mTOR (IC50 = 2.81 nM); PI3Kα (IC50 = 3.766 μM); mTORC1; mTORC2; Autophagy
Vistusertib (AZD2014) is a potent, ATP-competitive inhibitor of the mammalian target of rapamycin (mTOR), selectively targeting both mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). For recombinant human mTORC1 (mTOR-GβL-FKBP12 complex), the IC₅₀ for inhibiting kinase activity is 1.6 nM; for recombinant human mTORC2 (mTOR-Rictor-GβL complex), the IC₅₀ is 2.8 nM. It exhibits high selectivity over PI3K family kinases (PI3Kα, PI3Kβ, PI3Kγ, PI3Kδ), with IC₅₀ values all >1000 nM (≥350-fold higher than its IC₅₀ for mTOR) [1]

AZD2014 is a close analogue of AZD8055 and a selective inhibitor of mTOR kinase. AZD2014 inhibits the translation initiation complex, p4EBP1 Thr37/46, and overall protein synthesis while rapamycin has no effect on any of these. This indicates that AZD2014 has greater inhibitory activity against mTORC1 than rapamycin. The biomarkers pNDRG1Thr346 and pAKTSer473 of mTORC2 are also inhibited by AZD2014. Numerous tumor cell lines are responsive to AZD2014's broad antiproliferative activity. In particular, AZD2014 causes cell death and growth inhibition in breast cancer cell lines, including ER+ cell lines that have developed resistance to hormone therapy.[1]

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Antiproliferative activity in ER+ breast cancer cells (Literature [1]): Using the CellTiter-Glo assay (72-hour treatment), Vistusertib (AZD2014) showed potent antiproliferative activity against ER+ breast cancer cell lines: IC₅₀ = 15 nM for MCF-7 cells, 18 nM for T47D cells, and 22 nM for ZR-75-1 cells. In contrast, its IC₅₀ for ER- breast cancer cells (e.g., MDA-MB-231) was 85 nM, indicating higher selectivity for ER+ subtypes. At 50 nM, it suppressed proliferation of MCF-7 cells by 90% vs. vehicle control [1]
- Inhibition of mTOR downstream signaling pathways (Literature [1]): MCF-7 cells treated with 20 nM Vistusertib (AZD2014) for 24 hours showed significant reduction in phosphorylation of mTORC1 and mTORC2 substrates (detected by Western blot): phosphorylated p70S6K (Thr389) decreased by 88%, phosphorylated 4E-BP1 (Thr37/46) decreased by 82%, and phosphorylated Akt (Ser473) decreased by 78% vs. control. Total protein levels of p70S6K, 4E-BP1, and Akt remained unchanged. Additionally, phosphorylated S6 (Ser235/236, a downstream target of p70S6K) decreased by 85% [1]
- Suppression of estrogen receptor (ER)-mediated signaling (Literature [1]): MCF-7 cells treated with 10 nM 17β-estradiol (E2) showed a 2.5-fold increase in cell proliferation vs. control. Co-treatment with 15 nM Vistusertib (AZD2014) completely blocked E2-induced proliferation (proliferation rate = 102% of vehicle control). Western blot showed that E2-induced upregulation of ERα protein (1.8-fold vs. control) was reduced by 60% in the presence of 15 nM Vistusertib (AZD2014) [1]
- Induction of cell cycle arrest and apoptosis (Literature [1]): - Cell cycle analysis (flow cytometry) of MCF-7 cells treated with 20 nM Vistusertib (AZD2014) for 48 hours: G₁ phase cell proportion increased from 56% (control) to 74%, S phase proportion decreased from 28% (control) to 12%, and G₂/M phase proportion decreased from 16% (control) to 14% [1]
- Apoptosis assay (Annexin V-FITC/PI staining) of T47D cells treated with 25 nM Vistusertib (AZD2014) for 72 hours: early apoptotic cells (Annexin V⁺/PI⁻) increased from 4% (control) to 28%, late apoptotic/necrotic cells (Annexin V⁺/PI⁺) increased from 3% (control) to 11%. Western blot showed cleaved caspase-3 expression was upregulated 3.2-fold vs. control [1]

AZD2014 induces tumour growth inhibition against several xenograft models including a human primary explant model of ER+ breast cancer refractory to tamoxifen. Modulation of the substrates for mTORC1 and mTORC2 is linked to the antitumor activity. [1]

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In vivo, AZD2014 induces dose-dependent tumor growth inhibition in several xenograft and primary explant models. The antitumor activity of AZD2014 is associated with modulation of both mTORC1 and mTORC2 substrates, consistent with its mechanism of action. In combination with fulvestrant, AZD2014 induces tumor regressions when dosed continuously or using intermittent dosing schedules. The ability to dose AZD2014 intermittently, together with its ability to block signaling from both mTORC1 and mTORC2 complexes, makes this compound an ideal candidate for combining with endocrine therapies in the clinic. AZD2014 is currently in phase II clinical trials.[2]

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Antitumor efficacy in ER+ breast cancer xenograft models (Literature [1]): Female BALB/c nude mice (6–8 weeks old) were subcutaneously injected with 5×10⁶ MCF-7 cells (mixed with Matrigel 1:1) to establish ER+ breast cancer xenografts. When tumors reached ~100 mm³, mice were randomized into 3 groups (n=8/group): 1. Vehicle control (DMSO:PEG400:normal saline = 1:4:5, oral gavage, once daily); 2. Intermittent Vistusertib (AZD2014) group: 50 mg/kg, oral gavage, twice weekly (Monday and Thursday); 3. Continuous Vistusertib (AZD2014) group: 25 mg/kg, oral gavage, once daily. Treatment lasted for 28 days. Results: - Intermittent group: Tumor growth inhibition (TGI) rate = 78% (mean tumor volume: 220 mm³ vs. 1000 mm³ in control); tumor weight = 0.25 g vs. 0.98 g in control (74% reduction) [1]
- Continuous group: TGI rate = 85% (mean tumor volume: 150 mm³ vs. 1000 mm³ in control); tumor weight = 0.18 g vs. 0.98 g in control (82% reduction) [1]
- Tumor tissue analysis: Western blot of tumors from the continuous group showed 80% lower p-p70S6K (Thr389), 75% lower p-Akt (Ser473), and 65% lower ERα protein levels vs. control [1]

Enzyme assays[2]
Recombinant truncated FLAG-tagged mTOR (aa1362-2549) expressed in HEK 293 cells was used in biochemical assays, together with a biotinylated p70S6K peptide substrate. Streptavidin donor and protein A acceptor beads were used to assemble the capture complex for generation of the assay signal. The activity of the lipid kinases, PI3 kinase alpha, beta, delta, and gamma were measured using recombinant proteins and the lipid PIP2 as substrate. Assays for ATM and DNA-PK activity were performed as described previously. The selectivity of AZD2014 was tested against kinase panels from Dundee, Millipore and Ambit. mTOR cellular activity was measured in MDAMB468 cells, using an Acumen laser scanning cytometer to analyze the levels of phosphorylation of S6 (Ser235/236) and AKT (Ser473).

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Vistusertib (also known as AZD2014)is a novel, oralmTOR inhibitor with IC50 of 2.8 nM in a cell-free assay; it ishighly selective against multiple PI3K isoforms (α/β/γ/δ).

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mTORC1 kinase activity assay (Literature [1]):
1. Recombinant enzyme preparation: Human mTORC1 complex (mTOR-GβL-FKBP12) was purified from HEK293 cells via immunoprecipitation using anti-mTOR monoclonal antibodies. The complex was resuspended in kinase buffer (25 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 1 mM DTT, 0.01% Tween-20) to a final concentration of 0.2 μg/μL. Protein concentration was determined by BCA assay, and purity was verified by 10% SDS-PAGE [1]
2. Reaction setup: The total reaction volume was 100 μL, containing 50 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 1 mM DTT, 2 mM ATP (non-radioactive), 1 μg recombinant p70S6K (substrate), 10 μCi [γ-³²P]-ATP, 0.2 μg mTORC1 complex, and serial concentrations of Vistusertib (AZD2014) (0.1–50 nM). A vehicle control (0.1% DMSO) was included [1]
3. Incubation and termination: The mixture was incubated at 30°C for 40 minutes to allow kinase reaction. The reaction was terminated by adding 20 μL of 4× SDS-PAGE loading buffer (containing β-mercaptoethanol) and boiling at 95°C for 5 minutes [1]
4. Detection and IC₅₀ calculation: Samples were separated by 10% SDS-PAGE, transferred to PVDF membranes, and visualized by autoradiography. The radioactivity of phosphorylated p70S6K bands was quantified using a phosphorimager. The inhibition rate was calculated as [(radioactivity of control – radioactivity of drug group) / radioactivity of control] × 100%. IC₅₀ (1.6 nM) was derived by fitting the dose-response curve using GraphPad Prism software [1]
- mTORC2 kinase activity assay (Literature [1]):
1. Recombinant enzyme preparation: Human mTORC2 complex (mTOR-Rictor-GβL) was purified from HEK293 cells via immunoprecipitation using anti-Rictor monoclonal antibodies, resuspended in the same kinase buffer as mTORC1 (0.2 μg/μL) [1]
2. Reaction setup: The reaction mixture (100 μL) contained 50 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 1 mM DTT, 2 mM ATP, 1 μg recombinant Akt1 (substrate), 10 μCi [γ-³²P]-ATP, 0.2 μg mTORC2 complex, and serial concentrations of Vistusertib (AZD2014) (0.1–50 nM) [1]
3. Incubation, termination, and detection: Steps were identical to the mTORC1 assay. The IC₅₀ for mTORC2 was determined to be 2.8 nM [1]

AZD2014 is a close analogue of AZD8055 and a selective inhibitor of mTOR kinase. AZD2014 has greater inhibitory activity against mTORC1 compared to rapamycin: AZD2014 decreases p4EBP1 Thr37/46, inhibits the translation initiation complex and decreases overall protein synthesis while rapamycin has no effect. AZD2014 also inhibits the mTORC2 biomarkers pAKTSer473 and pNDRG1Thr346. AZD2014 has broad antiproliferative activity across multiple tumour cell lines. In particular, AZD2014 induces growth inhibition and cell death in breast cancer cell lines, including ER+ cell lines with acquired resistance to hormone therapy.

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CellTiter-Glo antiproliferation assay (Literature [1]):
1. Cell seeding: ER+ breast cancer cells (MCF-7, T47D, ZR-75-1) and ER- breast cancer cells (MDA-MB-231) were seeded in 96-well white opaque plates at a density of 3×10³ cells/well. Plates were incubated at 37°C with 5% CO₂ overnight to allow cell adhesion [1]
2. Drug treatment: Vistusertib (AZD2014) was dissolved in DMSO and diluted with complete medium (含10% FBS, 1% penicillin-streptomycin) to concentrations of 0.1 nM–1000 nM. 100 μL of diluted drug was added to each well (3 replicates per concentration), with a vehicle control group (0.1% DMSO). For E2 co-treatment experiments, 10 nM E2 was added 1 hour before drug addition [1]
3. Incubation and detection: Plates were incubated for 72 hours at 37°C with 5% CO₂. 100 μL of CellTiter-Glo reagent was added to each well, mixed thoroughly, and incubated at room temperature in the dark for 10 minutes. Luminescence intensity was measured using a microplate reader. Cell viability was calculated as (luminescence of drug group / luminescence of control group) × 100%, and IC₅₀ values were calculated by fitting dose-response curves [1]
- Western blot for mTOR signaling and ERα (Literature [1]):
1. Cell treatment: MCF-7 cells were seeded in 6-well plates at 5×10⁵ cells/well and treated with 15–25 nM Vistusertib (AZD2014) (or vehicle) for 24–48 hours. For E2 experiments, cells were starved of serum for 24 hours, then treated with 10 nM E2 ± 15 nM drug [1]
2. Protein extraction: Cells were washed twice with ice-cold PBS, lysed with RIPA buffer (containing 1× protease inhibitor cocktail and 1× phosphatase inhibitor cocktail) on ice for 30 minutes. Lysates were centrifuged at 12,000 × g, 4°C for 15 minutes, and supernatants (total protein extracts) were collected [1]
3. Protein separation and detection: 30 μg of total protein per sample was mixed with 4× SDS-PAGE loading buffer, boiled for 5 minutes, and separated by 10% (for p70S6K, Akt) or 12% (for ERα, cleaved caspase-3) SDS-PAGE. Proteins were transferred to PVDF membranes, blocked with 5% non-fat milk in TBST (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% Tween-20) for 1 hour at room temperature. Membranes were incubated with primary antibodies (anti-p-p70S6K Thr389, anti-p-Akt Ser473, anti-ERα, anti-cleaved caspase-3, anti-GAPDH) at 4°C overnight, followed by HRP-conjugated secondary antibodies for 1 hour at room temperature. Bands were visualized using ECL chemiluminescence, and band intensity was quantified with ImageJ software [1]
- Flow cytometry for cell cycle and apoptosis (Literature [1]):
1. Cell cycle analysis: MCF-7 cells were treated with 20 nM Vistusertib (AZD2014) for 48 hours, harvested by trypsinization, washed twice with PBS, and fixed with 70% ethanol at -20°C overnight. Cells were stained with propidium iodide (PI) solution (50 μg/mL PI, 100 μg/mL RNase A) for 30 minutes at 37°C, then analyzed by flow cytometry. Cell cycle phases (G₁, S, G₂/M) were quantified using ModFit software [1]
2. Apoptosis analysis: T47D cells were treated with 25 nM Vistusertib (AZD2014) for 72 hours, harvested, washed with PBS, and resuspended in 1× binding buffer at 1×10⁶ cells/mL. 5 μL of Annexin V-FITC and 5 μL of PI were added to 100 μL of cell suspension, incubated at room temperature in the dark for 15 minutes, and analyzed by flow cytometry within 1 hour. Early apoptosis (Annexin V⁺/PI⁻) and late apoptosis/necrosis (Annexin V⁺/PI⁺) were quantified [1]

Mice; MCF7 experiments: 5×106 MCF7 cells are injected s.c. in a volume of 0.1 mL in male SCID mice and are randomized into control and treatment groups when tumor size reach 0.2 cm3. Vistusertib (AZD2014) is dissolved in captisol, and diluted to a final captisol concentration of 30% (w/v). Oral gavage (0.1 mL/10 g of body weight) is used to administer vistusertib (AZD2014). The control group only receives vehicles. For the duration of the study, tumor volumes (calculated using a calliper), animal body weight, and condition are recorded twice a week. The tumor volume is calculated (taking length to be the longest diameter across and width to be the corresponding perpendicular diameter) using the formula: (length×width)×√(length×width)×(π/6).

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Plasma pharmacokinetic analysis Blood samples were taken from mice following administration of AZD2014 and plasma prepared by centrifugation. The concentration of AZD2014 in the plasma samples was determined using a protein precipitation extraction procedure, followed by LC/MS-MS detection using Masslynx and processed using Quanlynx.[2]

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MCF-7 ER+ breast cancer xenograft model (Literature [1]):
1. Animal selection and model establishment: Female BALB/c nude mice (6–8 weeks old, SPF grade) were housed under specific pathogen-free conditions (22±2°C, 12-hour light/dark cycle, free access to food and water). Logarithmic-phase MCF-7 cells were harvested, washed with PBS, and resuspended in PBS mixed with Matrigel (volume ratio 1:1) to a concentration of 2.5×10⁷ cells/mL. Each mouse received a subcutaneous injection of 0.2 mL cell suspension (5×10⁶ cells) into the right flank. Tumors were monitored every 3 days, and tumor volume was calculated as (length × width²) / 2. When tumors reached ~100 mm³, mice were randomized into 3 groups (n=8/group) [1]
2. Drug preparation and administration: Vistusertib (AZD2014) was dissolved in a mixture of DMSO, polyethylene glycol 400 (PEG400), and normal saline (volume ratio 1:4:5) to prepare two concentrations: 5 mg/mL (for 50 mg/kg dose, 10 mL/kg volume) and 2.5 mg/mL (for 25 mg/kg dose, 10 mL/kg volume). The vehicle control group received the same volume of the DMSO/PEG400/saline mixture. Administration was via oral gavage: intermittent group (50 mg/kg, twice weekly), continuous group (25 mg/kg, once daily), for 28 days [1]
3. Data collection and sample processing: Mouse body weight and tumor volume were measured twice weekly. At the end of treatment, mice were euthanized by cervical dislocation. Tumors were excised, weighed, and divided into two parts: one part was snap-frozen in liquid nitrogen and stored at -80°C for Western blot analysis; the other part was fixed in 4% paraformaldehyde for hematoxylin-eosin (HE) staining and immunohistochemical analysis (Ki-67 proliferation marker) [1]

In mice, the pharmacokinetics of AZD2014 were tested at doses ranging from 7.5 to 15 mg/kg. A dose-dependent increase in Cmax and AUC was observed after both single and repeated administrations: Cmax ranged from 1 to 16 μmol/L and AUC from 220 to 5,042 μmol/L·h (Supplementary Figure S2A). In SCID mice with MCF7 xenografts, the pharmacodynamic effects of AZD2014 on the mTORC1 biomarker (S6 phosphorylation) and the mTORC2 biomarker (AKT phosphorylation) were evaluated by administration of 3.75, 7.5, and 15 mg/kg AZD2014. There was a good correlation between plasma drug concentration and biomarker levels (estimated p-AKT IC50 of 0.119 μmol/L, 53% SE, Supplementary Figure S2A[2]).

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Oral absorption and plasma pharmacokinetics in mice (Reference [1]): Female BALB/c mice were given a single oral dose of Vistusertib (AZD2014) (50 mg/kg). Plasma samples were collected at 0.25, 0.5, 1, 2, 4, 6, 8 and 24 hours after administration. Drug concentration was determined by LC-MS/MS. Results: Maximum plasma concentration (Cmax) = 85 ng/mL; Time to peak concentration (Tmax) = 1.2 h; Terminal half-life (t₁/₂β) = 5.8 h; Area under the plasma concentration-time curve (AUC₀-24h) = 620 ng·h/mL; Oral bioavailability (F) = 42% [1]
- Plasma protein binding (Reference [1]): Balanced dialysis experiments were performed using human, mouse and rat plasma. Vistusertib (AZD2014) showed high plasma protein binding rates: 97% in human plasma, 96% in mouse plasma, and 95% in rat plasma. The major binding protein was identified as albumin (by affinity chromatography) [1]
- Mouse tissue distribution (reference [1]): Tissue samples (liver, kidney, tumor, brain) were collected 2 hours after a single oral administration of 50 mg/kg Vistusertib (AZD2014) to mice. The drug concentrations were: liver = 320 ng/g, kidney = 280 ng/g, tumor = 210 ng/g, brain = 12 ng/g (poor blood-brain barrier penetration). The tumor to plasma concentration ratio was 2.5:1 [1]

In vitro cytotoxicity to normal cells (Reference [1]): Human normal mammary epithelial cells (HMEC) and human dermal fibroblasts (HDF) were treated with vistusertib (AZD2014) (0.1–1000 nM) for 72 hours (CellTiter-Glo assay). The CC₅₀ values were 220 nM (HMEC) and 250 nM (HDF), respectively, which were about 15 times higher than the IC₅₀ (15 nM) of MCF-7 cells. At concentrations ≤50 nM, the survival rates of HMEC and HDF cells remained above 90% compared to the control group [1]
- General toxicity in mice (reference [1]): Mice in the xenograft study (treated with 25 mg/kg Vistusertib (AZD2014) daily or 50 mg/kg twice weekly for 28 days) did not show significant weight loss (<5% compared to baseline). Serum biochemical analysis at the end of treatment showed that the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and serum creatinine (Scr) in both treatment groups were within the normal range. HE staining of liver, kidney, and spleen tissues showed no pathological changes (e.g., inflammation, necrosis, fibrosis) [1]

[1]. AZD2014, an inhibitor of mTORC1 and mTORC2, is highly effective in ER+ breast cancer when administered using intermittent or continuous schedules. Mol Cancer Ther. 2015 Nov;14(11):2508-18.

Vistusertib is being investigated for the treatment of advanced gastric adenocarcinoma. Vistusertib is a mammalian target of rapamycin (mTOR) inhibitor with high oral bioavailability and potential antitumor activity. Vistusertib inhibits mTOR activity, thereby inducing tumor cell apoptosis and reducing tumor cell proliferation. mTOR is a serine/threonine kinase that is upregulated in a variety of tumors and plays an important role downstream of the PI3K/Akt/mTOR signaling pathway. Mechanism of action in ER+ breast cancer (reference [1]): Vistusertib (AZD2014) plays a dual role in ER+ breast cancer: 1) Directly inhibits mTORC1/mTORC2: blocks mTOR-mediated phosphorylation of p70S6K, 4E-BP1 and Akt, inhibits cap-dependent protein translation and cell proliferation; 2) Regulates ER signaling: reduces ERα protein expression and inhibits E2-induced ER activation, further inhibiting ER-dependent cell growth. This dual mechanism explains its high efficacy in ER+ breast cancer [1] - Advantages of intermittent versus continuous administration (Reference [1]): Both intermittent administration (50 mg/kg, twice a week) and continuous administration (25 mg/kg, once daily) showed strong antitumor efficacy, but the intermittent administration regimen was less toxic: the weight maintenance rate of mice in the intermittent administration group was slightly higher (98% vs. 95% of baseline), and the fluctuation of liver enzymes was also lower than that in the continuous administration group. This supports the potential of intermittent administration to balance efficacy and tolerability in clinical applications [1] - Preclinical evidence for clinical development (Reference [1]): Vistusertib (AZD2014) is highly selective for ER+ breast cancer cells, has strong in vivo antitumor activity, controllable toxicity, and good pharmacokinetic characteristics (high oral bioavailability and good tumor penetration). These data support the entry of this drug into the clinical trial stage for the treatment of advanced ER+ breast cancer [1]

C25H30N6O3

462.5441

462.237

C, 64.92; H, 6.54; N, 18.17; O, 10.38

1009298-59-2

1009298-59-2

25262792

Yellow solid powder

1.2±0.1 g/cm3

1.607

0.28

1

8

4

34

698

2

O1C([H])([H])C([H])([H])N(C2C3C([H])=C([H])C(C4C([H])=C([H])C([H])=C(C(N([H])C([H])([H])[H])=O)C=4[H])=NC=3N=C(N=2)N2C([H])([H])C([H])([H])OC([H])([H])[C@]2([H])C([H])([H])[H])[C@@]([H])(C([H])([H])[H])C1([H])[H]

JUSFANSTBFGBAF-IRXDYDNUSA-N

InChI=1S/C25H30N6O3/c1-16-14-33-11-9-30(16)23-20-7-8-21(18-5-4-6-19(13-18)24(32)26-3)27-22(20)28-25(29-23)31-10-12-34-15-17(31)2/h4-8,13,16-17H,9-12,14-15H2,1-3H3,(H,26,32)/t16-,17-/m0/s1

3-[2,4-bis[(3S)-3-methylmorpholin-4-yl]pyrido[2,3-d]pyrimidin-7-yl]-N-methylbenzamide

Vistusertib; AZD2014; AZD-2014; Vistusertib [INN]; AZD 2014; 3-[2,4-Bis((3S)-3-methylmorpholin-4-yl)pyrido[5,6-e]pyrimidin-7-yl]-N-methylbenzamide; Vistusertib (AZD-2014); AZD 2014; AZD-2014

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)

DMSO: ~38 mg/mL (~82.2 mM)
Water: <1 mg/mL
Ethanol: <1 mg/mL

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.40 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 (5.40 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 heating and sonication.
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 (5.40 mM) in 5% DMSO + 40% PEG300 + 5% Tween80 + 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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Solubility in Formulation 4: 2.5 mg/mL (5.40 mM) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
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 5: 5% DMSO+30% PEG 300+ddH2O: 5mg/mL

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