a small molecule inhibitor of the wnt pathway sm04690 as a potential disease modifying agent for the treatment of osteoarthritis of the knee

Accepted Manuscript 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: January 2017 Revised Date: 18 July 2017 Accepted Date: 30 August 2017 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 This is a PDF file of an unedited manuscript that has been accepted for publication As a service to our customers we are providing this early version of the manuscript The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain ACCEPTED MANUSCRIPT Title Page A Small Molecule Inhibitor of the Wnt Pathway (SM04690) As a Potential Disease Modifying Agent for the Treatment of Osteoarthritis of the Knee RI PT Running title: Wnt pathway inhibitor as potential DMOAD for OA SC Authors: Vishal Deshmukh1, Haide Hu1, Charlene Barroga1, Carine Bossard1, Sunil KC1, Luis Dellamary1, Joshua Stewart1, Kevin Chiu1, Maureen Ibanez1, Melinda Pedraza1, Tim Seo1, Long Do1, Shawn Cho1, Joseph Cahiwat1, Betty Tam1, Jeyanesh M AN U 10 R.S Tambiah1, John Hood1, Nancy E Lane2 , Yusuf Yazici1+ 11 + 12 14 15 Affiliations: Samumed, LLC, San Diego, CA, USA University of California, Davis, CA, USA EP 13 TE D corresponding author 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 AC C 16 ACCEPTED MANUSCRIPT (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) RI PT 23 27 28 + 29 Suite 160, San Diego, CA 92121; Tel: 858-926-2962; yusuf@samumed.com SC Main corresponding author: Yusuf Yazici, MD, Samumed LLC 9381 Judicial Drive, 30 M AN U 31 32 33 34 38 39 40 41 42 EP 37 AC C 36 TE D 35 43 44 45 ACCEPTED MANUSCRIPT 46 Abstract 47 Objectives RI PT 48 49 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 EP 59 Design AC C 58 TE D 57 M AN U SC 50 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 C.33.44.55.54.78.65.5.43.22.2.4 22.Tai lieu Luan 66.55.77.99 van Luan an.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.33.44.55.54.78.655.43.22.2.4.55.22 Do an.Tai lieu Luan van Luan an Do an.Tai lieu Luan van Luan an Do an ACCEPTED MANUSCRIPT 66 biochemical and histopathological techniques in a rodent acute cruciate ligament tear 67 and partial medial meniscectomy (ACLT+pMMx) OA model 69 RI PT 68 Results SC 70 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 77 79 Conclusions EP 78 TE D M AN U 71 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 82 83 AC C 80 Keywords: Osteoarthritis, Wnt signaling, chondrocyte, ACLT, DMOAD, small molecule 84 Stt.010.Mssv.BKD002ac.email.ninhd 77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77t@edu.gmail.com.vn.bkc19134.hmu.edu.vn.Stt.010.Mssv.BKD002ac.email.ninhddtt@edu.gmail.com.vn.bkc19134.hmu.edu.vn C.33.44.55.54.78.65.5.43.22.2.4 22.Tai lieu Luan 66.55.77.99 van Luan an.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.33.44.55.54.78.655.43.22.2.4.55.22 Do an.Tai lieu Luan van Luan an Do an.Tai lieu Luan van Luan an Do an ACCEPTED MANUSCRIPT 85 INTRODUCTION 86 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, 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 SC M AN U TE D EP 99 RI PT 87 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 AC C 100 Stt.010.Mssv.BKD002ac.email.ninhd 77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77t@edu.gmail.com.vn.bkc19134.hmu.edu.vn.Stt.010.Mssv.BKD002ac.email.ninhddtt@edu.gmail.com.vn.bkc19134.hmu.edu.vn C.33.44.55.54.78.65.5.43.22.2.4 22.Tai lieu Luan 66.55.77.99 van Luan an.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.33.44.55.54.78.655.43.22.2.4.55.22 Do an.Tai lieu Luan van Luan an Do an.Tai lieu Luan van Luan an Do an ACCEPTED MANUSCRIPT canonical Wnt/β-catenin-signaling pathway, binding of Wnt proteins to cell surface 108 receptors leads to stabilized β-catenin levels by inhibition of β-catenin phosphorylation 109 and proteasome-mediated degradation Stabilized β-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 M AN U SC RI PT 107 116 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 AC C EP TE D 117 Stt.010.Mssv.BKD002ac.email.ninhd 77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77t@edu.gmail.com.vn.bkc19134.hmu.edu.vn.Stt.010.Mssv.BKD002ac.email.ninhddtt@edu.gmail.com.vn.bkc19134.hmu.edu.vn C.33.44.55.54.78.65.5.43.22.2.4 22.Tai lieu Luan 66.55.77.99 van Luan an.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.33.44.55.54.78.655.43.22.2.4.55.22 Do an.Tai lieu Luan van Luan an Do an.Tai lieu Luan van Luan an Do an ACCEPTED MANUSCRIPT 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 RI PT 130 134 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 M AN U SC 135 139 141 METHODS TE D 140 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 and AC C EP 142 149 150 High-throughput screening Stt.010.Mssv.BKD002ac.email.ninhd 77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77t@edu.gmail.com.vn.bkc19134.hmu.edu.vn.Stt.010.Mssv.BKD002ac.email.ninhddtt@edu.gmail.com.vn.bkc19134.hmu.edu.vn C.33.44.55.54.78.65.5.43.22.2.4 22.Tai lieu Luan 66.55.77.99 van Luan an.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.33.44.55.54.78.655.43.22.2.4.55.22 Do an.Tai lieu Luan van Luan an Do an.Tai lieu Luan van Luan an Do an ACCEPTED MANUSCRIPT 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 β3 (TGFβ3; 20ng/ml, 159 Peprotech Inc., Rocky Hill, NJ) On Day 5, cells were fixed, stained with 1µg/mL 160 Rhodamine B and imaged using Cellomics CellInsight (ThermoFisher) 161 M AN U SC RI PT 151 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 TGFβ3 (20ng/ml) Cells were incubated for 21 days, with 166 media changes every 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 days AC C EP TE D 162 Stt.010.Mssv.BKD002ac.email.ninhd 77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77t@edu.gmail.com.vn.bkc19134.hmu.edu.vn.Stt.010.Mssv.BKD002ac.email.ninhddtt@edu.gmail.com.vn.bkc19134.hmu.edu.vn C.33.44.55.54.78.65.5.43.22.2.4 22.Tai lieu Luan 66.55.77.99 van Luan an.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.33.44.55.54.78.655.43.22.2.4.55.22 Do an.Tai lieu Luan van Luan an Do an.Tai lieu Luan van Luan an Do an ACCEPTED MANUSCRIPT 173 cells were treated with DMSO or SM04690 On Day 21, cells were fixed and stained 174 with Alcian Blue 175 Cartilage Matrix Degradation- Glycosaminoglycan (GAG) and Nitric Oxide (NO) 177 measurement 178 hMSCs were differentiated into chondrocytes using TGFβ3 for 21 days followed by 179 treatment with either TNFα (20ng/ml) + Oncostatin M (10ng/ml) or IL1β (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 (Biotek, Winooski, VT) 183 NO was measured using Greiss reagent kit (Promega) M AN U SC RI PT 176 184 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 EP AC C 192 TE D 185 Surgery-induced OA model Stt.010.Mssv.BKD002ac.email.ninhd 77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77t@edu.gmail.com.vn.bkc19134.hmu.edu.vn.Stt.010.Mssv.BKD002ac.email.ninhddtt@edu.gmail.com.vn.bkc19134.hmu.edu.vn C.33.44.55.54.78.65.5.43.22.2.4 22.Tai lieu Luan 66.55.77.99 van Luan an.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.33.44.55.54.78.655.43.22.2.4.55.22 Do an.Tai lieu Luan van Luan an Do an.Tai lieu Luan van Luan an Do an ACCEPTED MANUSCRIPT 97 Supplementary Figure 99 SM04690 induced chondrocyte differentiation from hMSCs RI PT 98 (a) Dose response quantification of the number of Rhodamine B stained chondrogenic 101 colonies from hMSCs treated with SM04690 or DMSO or TGFβ3 for days (n=4, Mean 102 ± 95% CI, *p=0.013, ***p