Adavivint (SM04690) has no effect on the SV40 luciferase reporter and is a strong carboxyl Wnt signaling activator with an EC50 of 19.5 nM as determined by high-field TCF/LEF reporter on SW480 porous media. Human mesenchymal stem cells (hMSC) aggregated at an EC50 of 10 nM. Adavivint (30 nM) shields chondrocytes from further damage [1].

Adavivint (SM04690) has no effect on the SV40 luciferase reporter and is a strong carboxyl Wnt signaling activator with an EC50 of 19.5 nM as determined by high-field TCF/LEF reporter on SW480 porous media. Human mesenchymal stem cells (hMSC) aggregated at an EC50 of 10 nM. Adavivint (30 nM) shields chondrocytes from further damage [1].

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SM04690 potently inhibited canonical Wnt signaling in SW480 colon cancer cells (which have constitutively active Wnt signaling) and in human mesenchymal stem cells (hMSCs), as evidenced by dose-dependent decreases in the expression of Wnt target genes (ASCL1, LEF1, TCF7L2, TCF7, C-MYC, AXIN2) at both mRNA and protein levels. It was approximately 150-500 times more potent than several other known Wnt pathway inhibitors (FH535, IWR-1, ICG001, iCRT14, KY02111, CX-4945) in a TCF/LEF reporter assay. [1]
Treatment of hMSCs with SM04690 (EC50 = 10 nM) for 5 days induced early chondrogenic condensation, as shown by a >40-fold increase in Rhodamine B-stained colonies compared to DMSO control. [1]
Treatment of hMSCs with SM04690 (30 nM) for 21 days under monolayer or 3D pellet culture conditions induced differentiation into mature chondrocytes. This was evidenced by increased staining for markers like Alcian Blue, Safranin O, Type II collagen, aggrecan, TIMP1, and CD44, as well as a significant increase in sulfated glycosaminoglycan (GAG) content. Gene expression analysis confirmed upregulation of chondrocyte-associated genes (SOX9, COMP, aggrecan, COL2A1, TGFβ1, TIMP1, CD44, COL10A1) and downregulation of osteoblast/tendon lineage markers (COL1A1, BGLAP, ALPL, BMP4, RUNX2). [1]
SM04690 (30 nM) protected cytokine-stimulated (TNFα/Oncostatin M or IL-1β) chondrocytes and hMSCs from catabolic breakdown in vitro. It significantly inhibited the cytokine-induced upregulation of matrix-degrading enzymes (MMP-1, MMP-3, MMP-13, IHH), reduced the secretion of GAGs, and decreased nitric oxide (NO) production. [1]

In effective cruciate ligament tear and partial medial meniscectomy osteoarthritis (ACLT + pMMx OA) mice, Adavivint (0.3 μg) improves cartilage healing and protection [1].

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In a rat model of osteoarthritis (OA) induced by anterior cruciate ligament transection and partial medial meniscectomy (ACLT+pMMx), a single intra-articular (IA) injection of SM04690 (0.3 µg) administered 1-week post-surgery promoted cartilage repair and protection when assessed 12-13 weeks later. Treated animals showed significantly increased articular cartilage thickness, smoother cartilage surface, and more intense Safranin O staining (indicative of proteoglycan content) compared to vehicle controls. [1]
Blinded histological evaluation using the Osteoarthritis Research Society International (OARSI) scoring system revealed a significant decrease (improvement) in OARSI scores in the SM04690-treated group compared to the vehicle group at 12 weeks post-treatment. [1]
Serum levels of the cartilage synthesis biomarker PIIANP were significantly increased at 3 weeks post-SM04690 treatment. Circulating levels of the cartilage degradation biomarker COMP were significantly reduced at 5 weeks post-treatment. [1]
Gene expression analysis of cartilage from treated rats (4 weeks post-injection) showed increased expression of chondrogenic markers (Col2a1, aggrecan, COMP) and significantly decreased expression of catabolic proteases (MMP1, MMP3, MMP13, ADAMTS5) compared to vehicle. No significant change was observed in the hypertrophy marker Col10a1. [1]
Immunohistochemistry showed a significant increase in the number of Doublecortin (Dcx)-positive articular chondrocytes in the superficial zone of cartilage from SM04690-treated rats at 12 weeks post-injection. [1]
Gene expression analysis in cartilage from the OA model confirmed in vivo Wnt pathway modulation by SM04690, showing decreased expression of Wnt pathway genes (e.g., GSK3β, Dvl1, Wnt3a, TCF7, Axin2, β-catenin) and increased expression of Wnt inhibitory genes (e.g., DKK1, WIF1). Decreased nuclear localization of β-catenin was also observed in articular chondrocytes. [1]

TCF/LEF Reporter Assay: A high-throughput screen was performed using SW480 colon cancer cells stably transduced with a TCF/LEF-luciferase lentiviral reporter. Cells were plated in 384-well plates (3000 cells/well) in medium with 1% fetal bovine serum. Compounds from a chemical library were transferred using an acoustic dispenser. After 48 hours of incubation, luminescence was measured using a luciferase substrate and a plate reader. Hits were counter-screened against SW480 cells expressing an SV40-driven luciferase reporter to eliminate non-specific compounds. SM04690 was identified and its EC50 was determined using sigmoidal dose-response curve fitting. [1]
Chondrogenic Differentiation Assay (Early Condensation): Human mesenchymal stem cells (hMSCs) were plated at 20,000 cells per well in 96-well plates in incomplete chondrocyte differentiation medium. Cells were treated with SM04690 or control compounds (TGFβ3, other Wnt inhibitors). After 5 days, cells were fixed, stained with 1 µg/mL Rhodamine B, and imaged. The number of Rhodamine B-stained colonies (chondrogenic nodules) was quantified using an automated imaging system. [1]
Chondrogenic Differentiation Assay (Maturation - Monolayer/3D): For monolayer differentiation, hMSCs were plated in 48-well plates (40,000 cells/well) in incomplete chondrocyte differentiation medium and treated with SM04690 or DMSO. Medium was changed every 5 days for 21 days. Cells were then fixed and immunostained for specific proteins (e.g., Type II collagen, aggrecan) or stained with Alcian Blue, Safranin O, or Toluidine Blue. For 3D pellet culture, 150,000 hMSCs were dispensed into conical tubes, centrifuged to form pellets, and treated in suspension with SM04690 for 21 days with medium changes. Pellets were then processed for histology (Safranin O staining) or digested for sulfated GAG quantification using a dimethylmethylene blue (DMMB) assay. [1]
Cartilage Catabolism Assay: hMSCs were first differentiated into chondrocytes using TGFβ3 for 21 days. The resulting chondrocytes were then treated with catabolic cytokines (TNFα [20 ng/mL] + Oncostatin M [10 ng/mL] or IL-1β [10 ng/mL]) in the presence or absence of SM04690 for 72 hours. Conditioned media and cell layers were collected. Secreted glycosaminoglycans (GAGs) in the media were measured using the DMMB assay and normalized to intracellular GAG content. Nitric oxide (NO) in the media was measured using a Griess reagent assay. Gene expression of catabolic enzymes (MMP1, MMP3, MMP13, IHH) was analyzed by quantitative real-time PCR (qRT-PCR). [1]

Osteoarthritis Model Induction and Drug Efficacy Study: Male Sprague-Dawley rats (10 weeks old) underwent surgical induction of osteoarthritis via severing of the anterior cruciate, medial collateral, and medial meniscotibial ligaments (ACLT+pMMx). One week after surgery, rats were randomized and received a single intra-articular (IA) injection into the knee joint of either SM04690 (0.3 µg in 50 µL volume) or vehicle control (n=12 per group). Thirteen weeks post-surgery (12 weeks post-treatment), knee joints were harvested, fixed in formalin, decalcified, embedded in paraffin, and sectioned. Frontal sections (5 µm thick) were stained with Safranin O/Fast Green for histological evaluation (OARSI scoring, cartilage thickness, staining intensity). Serum was collected at weeks 3, 4, and 6 post-treatment for biomarker analysis (PIIANP, COMP). [1]
Gene Expression Analysis in OA Model: In a separate study with a similar design, rats underwent ACLT+pMMx surgery and received a single IA injection of SM04690 (0.3 µg) or vehicle one week later (n=12 per group). At 5 weeks post-surgery (4 weeks post-treatment), articular cartilage was isolated from the knee joints for RNA extraction and subsequent qRT-PCR analysis of chondrocyte markers and protease genes. [1]

Following a single intra-articular injection of SM04690 (0.3 µg) into the knee joint of Sprague-Dawley rats, the drug exhibited a prolonged local retention time in knee joint tissues (cartilage and bone) and remained detectable for more than 180 days. Systemic exposure was extremely low, with plasma drug concentrations below the limit of quantitation (10 nM) at all detection time points. [1]

In rat studies, no significant side effects (e.g., weight loss, significant joint swelling, or pain or discomfort) were observed after a single intra-articular injection of SM04690 (0.3 µg). [1]

[1]. A small-molecule inhibitor of the Wnt pathway (SM04690) as a potential disease modifying agent for the treatment of osteoarthritis of the knee. Osteoarthritis Cartilage. 2018 Jan;26(1):18-27.

Lorecivivint is being investigated in the clinical trial NCT03246399 (a study of the safety, tolerability, and pharmacokinetics of a single intradiscal injection of SM04690 injection suspension in patients with degenerative disc disease). SM04690 (Adavivint) is being developed as a potential disease-modifying osteoarthritis drug (DMOAD). Its proposed dual mechanism of action involves inhibiting the Wnt signaling pathway to: 1) induce chondrogenesis (differentiation of mesenchymal stem cells into functional chondrocytes) to regenerate cartilage; and 2) protect existing cartilage by inhibiting the production of catabolic proteases (MMPs, ADAMTS) and reducing matrix degradation. [1] This study suggests that SM04690 represents a novel approach that targets intrinsic stem cells within the joint to promote repair, in contrast to strategies that only inhibit inflammatory cytokines or matrix-degrading enzymes. [1]

C29H24FN7O

505.56

505.202

C, 68.90; H, 4.79; F, 3.76; N, 19.39; O, 3.16

1467093-03-3

1467093-03-3;Adavivint HCl;

135565709

Light yellow to gray solid powder

1.4±0.1 g/cm3

1.713

5.39

3

6

6

38

816

0

FC1=CC=CC(=C1)C1=CN=CC2=C1N=C(C1C3C=C(C=CC=3NN=1)C1C=NC=C(C=1)NC(CC(C)C)=O)N2

AQDWDWAYVBQMAM-UHFFFAOYSA-N

InChI=1S/C29H24FN7O/c1-16(2)8-26(38)33-21-10-19(12-31-13-21)17-6-7-24-22(11-17)28(37-36-24)29-34-25-15-32-14-23(27(25)35-29)18-4-3-5-20(30)9-18/h3-7,9-16H,8H2,1-2H3,(H,33,38)(H,34,35)(H,36,37)

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.95 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (4.95 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (4.95 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)