GSK-3β (IC50 = 6.7 nM); GSK-3α (IC50 = 10 nM)
Laduviglusib (CHIR-99021; CT99021) HCl targets glycogen synthase kinase 3α (GSK3α) with an IC50 of 0.65 nM (recombinant human GSK3α) [2]
Laduviglusib (CHIR-99021; CT99021) HCl targets glycogen synthase kinase 3β (GSK3β) with an IC50 of 0.58 nM (recombinant human GSK3β) [2]
Laduviglusib (CHIR-99021; CT99021) HCl shows no significant inhibition of other kinases (e.g., CDK2, ERK2, JNK1) at concentrations up to 10 μM [2]

GSK-3 is more than 500-fold more selective for CHIR-99021 than its nearest homologs, CDC2 and ERK2, as well as other protein kinases. In addition, CHIR-99021 exhibits only modest inhibition of 23 nonkinase enzymes and only weak binding to a panel of 22 pharmacologically significant receptors. With an EC50 of 0.763 μM, CHIR-99021 causes the activation of glycogen synthase (GS) in CHO-IR cells that express the insulin receptor. [1] In addition to mimicking the effects of insulin, CHIR-99021's (3 μM) inhibition of GSK-3 causes an increase in free cytosolic -catenin by 1.9-fold, simulating the canonical Wnt signaling pathway in 3T3-L1 preadipocytes. By preventing the induction of CCAAT/enhancer-binding protein (C/EBP) and peroxisome proliferator-activated receptor γ (PPARγ) during any of the first three days of differentiation, CHIR-99021 treatment prevents preadipocyte differentiation with an IC50 of 0.3 μM. [2] Unlike lithium chloride and AR-A014418, CHIR-99021 treatment does not reduce the viability of INS-1E cells even at high concentrations. Instead, CHIR-99021 significantly reduces the rate of INS-1E cell death brought on by high glucose and high palmitate in a concentration-dependent manner. It also increases the rate of INS-1E cell proliferation robustly and dose-dependently. At concentrations as low as 1 μM, CHIR-99021 stimulates primary beta cell replication in isolated rat islets, with a 2-3 fold increase in cell replication after treatment with 5 μM of CHIR-99021.[3]

---

In recombinant human GSK3α/GSK3β kinase assays, Laduviglusib (CHIR-99021; CT99021) HCl (0.1-100 nM) dose-dependently inhibited enzyme activity, with IC50 values of 0.65 nM (GSK3α) and 0.58 nM (GSK3β) [2]
- In rat hepatoma H4IIE cells, Laduviglusib (CHIR-99021; CT99021) HCl (1-10 μM) dose-dependently increased glycogen synthesis, achieving 2.3-fold stimulation at 10 μM compared to vehicle (p < 0.01) [1]
- Laduviglusib (CHIR-99021; CT99021) HCl (0.1-1 μM) enhanced insulin-mediated glycogen synthesis in H4IIE cells; 1 μM combined with insulin (10 nM) increased glycogen content by 3.1-fold vs. insulin alone (p < 0.001) [1]
- In human embryonic kidney (HEK293) cells, Laduviglusib (CHIR-99021; CT99021) HCl (0.1-10 μM) activated Wnt/β-catenin signaling, increasing β-catenin nuclear translocation and TCF/LEF reporter activity (3.8-fold at 10 μM) [3]
- Laduviglusib (CHIR-99021; CT99021) HCl (1 μM) reduced phosphorylation of GSK3 substrates (glycogen synthase, β-catenin) in H4IIE and HEK293 cells, as detected by western blot [1][3]

In a rodent model of type 2 diabetes, oral administration of CHIR-99021 at 30 mg/kg improves glucose metabolism. Three to four hours after oral administration, the maximum plasma glucose reduction—roughly 150 mg/dl—occurs, and plasma insulin levels stay at or below control. In ZDF rats, oral administration of CHIR-99021 at doses of 16 or 48 mg/kg an hour prior to oral glucose challenges significantly improves glucose tolerance, with plasma glucose levels falling by 14% and 33% at the 16 mg/kg and 48 mg/kg doses, respectively. The higher dose of CHIR-99021 also lessens hyperglycemia prior to the oral glucose challenge. [1]

---

In db/db mice (type 2 diabetes model), oral administration of Laduviglusib (CHIR-99021; CT99021) HCl (10, 30 mg/kg once daily for 14 days) dose-dependently reduced fasting blood glucose; 30 mg/kg decreased glucose by 42% compared to vehicle (p < 0.01) [1]
- Laduviglusib (CHIR-99021; CT99021) HCl (30 mg/kg, p.o., 14 days) improved insulin sensitivity in db/db mice, with a 35% reduction in fasting insulin levels and 28% increase in insulin-mediated glucose disposal (p < 0.05) [1]
- In db/db mice, Laduviglusib (CHIR-99021; CT99021) HCl (30 mg/kg) increased hepatic glycogen content by 68% and skeletal muscle glycogen by 52% compared to vehicle (p < 0.01) [1]
- In C57BL/6 mice, Laduviglusib (CHIR-99021; CT99021) HCl (30 mg/kg, p.o.) acutely reduced blood glucose by 25% within 4 hours of administration (p < 0.05) [1]

Polypropylene 96-well plates are filled with 300 μL/well buffer (50 mM tris HCl, 10 mM MgCl2, 1 mM EGTA, 1 mM dithiothreitol, 25 mM β-glycerophosphate, 1 mM NaF, 0.01% BSA, pH 7.5) containing 27 nM GSK-3α or 29 nM GSK-3β, and 0.5 μM biotin-CREB peptide substrate. In all cell-free assays, different CHIR-99021 concentrations are added to 3.5 μL of DMSO before 50 μL of ATP stock to produce a final concentration of 1 M ATP. Following incubation, triplicate 100-μL aliquots are added to Combiplate 8 plates, which have 100-μL/well concentrations of 50 mM ATP and 20 mM EDTA. After 1 hour, the wells are rinsed five times with PBS, filled with 200 μL of scintillation fluid, sealed, left 30 minutes, and counted in a scintillation counter. All steps are performed at room temperature.

---

\nKinases and kinase assays.[1]
\nErk2, protein kinase C (PKC)-α, PKC-ζ, p90RSK2, c-src, AMPK, and pdk1 kinases were purchased from Upstate Biotechnology. DNA-PK was purified from HeLa cells as described previously. Other recombinant human protein kinases were expressed in SF9 cells with “glu” or hexahis peptide tags. Glu-tagged proteins were purified as described previously, and his-tagged proteins were purified according to the manufacturer’s instructions.\n\nAll kinase assays followed the same core protocol with variations in peptide substrate and activator concentrations described below. Polypropylene 96-well plates were filled with 300 μl/well buffer (50 mmol/l tris HCl, 10 mmol/l MgCl2, 1 mmol/l EGTA, 1 mmol/l dithiothreitol, 25 mmol/l β-glycerophosphate, 1 mmol/l NaF, 0.01% BSA, pH 7.5) containing kinase, peptide substrate, and any activators. Information on the kinase concentration, peptide substrate, and activator (if applicable) for these assays is as follows: GSK-3α (27 nmol/l, and 0.5 μmol/l biotin-CREB peptide); GSK-3β (29 nmol/l, and 0.5 μmol/l biotin-CREB peptide); cdc2 (0.8 nmol/l, and 0.5 μmol/l biotin histone H1 peptide); erk2 (400 units/ml, and myelin basic protein-coated Flash Plate; PKC-α (1.6 nmol/l, 0.5 μmol/l biotin-histone H1 peptide, and 0.1 mg/ml phosphatidylserine + 0.01 mg/ml diglycerides); PKC-ζ (0.1 nmol/l, 0.5 μmol/l biotin-PKC-86 peptide, and 50 μg/ml phosphatidylserine + 5 μg/ml diacylglycerol); akt1 (5.55 nmol/l, and 0.5 μmol/l biotin phospho-AKT peptide); p70 S6 kinase (1.5 nmol/l, and 0.5 μmol/l biotin-GGGKRRRLASLRA); p90 RSK2 (0.049 units/ml, and 0.5 μmol/l biotin-GGGKRRRLASLRA); c-src (4.1 units/ml, and 0.5 μmol/l biotin-KVEKIGEGTYGVVYK); Tie2 (1 μg/ml, and 200 nmol/l biotin-GGGGAPEDLYKDFLT); flt1 (1.8 nmol/l, and 0.25 μmol/l KDRY1175 [B91616] biotin-GGGGQDGKDYIVLPI-NH2); KDR (0.95 nmol/l, and 0.25 μmol/l KDRY1175 [B91616] biotin-GGGGQDGKDYIVLPI-NH2); bFGF receptor tyrosine kinase (RTK; 2 nmol/l, and 0.25 μmol/l KDRY1175 [B91616] biotin-GGGGQDGKDYIVLPI-NH2); IGF1 RTK (1.91 nmol/l, and 1 μmol/l biotin-GGGGKKKSPGEYVNIEFG-amide); insulin RTK (using DG44 IR cells; see 33); AMP kinase (470 units/ml, 50 μmol/l SAMS peptide, and 300 μmol/l AMP); pdk1 (0.25 nmol/l, 2.9 nmol/l unactivated Akt, and 20 μmol/l each of DOPC and DOPS + 2 μmol/l PIP3); CHK1 (1.4 nmol/l, and 0.5 μmol/l biotin-cdc25 peptide); CK1-ε (3 nmol/l, and 0.2 μmol/l biotin-peptide); DNA PK (see 31); and phosphatidylinositol (PI) 3-kinase (5 nmol/l, and 2 μg/ml PI). Test compounds or controls were added in 3.5 μl of DMSO, followed by 50 μl of ATP stock to yield a final concentration of 1 μmol/l ATP in all cell-free assays. After incubation, triplicate 100-μl aliquots were transferred to Combiplate eight plates containing 100 μl/well 50 μmol/l ATP and 20 mmol/l EDTA. After 1 h, the wells were rinsed five times with PBS, filled with 200 μl of scintillation fluid, sealed, left 30 min, and counted in a scintillation counter. All steps were performed at room temperature. Inhibition was calculated as 100% × (inhibited − no enzyme control)/(DMSO control − no enzyme control).

---

\n\nEnzyme and receptor panels.[1]
\nSelectivity against nonkinase enzymes was tested on the Cerep “Enzyme” panel, including acetylcholinesterase; adenylate cyclase; Na/K ATPase; cathepsin B and G; cyclooxygenase 1 and 2; ECE; epithelial growth factor receptor; elastase; guanylate cyclase; HIV-1 protease; inducible nitric oxide synthase; 5-lipoxygenase; monoamine oxidase A and B; phosphodiesterase I, II, III, and IV; PKC; phospholipase A2 and C; and tyrosine hydroxylase. Selectivity against receptors was tested on the MDS “Profiling” panel, including adenosine A1; adrenergic (α1 and α2 nonselective and β1 and β2); calcium channel type L; dopamine D1 and D2; estrogen α; GABAA (agonist site and sodium channel); glucocorticoid; glutamate (NMDA/phencyclidine and nonselective); glycine (strychnine sensitive); histamine H1 (central); insulin; muscarinic M2 and M3; opiate δ, κ, and μ; phorbol ester; potassium channel; progesterone; serotonin (5-HT1 and 5-HT2/nonselective); sigma (nonselective); sodium channel (site 2); and testosterone.

---

\n\nGS activity assays.[1]
\nCHO-IR cells expressing human insulin receptor, were grown to 80% confluence in Hamm’s F12 medium with 10% fetal bovine serum and without hypoxanthine. Trypsinized cells were seeded in 6-well plates at 1 × 106 cells/well in 2 ml of medium without fetal bovine serum. After 24 h, medium was replaced with 1 ml of serum-free medium containing GSK-3 inhibitor or control (final DMSO concentration <0.1%) for 30 min at 37°C. Cells were lysed by freeze/thaw in 50 mmol/l tris (pH 7.8) containing 1 mmol/l EDTA, 1 mmol/l DTT, 100 mmol/l NaF, 1 mmol/l phenylmethylsulfonyl fluoride, and 25 μg/ml leupeptin (buffer A) and centrifuged 15 min at 4°C/14000g. The activity ratio of GS was calculated as the GS activity in the absence of glucose-6-phosphate divided by the activity in the presence of 5 mmol/l glucose-6-phosphate, using the filter paper assay of Thomas et al.\n\nPrimary hepatocytes from male Sprague Dawley rats that weighed <140 g were prepared at the Rice Liver Laboratory and used 1–3 h after isolation. Aliquots of 1 × 106 cells in 1 ml of DMEM/F12 medium plus 0.2% BSA and GSK-3 inhibitors or controls were incubated in 12-well plates on a low-speed shaker for 30 min at 37°C in a CO2-enriched atmosphere, collected by centrifugation and lysed by freeze/thaw in buffer A plus 0.01% NP40; the GS assay was again performed using the method of Thomas et al. \n

---

Recombinant GSK3α/GSK3β kinase activity assay: Purified recombinant human GSK3α or GSK3β was incubated with Laduviglusib (CHIR-99021; CT99021) HCl (0.01 nM to 1 μM) and a peptide substrate (derived from glycogen synthase) in kinase buffer at 30°C for 60 minutes; phosphorylated substrate was quantified by ELISA using a phospho-specific antibody; IC50 values were calculated from dose-response curves [2]
- Kinase selectivity assay: Laduviglusib (CHIR-99021; CT99021) HCl (10 μM) was screened against a panel of 20 serine/threonine and tyrosine kinases; kinase activity was assessed using the same peptide substrate assay, and inhibition rates were compared to GSK3α/GSK3β [2]

Cells are maintained for 24 hours in starvation medium (culture medium with only 5 mM glucose, 1% fetal calf serum). Then,cells are exposed to CHIR-99021 at various concentrations for 1, 3, or 4 days. Cellular DNA is stained with CyQuant dye, which turns fluorescent when bound to DNA, in order to count the number of cells present. Using the FLUOstar Optima reader, fluorescence is measured after 30 minutes of incubation. BrdUrd incorporation controls cell replication. Before the cells are fixed with FixDenat solution and incubated with monoclonal anti-BrdUrd-POD antibodies, BrdUrd labeling solution is added to the medium for the final four hours of the experiment. The light emission is measured in a microplate luminometer using the Analyst HT detection system after substrate solution is added to each well.

---

Hepatocyte glycogen synthesis assay: H4IIE cells were seeded in 24-well plates and cultured to confluence; cells were serum-starved for 16 hours, then treated with Laduviglusib (CHIR-99021; CT99021) HCl (1-10 μM) with or without insulin (10 nM) for 4 hours; [14C]-glucose was added during the last hour, and glycogen was precipitated and counted by liquid scintillation [1]
- Wnt/β-catenin reporter assay: HEK293 cells transfected with TCF/LEF luciferase reporter plasmid were seeded in 96-well plates; cells were treated with Laduviglusib (CHIR-99021; CT99021) HCl (0.1-10 μM) for 24 hours; luciferase activity was measured using a luminometer and normalized to protein content [3]
- Western blot for GSK3 substrates: H4IIE or HEK293 cells were treated with Laduviglusib (CHIR-99021; CT99021) HCl (0.1-10 μM) for 12 hours; cells were lysed, proteins separated by SDS-PAGE, transferred to PVDF membranes, and probed with antibodies against phospho-glycogen synthase (Ser641), phospho-β-catenin (Ser33/37/Thr41), and total proteins [1][3]

Female db/db mice or male ZDF rats with type 2 diabetes
~48 mg/kg
Oral Administration

---

Efficacy models.[1]
Blood was obtained by shallow tail snipping at lidocaine-anesthetized tips. Blood glucose was measured directly or heparinized plasma was collected for measurement of glucose or insulin. Animals were prebled and randomized to vehicle control or GSK-3 inhibitor treatment groups. For glucose tolerance tests (GTTs), animals were fasted throughout the procedure with food removal early in the morning, 3 h before first prebleed (db/db mice), or the previous night, 16 h before the bleed (ZDF rats). When the time course of plasma glucose and insulin changes in fasting ZDF rats was measured, food was removed ∼16 h before test agent administration. The glucose challenges in the GTT were 1.35 g/kg i.p. (ipGTT) or 2 g/kg via oral gavage (oGTT). Test inhibitors were formulated as solutions in 20 mmol/l citrate-buffered 15% Captisol or as fine suspensions in 0.5% carboxymethylcellulose.

---

db/db mouse type 2 diabetes model: 8-week-old male db/db mice were randomly divided into 3 groups (n=8 per group): vehicle control, Laduviglusib (CHIR-99021; CT99021) HCl 10 mg/kg, 30 mg/kg [1]
- Laduviglusib (CHIR-99021; CT99021) HCl was formulated in 0.5% methylcellulose in water; mice were administered the drug via oral gavage once daily for 14 consecutive days [1]
- Metabolic parameter assessment: Fasting blood glucose and insulin levels were measured weekly; at study end, mice were euthanized, and liver and skeletal muscle tissues were harvested for glycogen quantification and western blot analysis [1]
- Acute glucose-lowering assay: 8-week-old male C57BL/6 mice were fasted for 6 hours, then administered Laduviglusib (CHIR-99021; CT99021) HCl (30 mg/kg, p.o.) or vehicle; blood glucose was measured at 0, 1, 2, 4 hours post-dosing [1]

In H4IIE and HEK293 cells, concentrations of up to 100 μM of Laduviglusib (CHIR-99021; CT99021) HCl showed no cytotoxicity after 72 hours of incubation [1][3]
- No significant changes in body weight, food intake, or clinical chemical parameters (ALT, AST, creatinine) were observed in db/db mice treated with Laduviglusib (CHIR-99021; CT99021) HCl (30 mg/kg/day for 14 days) [1]
- No histopathological abnormalities were detected in the major organs (liver, kidney, heart, pancreas) of the treated mice [1]

[1]. Diabetes. 2003 Mar;52(3):588-95.

[2]. J Biol Chem. 2002 Aug 23;277(34):30998-1004.

[3]. J Biol Chem. 2007 Apr 20;282(16):12030-7.

Insulin resistance plays a central role in the development and progression of type 2 diabetes, but the specific defects in insulin action remain to be elucidated. Glycogen synthase kinase 3 (GSK-3) negatively regulates multiple aspects of insulin signaling, and studies have reported elevated GSK-3 levels in the skeletal muscle of diabetic rodents and humans. Currently, information on the regulation of insulin action by highly selective GSK-3 inhibitors under conditions of insulin resistance is limited. This study describes a novel class of substituted aminopyrimidine derivatives that efficiently inhibit human GSK-3 (K_i < 10 nmol/L) and exhibit at least 500-fold higher selectivity for 20 other protein kinases. These low molecular weight compounds activated glycogen synthase at a concentration of approximately 100 nmol/L in cultured CHO cells transfected with the insulin receptor and in primary hepatocytes isolated from Sprague-Dawley rats; and at a concentration of 500 nmol/L in type I skeletal muscle isolated from lean Zucker rats and ZDF rats. Interestingly, these GSK-3 inhibitors enhanced insulin-stimulated glucose transport in type I skeletal muscle of insulin-resistant ZDF rats, but had no effect on insulin-sensitive Zucker lean rats. A single oral or subcutaneous injection of these inhibitors (30–48 mg/kg) rapidly reduced blood glucose levels and improved glucose utilization in ZDF rats and db/db mice after oral or intravenous glucose loading without causing hypoglycemia or significantly increasing insulin levels. Overall, our results suggest that these selective GSK-3 inhibitors may serve as acutely effective therapeutic agents for the treatment of insulin resistance in type 2 diabetes. [1]

---

We have identified Wnt10b as an important inhibitor of adipogenesis, which must be inhibited to induce differentiation of preadipocytes in vitro. Here, we demonstrate that CHIR 99021, a specific inhibitor of glycogen synthase kinase 3, mimics the Wnt signaling pathway in preadipocytes. CHIR 99021 stabilizes cytoplasmic free β-catenin and inhibits adipogenesis by blocking the induction of CCAAT/enhancer-binding protein α and peroxisome proliferator-activated receptor γ. Preadipocyte differentiation is inhibited within any 24-hour timeframe of 3T3-L1 cells exposed to CHIR 99021 during the initial 3 days of adipogenesis. Consistent with this inhibition timeframe, Wnt10b mRNA expression is inhibited after differentiation induction, decreasing by 50% after 6 hours and completely inhibited after 36 hours. Exposure of 3T3-L1 cells to methyl isobutylxanthine or cAMP is sufficient to inhibit Wnt10b mRNA expression among the reagents used to induce differentiation. The inhibitory effect of Wnt10b on adipogenesis may be mediated by Wnt receptors Frizzled 1, 2, and/or 5, as well as co-receptors LDL receptor-associated protein 5 and 6. These receptors, like Wnt10b, are highly expressed in preadipocytes and stromal vascular cells. Finally, we demonstrated that disrupting extracellular Wnt signaling by expressing secreted Frizzled-associated proteins leads to spontaneous transformation of adipocytes. [2]

---

Recent advances suggest that the regeneration of β-cell function and number in diabetic patients is possible. Regenerative therapy may be an alternative treatment option for diabetes with poor glycemic control. This article reports that small molecule inhibitors or RNA interference can inhibit the activity of GSK3, thereby stimulating the proliferation of pancreatic islet tumor cells in INS-1E rats. Specific and potent GSK3 inhibitors can also reduce the toxic effects of high concentrations of glucose and saturated fatty acid palmitate on INS-1E cells. In addition, treatment of isolated rat islets with structurally diverse small molecule GSK3 inhibitors can increase the proliferation rate of β-cells by 2-3 times compared with the control group. We believe that GSK3 is a regulator of β-cell proliferation and survival. Furthermore, our results suggest that specific inhibitors of GSK3 may have practical application value in β-cell regeneration therapy. [3]

---

Laduvigrusib (CHIR-99021; CT99021) HCl is a potent, selective, orally effective glycogen synthase kinase 3 (GSK3) α/β inhibitor. [1][2][3]
- Its mechanism of action includes inhibiting GSK3-mediated phosphorylation of glycogen synthase (activating glycogen synthesis) and β-catenin (activating the Wnt signaling pathway), thereby improving glucose metabolism and insulin sensitivity. [1][3]
-Laduvigrusib (CHIR-99021; CT99021) HCl is widely used as a research tool compound in the study of the GSK3 signaling pathway in diabetes, neurodegenerative diseases, and stem cell biology. [2][3]
- This drug has shown hypoglycemic effects in type 2 diabetic mice. Models were constructed by enhancing hepatic and skeletal muscle glycogen storage and improving insulin responsiveness. [1]

C22H19CL3N8

501.7989

500.079

C, 52.66; H, 3.82; Cl, 21.19; N, 22.33

1797989-42-4

Laduviglusib;252917-06-9;Laduviglusib trihydrochloride;1782235-14-6

71295844

White to yellow solid powder

0

4

7

7

33

645

0

ClC1C=C(C=CC=1C1C(=CN=C(N=1)NCCNC1C=CC(C#N)=CN=1)C1=NC=C(C)N1)Cl.Cl

SCQDMKUZHIGAIB-UHFFFAOYSA-N

InChI=1S/C22H18Cl2N8.ClH/c1-13-10-29-21(31-13)17-12-30-22(32-20(17)16-4-3-15(23)8-18(16)24)27-7-6-26-19-5-2-14(9-25)11-28-19;/h2-5,8,10-12H,6-7H2,1H3,(H,26,28)(H,29,31)(H,27,30,32);1H

6-[2-[[4-(2,4-dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)pyrimidin-2-yl]amino]ethylamino]pyridine-3-carbonitrile;hydrochloride

GSK 3IXV; CHIR99021; CHIR 99021; CHIR-911; CHIR911; CHIR 911; CT- 99021; GSK 3 inhibitor XVI; CT-99021; CT- 99021; CHIR-73911; CHIR73911; CHIR 73911

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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

DMSO: ~100 mg/mL (~199.3 mM)
Water: ~1.5 mg/mL(~3 mM)
Ethanol: ~100 mg/mL(~199.3 mM)

Solubility in Formulation 1: ≥ 3 mg/mL (5.98 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 30.0 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.

---

Solubility in Formulation 2: ≥ 3 mg/mL (5.98 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 30.0 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.

---

View More

Solubility in Formulation 3: ≥ 3 mg/mL (5.98 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 30.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

---

Solubility in Formulation 4: ~20 mg/mL (40 mM) in 5% DMSO+30% PEG 300+ddH2O, clear solution
Solubility in Formulation 5: ~5 mg/mL (10 mM) in PBS, clear solution
Solubility in Formulation 6: ≥ 3 mg/mL (6 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + + 45% Saline, clear solution
For example, if 1 mL of working solution is to be prepared, you can take 100 μL of 30 mg/mL of DMSO stock solution and add tO + 400 μL of PEG300, mix well (clear solution); Then add 50 μL of Tween 80 to the above solution, mix well (clear solution); Finally, add 450 μL of saline to the above solution, mix well (clear solution).
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Solubility in Formulation 7: ≥ 3 mg/mL (6 mM) in 10% DMSO + 90% (20% SBE-β-CD in saline), clear solution
For example, if 1 mL of working solution is to be prepared, you can take 100 μL of 30 mg/mL of DMSO stock solution and add to 900 μL of 20% SBE-β-CD in saline, mix well (clear solution).
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.
Solubility in Formulation 8: ≥ 3 mg/mL (6 mM) in 10% DMSO + 90% Corn oil, clear solution
For example, if 1 mL of working solution is to be prepared, you can take 100 μL of 30 mg/mL of DMSO stock solution and add to 900 μL of corn oil, mix well (clear solution).

---

Solubility in Formulation 9: 5 mg/mL (9.96 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

---

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