel comprising 84 Wnt pathway genes. Decreased
418 expression (p<0.05) of several Wnt pathway genes (e.g. GSK3ß, Dvl1, Wnt3a, TCF7,
419 Axin 2, p-catenin), and upregulation in Wnt inhibitory genes (e.g. DKK1, WIF1) was
420 observed in the cartilage of animals treated with SM04690 as compared to vehicle
421 controls (Supplementary Figure 9b-d). Finally, decreased expression and nuclear
422 localization of p-catenin was observed in articular chondrocytes in SM04690- treated
423 rats as compared to vehicle-treated rats (Supplementary Figure 10a-c).
425 DISCUSSION
427 Wnt signaling has been shown to be a pivotal pathway in OA that modulates bone and
428 chondrocyte lineage specification, protease production and joint homeostasis. SM04690
429 is being developed as a novel small molecule inhibitor of the Wnt pathway. In this study,
430 we used several in vitro assays for Wnt signaling, chondrogenesis and cartilage
431 catabolism to test the effects of SM04690 on these processes. In vitro, SM04690 was
432 ~50-500-fold more potent than published Wnt inhibitors across multiple cellular assays.
433 Functional inhibition of Wnt signaling in cartilage in the rat ACLT+pMMx model
434 demonstrated by both gene expression and inhibited p-catenin nuclear localization,
435 provided evidence for the ability of SM04690 to modulate the Wnt pathway in vivo, in
436 the context of OA.
438 The appearance of Rhodamine B staining with SM04690- treated hMSCs and mouse
439 stem cells in vitro indicated significant and dose-dependent induction of early
440 chondrocyte condensation, followed by differentiation into mature chondrocytes that
441 expressed both Type II collagen and Aggrecan that are required for the specialized
442 extracellular matrix rich in highly sulfated GAGs necessary to provide elastic
443 biomechanical properties for motion and weight bearing30. In the ACLT+pMMx model,
444 SM04690 treatment led to increased cartilage as demonstrated by Safranin O staining,
445 GAG measurement and serum biomarker PIIANP. An increased number of Dcx positive
446 articular chondrocytes as well as increased expression of chondrogenic genes in the
447 cartilage, as compared to vehicle controls, were consistent with induction of
448 chondrocyte differentiation in SM04690- treated rats. These data demonstrated the
449 ability of SM04690 to potentially regenerate functional chondrocytes and hence restore
450 cartilage in vivo.
452 Enzymatic tissue degradation plays a significant role in the progression of OA and
453 expression of proteases is increased in articular cartilage in response to joint injury38, 39
454 SM04690 inhibited the expression of catabolic enzymes in cytokine stimulated
455 chondrocytes and hMSCs in vitro, as well as in the ACLT+pMMx animals, compared to
456 vehicle controls, thus demonstrating specific control and regulation of catabolic
457 enzymes in the context of OA. SM04690 also decreased cytokine induced GAG
458 breakdown and NO production in vitro, and levels of circulating COMP in vivo, together
459 providing strong evidence that SM04690 prevented matrix breakdown by proteases
460 under the pathological conditions of OA. While additional studies are needed to
461 evaluate SM04690 effects on protease production and matrix catabolism in primary OA
462 patient-derived chondrocytes, these data suggested that SM04690 may have potential
463 cartilage protective effects.
465 Changes in gene expression, biomarkers and restored cartilage measured in the rat
466 ACLT+pMMx model experiments by histological OARSI scores indicated improvements
467 in joint morphology and attenuated OA phenotype with SM04690 treatment, despite the
468 ongoing absence of joint ligaments responsible for inducing the model. The observed
469 efficacy may be a result of a combination of mechanisms, including MSC differentiation
470 into chondrocytes, and cartilage protective effects of SM04690, however, the relative
471 contribution of each is unknown. In vivo pharmacokinetic data supported the use of a
472 single SM04690 IA injection for this study and the demonstrated efficacy of the
473 compound in human cells in vitro supported the rationale to pursue clinical development
474 of SM04690. In a phase 1 first-in-human clinical trial (N=61), SM04690 appeared safe
475 and well-tolerated (in press).
477 In the search for an effective treatment for OA, development of DMOADs has focused
478 on either slowing down the progression of the disease by targeting either matrix-
479 degrading enzymes (using MMP/ADAMTS inhibitors)42 or inflammatory mediators such
480 as IL1 p and TNFa44. Expression of factors such as FGF-18 and TGF-p to promote
481 chondrocyte proliferation and maturation, or stem cell based transplants have been
482 tested, but challenges remain with translation of these techniques into the clinic45, 46
483 Targeting the resident stem cell population in the articular region with an effective
484 therapy remains a promising approach29, 46.The potential to stimulate cartilage repair via
485 chondrocyte differentiation and reduce cartilage destroying proteases, could lead to a
486 beneficial treatment effect with SM04690. These data support the further development
487 of SM04690 as an injectable small molecule potential DMOAD for the treatment of knee
488 OA.
491 Author contributions:
493 Conception and design (J.H., C.Ba., S.K, H.H., V.D., L.D., J.S., N.L and Y.Y.)
494 Acquisition, analysis and interpretation of data (V.D., H.H., C.Ba., S.K., L.D., J.S., K.C.,
495 C.Bo, M.I., M.P., T.S., J.C., L.D., S.C., B.T., J.T,J.H., N.L. and Y.Y.)
496 Drafting the article (V.D., J.H. N.L and Y.Y.)
497 Final approval of the article (V.D., H.H., C.Ba., S.K., L.D., J.S., K.C., C.Bo, M.I., M.P.,
498 T.S., J.C., L.D., S.C., B.T., J.T., J.H., N.L. and Y.Y.)
500 Financial support:
502 Financial support for this study and its publication was provided by Samumed, LLC.
504 Conflict of Interest:
506 Samumed LLC salary and equity- V.D., C.Ba., S.K., L.D., J.S., K.C., C.Bo., M.I., M.P.,
507 T.S., J.C., L.D., S.C., B.T., J.T., and Y.Y.
508 Samumed LLC equity- H.H. and J.H.
509 Samumed consultant- N.L.
511 Acknowledgements:
513 We thank Sarah Kennedy, PhD, David Herman, PhD, Timothy Phalen, PhD, Benoit
514 Melchior, PhD and Hutch Humphreys for critical revision of the manuscript and
515 Christopher Swearingen, PhD for assistance with statistical analysis.
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1 Figure 1
2 SM04690 was a potent and specific inhibitor of Wnt signaling
3 SW480 cells and hMSCs treated with SM04690 or DMSO for 48hrs (a) Dose response
4 of TCF/LEF promoter driven- or SV40 promoter driven- luciferase reporters in SW480
5 cells (n=4, Mean ± 95% CI). (b) Expression of genes in the Wnt signaling pathway in
6 SW480 cells as measured by qRT-PCR. Fold change relative to DMSO (n=3, Mean ±
7 95% CI, Ascll ***p<0.0001, LEF1 ***p=0.0001, TCF7L2 **p=0.0038, TCF7 ***p=0.0005,
8 c-myc ***p=0.0003, Axin2 *p=0.014, t-test). (c) Expression of proteins in the Wnt
9 signaling pathway in SW480 cells as measured by Western blot. (d) Expression of
10 genes in the Wnt signaling pathway in hMSCs as measured by qRT-PCR. Fold change
11 relative to DMSO (n=3, Mean ± 95% CI, Ascl1 **p=0.002, LEF1 *p=0.017, TCF7L2
12 **p=0.004, TCF7 **p=0.0053, c-myc ***p<0.0001, Axin2 ***p=0.0008, t-test). (e)
13 Expression of proteins in the Wnt signaling pathway in hMSCs as measured by Western
14 blot.
22 Supplementary Figure 1
23 SM04690 was a potent and specific inhibitor of Wnt signaling
24 (a, b) SW480 cells and hMSCs treated with SM04690 or various Wnt pathway inhibitors
25 or DMSO for 48hrs. (a) Dose response of TCF/LEF promoter driven- luciferase reporter
26 in SW480 cells (n=4, Mean ± 95% CI). (b) EC50 values for Wnt inhibitors in (a). (c, d)
27 Gene expression of Wnt pathway genes in (c) hMSCs and (d) IEC6 cells, stimulated
28 either with Wnt3a (200 ng/ml) or CHIR99021 (4 pM) and treated with SM04690 (30nM)
29 or CX-4945 (3pM) or KY02111 (10pM) for 16hrs. Fold change relative to unstimulated
30 control (n=4, Mean ± 95% CI, *p<0.05, ***p<0.01, ***p<0.001, one-way ANOVA).
42 SM04690 was a potent and specific inhibitor of Wnt signaling
43 (a, b) Expression of genes in the non-canonical Wnt pathway in hMSCs following
44 treatment with DMSO or SM04690 (30nM) or CX-4945 (10 pM) or KY02111 (10pM) for
45 48hrs in the (a) absence or (b) presence of Wnt5a (100ng/ml), measured by qRT-PCR.
46 Expression represented as ddCt relative to DMSO (n=4, Mean ± 95% CI, *p<0.05,
47 **p<0.01, one-way ANOVA).
60 SM04690 was a potent and specific inhibitor of Wnt signaling
61 Expression of genes in the BMP pathway in hMSCs following treatment with DMSO or
62 SM04690 (30nM, 100nM) for 6hrs, 40hrs or 72hrs measured by qRT-PCR. Expression
63 represented as ddCt relative to DMSO (n=4, Mean ± 95% CI, *p<0.05, **p<0.01,
64 ***p<0.001, t-test vs DMSO at each timepoint).
77 Figure 2
78 SM04690 induced chondrocyte differentiation from hMSCs
79 (a) hMSCs treated with SM04690 (30nM) or DMSO or TGFp3 (20ng/ml) for 5 days and
80 stained with Rhodamine B (scale bars, 200 pm). (b) Quantification of the number of
81 Rhodamine B stained colonies in (a) (n=6, Mean ± 95% CI, ***p<0.0001, t-test). (c)
82 hMSCs treated with SM04690 (30nM) or DMSO or TGFp3 (20ng/ml) for 3 days and
83 stained for Sox9 (scale bars, 200 pm). (d) Quantification of the number of Sox9 positive
84 cells in (c) (n=36 images, Mean ± 95% CI, ***p<0.0001, t-test). (e) hMSCs treated with
85 SM04690 (30nM) or DMSO in 3-D sphere culture for 21 days and stained with Safranin
86 O for mature chondrocytes (scale bars, 200 pm). (f) hMSCs treated with SM04690
87 (30nM) or DMSO for 21 days and stained for various markers for mature chondrocytes
88 (scale bars, 200 pm). (g) Total sulphated GAG in hMSCs treated with SM04690 (30nM)
89 or DMSO for 21 days measured by DMMB assay. Total GAG levels relative to the
90 weight of the cell aggregates shown (n=3, Mean ± 95% CI, *p=0.041, t-test).
99 SM04690 induced chondrocyte differentiation from hMSCs
100 (a) Dose response quantification of the number of Rhodamine B stained chondrogenic
101 colonies from hMSCs treated with SM04690 or DMSO or TGFp3 for 5 days (n=4, Mean
102 ± 95% CI, *p=0.013, ***p<0.0001, one-way ANOVA). (b) Expression of Sox9 in hMSCs
103 following treatment with various doses of SM04690 or DMSO for 3 days as measured
104 by Western blot. Tubulin binding protein (TBP) serves as a loading control.
110 111 112
116 Figure 3
117 SM04690 induced chondrocyte differentiation from hMSCs
118 hMSCs, ATDC5 cells and primary mouse calvarial cells treated with DMSO or SM04690
119 (30nM) or TGFp3 for 21 days (a) Gene expression of mature chondrocyte markers in
120 treated hMSCs measured by qRT-PCR. Fold change relative to DMSO (n=3, Mean ±
121 95% CI, SOX9 **p=0.002, COMP ***p<0.0001, ACAN **p=0.009, COL2A1 ***p=0.0001,
122 TGFp1 ***p=0.0009, TIMP1 *p=0.046, CD44 **p= 0.0031, COL10A1 ***p<0.0001, t-
123 test). (b) Expression of osteocyte and tendon/ligament markers in treated hMSCs
124 measured by qRT-PCR. Fold change relative to DMSO (n=3, Mean ± 95% CI, COL1A1,
125 BGLAP, ALPL, BMP4: ***p<0.0001, RUNX2 **p=0.0009, t-test). (c) Safranin O and
126 Alcian Blue staining of treated ATDC5 cells (scale bars, 200 pm). (d) Alcian Blue
127 staining of treated primary mouse calvaria cells (scale bar, 50 pm). (e) Quantification of
128 chondrocytes in (d) (n=3, Mean ± 95% CI, *p=0.044, t-test).
137 SM04690 induced chondrocyte differentiation from hMSCs
138 hMSCs treated with SM04690 (30nM or 10nM) or CX-4945 (10 pM) or KY02111 (10
139 pM) or DMSO for 21 days and gene expression measured by qRT-PCR (a) Gene
140 expression of mature chondrocyte markers in treated hMSCs. Fold change relative to
141 DMSO (n=3, Mean ± 95% CI, *p<0.05, **p<0.01, ***p<0.0001, one-way ANOVA). (b)
142 Expression of osteocyte and tendon/ligament markers in treated hMSCs. Fold change
143 relative to DMSO (n=3, Mean ± 95% CI, *p<0.05, p<0.01, ***p<0.001, one-way
144 ANOVA).
156 Supplementary Figure 6
157 SM04690 induced chondrocyte differentiation from hMSCs
158 (a) Dose response quantification of the number of Rhodamine B stained chondrogenic
159 colonies from hMSCs treated with Wnt pathway inhibitors (n=3, Mean ± 95% CI). (b)
160 EC5o values for compounds in (a). (c) Dose response quantification of Alcian blue
161 stained chondrocytes from hMSCs treated with DMSO or SM04690 or TGFp3 or Wnt
162 pathway inhibitors for 21 days (n=6, Mean ± 95% CI). (d) EC50 values for compounds in
163 (c). (e) Representative images of chondrocytes from (c). (f) Dose response
164 quantification of Safranin O stained chondrocytes from hMSCs treated with DMSO or
165 SM04690 or TGFp3 or Wnt pathway inhibitors for 21 days (n=6, Mean ± 95% CI). (g)
166 EC50 values for compounds in (f). (h) Representative images of chondrocytes from (f).
175 Figure 4
176 SM04690 protected chondrocytes from catabolic breakdown in vitro
177 Chondrocytes treated with cytokines- TNFa (20ng/ml) + Oncostatin M (10ng/ml) or IL1p
178 (10ng/ml) and SM04690 (30nM) or DMSO control for 72hrs (a, b) Gene expression of
179 proteases (MMP1, MMP3, MMP13, IHH) in chondrocytes measured by qRT-PCR. Fold
180 change relative to unstimulated control (n=3, Mean ± 95% CI, TNFa + Oncostatin M:
181 MMP1 *p=0.04, MMP3 *p=0.014, MMP13 **p=0.0046, IHH *p=0.022, IL1p: MMP1
182 *p=0.019, MMP3 *p=0.017, MMP13 *p=0.015, IHH *p=0.026, t-test). (c) Levels of
183 secreted GAG expressed as a ratio of intracellular GAG, measured by the DMMB
184 assay. (n=6, Mean ± 95% CI, TNFa+OM ***p<0.0001, IL1p *p=0.032, t-test). (d) Levels
185 of secreted Nitric Oxide (NO) measured using the Griess reagent assay. (n=6, Mean ±
186 95% CI, TNFa+OM ***p<0.0001, IL1p **p=0.005, t-test).
195 Supplementary Figure 7
196 SM04690 protected hMSCs and chondrocytes from catabolic breakdown in vitro
197 (a, b) Gene expression of proteases (MMP1, MMP3, MMP13, IHH) in hMSCs treated
198 with either (a) TNFa (20ng/ml) + Oncostatin M (10ng/ml) or (b) IL1p (10ng/ml) and
199 SM04690 (30nM) or DMSO for 72hrs measured by qRT-PCR. Fold change relative to
200 unstimulated control (n=3, Mean ± 95% CI, TNFa + Oncostatin M: MMP1 ***p=0.0004,
201 MMP3 p=0.148, MMP13 **p=0.01, IHH **p=0.0075, IL1p: MMP1 p=0.077, MMP3
202 p=0.141, MMP13 p=0.158, IHH *p=0.014, t-test). (c, d) Gene expression of proteases
203 (MMP1, MMP3, MMP13) in human chondrocytes treated with either (c) TNFa (20ng/ml)
204 + Oncostatin M (10ng/ml) or (d) IL1p (10ng/ml) and SM04690 (30nM) or CX-4945 (10
205 pM) or KY02111 (10 pM) or DMSO for 72hrs as measured by qRT-PCR. Fold change
206 relative to unstimulated control (n=3, Mean ± 95% CI, *p<0.05, **p<0.01, ***p<0.001,
207 one-way ANOVA).
210 211 212
215 Figure 5
216 SM04690 promoted cartilage repair and protection in the rat ACLT+pMMx OA model.
217 ACLT+pMMx rats treated with either vehicle or SM04690 (0.3|jg) (a) Representative
218 images of medial tibial plateau of the knee joint stained with Safranin O-Fast Green
219 from naive or vehicle-treated or SM04690 (0.3jg) treated rats 12 weeks from treatment.
220 (b) The medial joint score based on the OARSI scoring system (n= 12 rats, Mean ± 95%
221 CI, **p=0.008, Mann-Whitney U test). (c) Circulating PIIANP levels following treatment
222 (n= 12 rats, Mean ± 95% CI, week 3 *p=0.018, week 5 p=0.13, t-test). (d) Gene
223 expression of chondrocyte markers in the cartilage of rats 4 weeks from treatment
224 measured by qRT-PCR (n= 7 for vehicle, n= 8 for treatment, Mean ± 95% CI, Col2a1
225 *p=0.024, COMP ***p=0.0007, aggrecan ***p=0.0014, t-test). (e) Representative images
226 of the superficial zone of articular cartilage from the ACLT+pMMx model stained for
227 Doublecortin (Dcx). (f) Quantification of Dcx expressing chondrocytes in (e) (n= 9
228 rats/group for vehicle, n=12 rats/group for treatment, Mean ± 95% CI, **p=0.001, t-test).
229 (g) Gene expression of proteases in the cartilage of rats 4 weeks from treatment
230 measured by qRT-PCR (n= 7 for vehicle, n= 8 for treatment, Mean ± 95% CI, MMP1
231 ***p=0.0006, MMP3 ***p=0.0006, MMP13 **p=0.0046, ADAMTS5 *p=0.016, t-test). (h)
232 Circulating COMP levels following treatment (n= 12 rats, Mean ± 95% CI, week 2
233 p=0.19, week 5 **p=0.0064, t-test).
238 Supplementary Figure 8
239 SM04690 promoted cartilage repair and protection in the ACLT OA model in rats.
240 (a) Pharmacokinetics of SM04690 in rat cartilage, bone and plasma following a single IA
241 injection of SM04690 (0.3|jg). (b) The medial joint score in the ACLT + pMMx model,
242 based on the OARSI scoring system (n= 12 rats, Mean ± 95% CI, ***p=0.0005, Mann-
243 Whitney U test) (c) Cartilage thickness measurement and (d) Safranin O staining
244 intensity of knee sections from the ACLT+pMMx model (n=12 rats, Mean ± 95% CI,
245 **p=0.0042, ***p<0.0001 respectively, t-test).
257 Supplementary Figure 9
258 SM04690 inhibited Wnt signaling in the ACLT+pMMx OA model in rats.
259 ACLT+pMMx rats treated with either vehicle or single IA injection of SM04690 (0.3jg)
260 and cartilage analyzed 4 weeks from treatment for gene expression by qRT-PCR. (a)
261 Col10a1 expression. (b) Expression of Wnt pathway genes, represented on a color
262 scale as shown. Expression of Wnt pathway genes (c) Axin2 and (d) p-catenin (n= 7 for
263 vehicle, n= 8 for treatment, Mean ± 95% CI, Col10a p=0.48, Axin 2 *p=0.017, p-catenin
264 *p=0.04, t-test).
276 Supplementary Figure 10
277 SM04690 inhibited Wnt signaling in the ACLT+pMMx OA model in rats.
278 ACLT+pMMx rats treated with either vehicle or SM04690 (0.3pg) and analyzed by IHC
279 12 weeks from treatment. (a) Representative images of the superficial zone of articular
280 cartilage stained for p-catenin. (b, c) Quantification of staining intensity of p-catenin in
281 (b) the total cell and in (c) the nucleus of the cells, from images in (a) (n= 6 rats/group,
282 Mean ± 95% CI, **p=0.0091 and *p=0.044 respectively, t-test).
Relative Expression CT
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