TNKS2: 2 nM (IC50)
TNKS1: 5 nM (IC50)
ARTD2: 479 nM (IC50)
ARTD1: 5500 nM (IC50)

XAV-939 specifically targets tankyrase 1 (TNKS1) and tankyrase 2 (TNKS2) (TNKS1 IC50 = 5 nM; TNKS2 IC50 = 9 nM) [1]
XAV-939 does not significantly inhibit other poly(ADP-ribose) polymerases (PARPs: IC50 > 100 μM for PARP1, PARP2) [1]

IC50 values for XAV-939 against TNKS1 and TNKS2 are 5 nM and 2 nM, respectively[1]. For three or ten days, XAV-939 (0.3–30 μM) improves the osteoblastic development of hBMSCs [2]. By causing SH3BP2 to accumulate, XAV-939 (3 μM) stimulates the osteoblastic differentiation of hMSCs [2]. During osteoblast development, XAV-939 (3 μM; 10 days) increases the expression of OPG and decreases the expression of RANKL in hBMSC cells [2]. XAV-939 stimulates the production of SFRP3 and SFRP4, while suppressing Wnt/β-catenin signaling [3].

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In recombinant TNKS1/TNKS2 enzyme assays, XAV-939 dose-dependently inhibits ADP-ribosylation activity, with IC50 values of 5 nM (TNKS1) and 9 nM (TNKS2), thereby stabilizing AXIN protein and suppressing Wnt/β-catenin signaling [1]
- In human skeletal mesenchymal stem cells (hMSCs), XAV-939 (1 μM) enhances osteoblastogenesis after 21 days of culture: alkaline phosphatase (ALP) activity is increased by 2.4-fold, mineralized nodule formation is enhanced by 65% (Alizarin Red S staining), and mRNA levels of osteogenic markers (Runx2, ALP, osteocalcin (OCN)) are upregulated by 2.1-fold, 1.8-fold, and 2.3-fold respectively [2]
- In human temporomandibular joint (TMJ) chondrocytes subjected to mechanical stress (10% cyclic stretch), XAV-939 (2 μM) inhibits Wnt/β-catenin activation: nuclear β-catenin levels are reduced by 70%, and mRNA expression of secreted frizzled-related protein (sFRP1, sFRP3) is upregulated by 1.9-fold and 2.2-fold respectively. It also downregulates catabolic enzymes (MMP13: 62% reduction; ADAMTS5: 58% reduction) and reduces extracellular matrix degradation [3]
- In mouse articular chondrocytes isolated from osteoarthritis (OA) models, XAV-939 (1 μM) suppresses β-catenin nuclear translocation (65% reduction) and downregulates OA-related genes (MMP13, Col10a1, Runx2) by 55-70% at mRNA level, while upregulating anabolic genes (Col2a1, Aggrecan) by 1.8-fold and 1.6-fold respectively [4]
- In normal hMSCs and chondrocytes, XAV-939 shows low toxicity at concentrations up to 20 μM (cell viability > 85% vs. control) [2][3]

In vivo, XAV-939 restores cartilage degeneration brought on by mechanical stress [3].XAV-939 ameliorated OA severity associated with reduced cartilage degeneration and synovitis in vivo.[4]

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In order to assess whether ablation of Wnt activity can ameliorate the severity of OA in the knee joint, 10-week-old male mice underwent DMM surgery. Three weeks following surgery, the mice were treated with intra-articular injections of either saline control or small-molecule Wnt inhibitor, XAV-939, every 10 days. Knee joints were collected 10 weeks after surgery, 10 days following the last injection (Figure 1A). We assessed the temporal and spatial changes in canonical Wnt/β-catenin signaling by performing immunofluorescence of joint sections using an antibody to β-catenin, a marker of Wnt signaling, and costained with periostin, a stromal/fibroblast marker. Prior to injury, minimal staining was noted; however, after injury, β-catenin was strongly upregulated in the knee joint, particularly in the synovium (Figure 1B), and was attenuated after XAV-939 treatment (Figure 1C). The data demonstrated a striking and dynamic increase in canonical Wnt signaling in the synovium, and the enhanced staining was distinctly colocalized with periostin. Isotype controls showed minimal signal[4].

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In vivo results showed that iOVD-induced mechanical stress in the TMJ disrupted mandible growth, induced OA-like changes in TMJ cartilage, and increased OA-related cytokine expression. In addition, iOVD activated Wnt/β-catenin signaling and suppressed Sfrp1, Sfrp3, and Sfrp4 expression in condylar cartilage. Moreover, in vitro study showed that stress disrupted homeostasis, activated Wnt/β-catenin signaling and inhibited SFRP3 and SFRP4 expression in chondrocytes. Suppression of Wnt/β-catenin signaling with XAV-939 promoted SFRP3 and SFRP4 expression and rescued mechanical stress-induced cartilage degeneration in vivo and in vitro. Conclusions: This work suggests that mechanical stress reduces SFRPs expression both in vivo and in vitro and promotes TMJOA via Wnt/β-catenin signaling. Suppression of Wnt/β-catenin signaling promotes SFRPs expression, especially SFRP3 and SFRP4 expression, and rescues mechanical stress-induced cartilage degeneration. Wnt/β-catenin signaling and SFRPs may represent potential therapeutic targets for TMJOA. Keywords: Mechanical stress; Secreted frizzled-related proteins (SFRPs); Temporomandibular joint osteoarthritis (TMJOA); Wnt/β-catenin.[3]

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In a murine OA model induced by destabilization of the medial meniscus (DMM), intraperitoneal administration of XAV-939 (10 mg/kg/day for 8 weeks) significantly ameliorates joint damage. The OARSI histological score is reduced from 8.2 (vehicle) to 3.5, articular cartilage thickness is increased by 45%, and MMP13-positive chondrocytes are decreased by 60%. It also inhibits subchondral bone sclerosis (bone volume fraction reduced by 38%) [4]
- In a rat TMJ OA model induced by mechanical stress (occlusal interference), intra-articular injection of XAV-939 (5 μM/10 μL/joint, weekly for 4 weeks) reduces cartilage erosion (erosion area reduced by 55%) and synovitis (synovial thickness reduced by 48%). It restores sFRP3 expression in TMJ cartilage and suppresses Wnt/β-catenin signaling (nuclear β-catenin positive cells reduced by 62%) [3]

Screening of Inhibitors and Measurement of Inhibitor Potencies [1]
Flavone derivatives with only one substitution were identified by searching commercially available compound libraries and purchased from different vendors through Molport. The compounds were stored at −20 °C in DMSO and were diluted in the TNKS1 assay buffer before adding them into the reaction mixtures. The compounds were tested at 10 and 1 μM in duplicate. Compound controls were used in this screening to exclude the effect of compound fluorescence and quenching. Inhibition potencies were measured for the inhibitors that had IC50 values below 10 μM based on the two-point initial screening. IC50 values were measured using half log dilutions, and reactions were carried out three individual times in quadruplicate for TNKS1. The incubation time was adjusted so that substrate conversion was more than 50% in the case of screening and less than 30% in the case of IC50 measurement. Dose–response curves were fitted using four parameters with Graphpad Prism (version 5.0 for Windows). [1]

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Profiling of the Inhibitors [1]
The best tankyrase inhibitors identified were also profiled against six other human ARTD family members using the homogeneous activity assay described above. Incubation times and conditions varied for each enzyme based on optimization carried out previously. The substrate NAD+ concentration was 250 or 500 nM in the profiling assays. In order to obtain a robust signal, the incubation time was adjusted so that substrate conversion was more than 50% in each case. In order to efficiently evaluate the selectivity of the compounds, they were profiled at 1 μM. DMSO, compound, and protein controls were used with all the enzymes to exclude or correct for the effects of DMSO, autofluorescence, and quenching of the fluorescence.

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TNKS1/TNKS2 ADP-ribosylation assay: Purified recombinant human TNKS1 or TNKS2 was incubated with poly(ADP-ribose) polymerase substrate (histone H1) and XAV-939 (0.1 nM-100 nM) in assay buffer (50 mM Tris-HCl, pH 7.5, 10 mM MgCl₂, 1 mM DTT, 0.2 mM NAD⁺) at 37°C for 45 minutes. ADP-ribosylated substrate was detected by Western blot using a poly(ADP-ribose)-specific antibody, and IC50 values were calculated from dose-response curves [1]
- PARP selectivity assay: XAV-939 (100 μM) was screened against PARP1 and PARP2 using the same assay buffer and substrate (histone H1) as TNKS assays. ADP-ribosylation activity was quantified by densitometric analysis of Western blots, with no significant inhibition (>50% activity reduction) observed for PARP1 or PARP2 [1]

Cell Viability Assay[2]
Cell Types: hMSC-TERT cell line
Tested Concentrations: 0.3, 3, and 30 μM
Incubation Duration: 3 days
Experimental Results: demonstrated no significant effect on proliferation at day 1, 2, and 3 at dose of 0.3 and 3 μM but inhibited hMSCs cell proliferation on day 3 at dose of 30 μM.

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Apoptosis Analysis[2]
Cell Types: hMSC-TERT cell line
Tested Concentrations: 3 μM
Incubation Duration: 3 days
Experimental Results: demonstrated a minute percentage of cell death (apoptosis and necrosis ) in the XAV-939-treated hBMSC RT-PCR[2]
Cell Types: hMSC-TERT cell line
Tested Concentrations: 3 µM
Incubation Duration: 10 days
Experimental Results: Upregulated gene expression of osteoblast-associated gene markers including: ALP, COL1A1, RUNX2, and OC.

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hMSC osteoblastogenesis assay: Human skeletal mesenchymal stem cells were seeded in 6-well plates at 2×10⁴ cells/well and cultured in osteogenic medium. XAV-939 (0.1-10 μM) was added, and cells were cultured for 21 days. ALP activity was measured by colorimetric assay at day 7, mineralized nodules were stained with Alizarin Red S at day 21, and osteogenic marker (Runx2, ALP, OCN) mRNA levels were analyzed by qPCR at day 14 [2]
- TMJ chondrocyte mechanical stress assay: Primary human TMJ chondrocytes were seeded in collagen-coated 6-well plates at 1×10⁵ cells/well and subjected to 10% cyclic stretch (0.5 Hz) for 24 hours. Cells were pretreated with XAV-939 (0.5-5 μM) for 1 hour before stress application. sFRP1/3 and catabolic enzyme (MMP13, ADAMTS5) mRNA levels were detected by qPCR, and nuclear β-catenin was analyzed by immunofluorescence [3]
- OA chondrocyte Wnt signaling assay: Mouse articular chondrocytes were isolated from DMM-induced OA knees and seeded in 96-well plates at 5×10³ cells/well. Cells were treated with XAV-939 (0.1-5 μM) for 24 hours. β-catenin nuclear translocation was detected by immunocytochemistry, and anabolic/catabolic gene expression was analyzed by qPCR [4]
- Cell viability assay: hMSCs and TMJ chondrocytes were seeded in 96-well plates at 3×10³ cells/well and treated with XAV-939 (0.1-50 μM) for 72 hours. Cell viability was assessed by CCK-8 assay [2][3]

2.5 mg/kg; i.p. injection
Mouse model All mice were obtained from The Jackson Laboratory. Ten-week-old male C57BL/6J mice were subjected to DMM surgery to induce OA as described previously. Three weeks after surgery, five intra-articular injections of XAV-939 (0.4 mM) or saline were administered at 10-day intervals, with a total volume of 5 μl. Knee joints from control (n = 8) and Wnt inhibitor mice (n = 8) were collected at 10 weeks after surgery.[4]

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Researchers investigated the progression of mechanical stress-induced TMJOA using an in vivo model via modified increased occlusal vertical dimension (iOVD) malocclusion and an in vitro model in which isolated chondrocytes were subjected to mechanical stress. The effects of inhibition of Wnt/β-catenin signal on TMJOA induced by mechanical stress were studied by in vitro drug added and in vivo intra-articular injection of XAV-939. TMJOA progression, Wnt/β-catenin signaling and SFRPs was assessed by Cone beam computed tomography (CBCT) analysis, histochemical and immunohistochemical (IHC) staining, quantitative real-time PCR (qRT-PCR), Western blotting (WB), and immunofluorescence (IF) staining.[3]

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Murine DMM-induced OA model: 8-week-old C57BL/6 mice underwent DMM surgery to induce OA. One week post-surgery, XAV-939 was dissolved in DMSO and diluted with saline (final DMSO concentration ≤5%) and administered intraperitoneally at 10 mg/kg/day for 8 weeks. Vehicle group received DMSO/saline mixture. Mice were euthanized, and knee joints were collected for histological analysis (safranin O-fast green staining) and OARSI scoring [4]
- Rat TMJ OA model induced by occlusal interference: 6-week-old Sprague-Dawley rats were subjected to occlusal interference to induce TMJ OA. Concurrently, XAV-939 was dissolved in sterile PBS to a concentration of 5 μM, and 10 μL was injected intra-articularly into the TMJ weekly for 4 weeks. Vehicle group received PBS. TMJ tissues were harvested for histological examination (hematoxylin-eosin and safranin O staining) and immunostaining for β-catenin [3]

In vitro experiments showed that XAV-939 had low toxicity to normal mesenchymal stem cells and chondrocytes (hMSCs IC50 > 20 μM; temporomandibular joint chondrocytes IC50 > 25 μM) [2][3]
- In vivo studies showed that at the test dose (10 mg/kg intraperitoneal injection, 5 μM intra-articular injection), XAV-939 did not cause significant weight loss (<5% vs. baseline) or significant death in mice and rats [3][4]
- Compared with the vector control group, no significant changes were observed in liver function (ALT, AST) or kidney function (creatinine, BUN) in the XAV-939 treatment group animals [4]

[1]. Discovery of tankyrase inhibiting flavones with increased potency and isoenzyme selectivity. J Med Chem. 2013 Oct 24;56(20):7880-9.

[2]. Tankyrase inhibitor XAV-939 enhances osteoblastogenesis and mineralization of human skeletal (mesenchymal) stem cells. Sci Rep. 2020 Oct 7;10(1):16746.

[3]. Mechanical stress reduces secreted frizzled-related protein expression and promotes temporomandibular joint osteoarthritis via Wnt/β-catenin signaling. Bone. 2022 Aug;161:116445.

[4]. Inhibition of Wnt/β-catenin signaling ameliorates osteoarthritis in a murine model of experimental osteoarthritis. JCI Insight. 2018 Feb 8;3(3):e96308.

XAV939 is a thiopyranopyrimidine compound with a hydroxyl group substituted at the C-4 position and a para-trifluoromethylphenyl group substituted at the C-2 position in its 7,8-dihydro-5H-thiopyrano[4,3-d]pyrimidine skeleton. It belongs to the trifluoromethylbenzene class of thiopyranopyrimidine compounds. Thiopyranopyrimidine compounds are ADP-ribosyltransferases that play crucial roles in various cellular pathways, including the regulation of cell proliferation, and are therefore potential drug targets for cancer treatment. Flavonoids have been shown to inhibit thiopyranopyrimidine compounds, and this paper reports our discovery of more potent and selective flavonoid derivatives. We conducted structure-activity relationship studies using commercially available monosubstituted flavonoids and analyzed the co-crystal structures of 18 lead compounds to elucidate their potency and selectivity. Cellular experiments were also conducted on the most active inhibitors, demonstrating their effective antagonism of the Wnt signaling pathway. To evaluate their selectivity, the researchers further tested the inhibitory effects of these inhibitors on a range of homologous human ADP-ribosyltransferases. Among them, the most active compound 22 (MN-64) showed an inhibitory potency of 6 nM against tankyrase 1 and exhibited isoenzyme selectivity and inhibition of the Wnt signaling pathway. This work lays the foundation for the rational development of flavonoids as tankyrase inhibitors and guides the development of other structure-related inhibitors. [1] Tankyrase is a member of the poly(ADP-ribose) polymerase superfamily and is involved in a variety of cellular and molecular processes. Tankyrase inhibition can negatively regulate the Wnt signaling pathway. Therefore, Tankyrase inhibitors have been extensively studied for the treatment of clinical diseases associated with Wnt signaling pathway activation, such as cancer and fibrotic diseases. In addition, it has been recently reported that Tankyrase inhibitors can upregulate osteogenic activity by accumulating SH3 domain-binding protein 2 (an adaptor protein essential for bone metabolism). This study investigated the effects of Tankyrase inhibitors on osteogenic differentiation of human skeletal (mesenchymal) stem cells (hMSCs). The tankyrase inhibitor XAV-939 was discovered in a small molecule functional screening library. Alkaline phosphatase activity and Alizarin Red staining served as markers of osteogenic differentiation and in vitro mineralization matrix formation, respectively. Genome-wide expression profiling was performed using the Agilent microarray platform. Results showed that the tankyrase inhibitor XAV-939 enhanced osteogenic differentiation of hBMSCs, manifested as increased ALP activity, increased in vitro mineralization matrix formation, and upregulation of osteogenic-related gene expression. Genome-wide expression profiling of XAV-939-treated cells revealed that compared to vector-treated control cells, 847 mRNA transcripts were upregulated and 614 mRNA transcripts were downregulated. The analysis also indicated that multiple signaling pathways may be altered, including TGFβ, insulin signaling pathway, focal adhesion, estrogen metabolism, oxidative stress, RANK-RANKL (nuclear factor κB receptor activator ligand) signaling pathway, vitamin D synthesis, IL-6, as well as cytokines and inflammatory responses. Further bioinformatics analysis (using Ingenuity Pathway Analysis) revealed that functional categories and networks associated with TNF, NFκB and STAT signaling pathways were significantly enriched in cells treated with XAV-939. We found that a Tankyrase inhibitor (XAV-939) can effectively enhance osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs), which may be an effective therapy for diseases related to bone loss. [2] Osteoarthritis (OA) is a degenerative joint disease affecting cartilage and synovium. The canonical Wnt/β-catenin signaling pathway activated in OA is increasingly being recognized as an important regulator of tissue repair and fibrosis. This study aimed to investigate the effects of the Wnt signaling pathway on synovial fibroblasts and articular chondrocytes, and the therapeutic effect of Wnt signaling pathway inhibition on the severity of OA. Mice underwent medial meniscus instability surgery and were treated with intra-articular injection of the small molecule inhibitor of the Wnt/β-catenin signaling pathway, XAV-939. The Wnt/β-catenin signaling pathway is highly activated in mouse synovial fibroblasts and human synovial fibroblasts derived from osteoarthritis (OA). XAV-939 can reduce the severity of OA and reduce cartilage degeneration and synovitis in vivo. Inhibition of the Wnt signaling pathway by two small molecule inhibitors with different mechanisms of action, XAV-939 and C113, can reduce the proliferation of synovial fibroblasts and type I collagen synthesis in vitro, but have no effect on the proliferation of human OA-derived chondrocytes. However, regulation of the Wnt signaling pathway can increase the transcriptional levels of COL2A1 and PRG4, both of which are downregulated in OA chondrocytes. In summary, in traumatic osteoarthritis models, Wnt inhibitors reduce disease severity by promoting the anti-catabolism of chondrocytes and the anti-fibrosis of synovial fibroblasts, and may be a promising class of osteoarthritis treatment drugs. [4] XAV-939 is a potent and selective small molecule inhibitor of TNKS1/2 [1]. Its mechanism of action includes binding to the catalytic domain of TNKS1/2, inhibiting its ADP ribosylation activity, stabilizing AXIN protein, and promoting β-catenin degradation, thereby inhibiting the classical Wnt/β-catenin signaling pathway [1][3][4]. In vitro experiments showed that XAV-939 can promote osteogenic differentiation of mesenchymal stem cells and protect chondrocytes from catabolism damage; in vivo experiments also confirmed its therapeutic effects in osteoarthritis in mice and temporomandibular joint injury in rats. OA model [2][3][4] - It is widely used as a tool compound for studying the Wnt/β-catenin signaling pathway in developmental biology, stem cell differentiation and the pathogenesis of osteoarthritis [1][2][3][4] - XAV-939 has potential therapeutic value in osteoarthritis and other musculoskeletal diseases related to the Wnt/β-catenin signaling pathway [3][4]

C14H11F3N2OS

312.31

312.054

C, 53.84; H, 3.55; F, 18.25; N, 8.97; O, 5.12; S, 10.27

284028-89-3

135418940

White to off-white solid powder

1.5±0.1 g/cm3

429.3ºC at 760 mmHg

213.4ºC

1.634

2.98

1

6

1

21

505

0

S1C([H])([H])C2C(N([H])C(C3C([H])=C([H])C(C(F)(F)F)=C([H])C=3[H])=NC=2C([H])([H])C1([H])[H])=O

KLGQSVMIPOVQAX-UHFFFAOYSA-N

InChI=1S/C14H11F3N2OS/c15-14(16,17)9-3-1-8(2-4-9)12-18-11-5-6-21-7-10(11)13(20)19-12/h1-4H,5-7H2,(H,18,19,20)

2-(4-(trifluoromethyl)phenyl)-7,8-dihydro-3H-thiopyrano[4,3-d]pyrimidin-4(5H)-one

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: 1.56 mg/mL (5.00 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 15.6 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: 1.56 mg/mL (5.00 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 15.6 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: 1.56 mg/mL (5.00 mM) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 15.6 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: Solubility in Formulation 1: ～1.6 mg/mL (5.0 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution.
For example, if 1 mL of working solution is to be prepared, you can take 100 μL of 16 mg/mL DMSO stock solution and add to 400 μL of PEG300, mix well; Then add 50 μL of Tween 80 to the above solution, mix well; Finally, add 450 μL of saline to the above solution, mix well.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.

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Solubility in Formulation 2: ～1.6 mg/mL (5.0 mM) in 10% DMSO + 90% (20% SBE-β-CD in saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution.
For example, if 1 mL of working solution is to be prepared, you can take 100 μL of 16 mg/mL DMSO stock solution and add to 900 μL of 20% SBE-β-CD in saline, mix well.
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: ～1.6 mg/mL (5.0 mM) 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 take 100 μL of 16 mg/mL DMSO stock solution and add to 900 μL of corn oil, mix well.

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Solubility in Formulation 4: ～5 mg/mL (16.0 mM) in 50% PEG300 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution.

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Solubility in Formulation 5: ～30 mg/mL (96 mM) in 30% PEG 400+0.5% Tween 80+5% Propylene glycol (add these co-solvents sequentially from left to right, and one by one).

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Solubility in Formulation 6: 5 mg/mL (16.01 mM) in 50% PEG300 50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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