ASK1 (pIC50 = 8.3)
Selonsertib (also named GS-4997) targets Apoptosis Signal-regulating Kinase 1 (ASK1/MAP3K5/MAP Kinase Kinase Kinase 5); [1]
Selonsertib is a selective inhibitor of ASK1 [4]

Selonsertib (GS-4997) is a clinical-stage ASK1 inhibitor that has been assessed as a potential treatment for kidney fibrosis and diabetic nephropathy[1]. A once-daily oral ASK1 inhibitor with high selectivity and potency, selonsertib (GS-4997), competes with ATP in the ASK1 catalytic kinase domain.

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1. Selonsertib (5 µM) prevented LPS-primed JNK and DRP1 mitochondrial translocation in RAW264.7 cells, alleviated LPS-induced mitochondrial damage in macrophages (including reduced mitochondrial oxidative stress, restored mitochondrial membrane potential (Δψm), and inhibited mitochondrial permeability transition pore (mPTP) opening), and suppressed the release of inflammatory cytokines (TNF-α and IL-1α) from RAW264.7 cells after LPS exposure [4]
2. A lead ASK1 inhibitor (structural analog of Selonsertib) identified by structure-based design displayed robust MAP3K pathway inhibition in in vitro assays [3]

An oral bioavailable inhibitor of apoptosis signal-regulating kinase 1 (ASK1) that may have anti-inflammatory, anti-tumor, and anti-fibrotic properties. The ATP-competitive ASK1 inhibitor GS-4997 targets and binds to the catalytic kinase domain of ASK1 after oral administration, preventing its phosphorylation and activation. This stops downstream kinases, including p38 MAPK and c-Jun N-terminal kinases (JNKs), from becoming phosphorylated. GS-4997 inhibits cellular proliferation, down-regulates the expression of fibrosis-related genes, and suppresses excessive apoptosis by preventing the activation of ASK1-dependent signal transduction pathways[2]. Inflammatory cytokines are also prevented from being produced.

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1. Selonsertib pretreatment significantly ameliorated LPS/GalN-induced acute liver failure (ALF) in mice: reduced hepatic necrosis, decreased serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBIL) and inflammatory cytokines in a dose-dependent manner (15 mg/kg, 30 mg/kg, 60 mg/kg); the therapeutic effect was only effective early after LPS/GalN administration (pretreatment or treatment at 0.5 h/1 h post-injection, ineffective at 2 h/4 h post-injection) [4]
2. Selonsertib attenuated JNK-mediated DRP1 mitochondrial translocation in murine liver tissue and primary hepatic macrophages, re-established mitochondrial fusion-fission balance, rescued mitochondrial damage in macrophages, and relieved inflammatory injury in ALF mice [4]
3. A lead ASK1 inhibitor (structural analog of Selonsertib) reduced infarct size in an isolated perfused heart model of cardiac ischemia/reperfusion injury [3]
4. Phase 2 clinical trial design: Selonsertib (oral, once daily) was investigated in ~300 patients with T2DM and stage 3/4 diabetic kidney disease (DKD) receiving standard care; primary endpoint was change in estimated glomerular filtration rate (eGFR) at 48 weeks, key secondary endpoint was change in albuminuria [1]

ASK1 Enzyme Inhibition Assay [3]
Material: ASKI recombinant protein and HTRF® KinEASE™ STK substrate 3 kit were used. The kinase assay was conducted in 250mM Hepes buffer containing NaN3 0.1%, 0.05% BSA, 0.5mM Orthovanedate, 5mM MgCl2,1mM DTT, 1% DMSO (after compound addition) at pH 7.0 according to the protocol provided by the vendor. [3]
Methods: IC50 determination – the IC50 value for each compound was determined in the presence of compound (various concentrations, from 0 to 10uM) and a fixed amount of ATP (200uM, final concentration), substrate STK3 (1uM, final concentration). The enzymatic reaction was initiated by adding ASK1 (10nM, final concentration). The assay was conducted at room temperature (~ 22°C). After 60 min, the enzymatic reaction was stopped using the stop reagents provided by the CisBio kit. All the reagents were dispensed using a Multidrop Combi reagent dispenser into white 384 SV Greiner plates. The release of product was detected using a BMG PHERAstar plate reader at 337 nm (excitation wavelength) and measuring the ratio of fluorescence 665/620 nm (emission wavelengths). [3]

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Human and Rat Liver Microsomal stability assay: [3]
Pooled rat or human liver microsomes were quickly thawed before further processing. The reactions were initiated on a Tecan liquid handler by adding room temperature NADPH solution into pre-warmed wells containing test compound or control compound as well as liver microsomes. The final incubation solutions (20 μL) contained 1 μM test compound or control, liver microsomes (0.5 mg/mL), 2 mM NADPH, and 50 mM potassium phosphate buffer (pH 7.4). Plates were incubated at 37°C for 0, 5, 15 and 30 minutes. The reactions were terminated by the addition of 40 μL of 0.1 M trichloroacetic acid (TCA) quenching solution. The plates were centrifuged at 4500g for 20 min. The peak areas of test compound or controls in the supernatants were measured by LC-MS/MS. A Waters Quattro Premier UPLC-MS/MS System consisting of an Acquity Binary Solvent manager; Waters Acquity sample manager and Waters Acquity Sample Organizer was used for liver microsomal sample analysis. Waters MassLynx software was used for instrument control, data acquisition, data processing and chromatogram/spectrum browsing. Liquid chromatography was performed using a Waters BEH C18, 1.7 μm, ID 2.1 x 50 mm at 50°C column eluting with mobile phase A: 0.04% formic acid in water and mobile phase B: 0.04% formic acid in acetonitrile with a flow rate of 0.35 mL min-1 and a gradient of 1% to 99% B over 1.0 min. The metabolic half life (t1/2) values were calculated using Equation 1, where A is the initial peak area expressed as 100%, k is the firstorder rate constant, and t is time in minutes.[3]

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Selonsertib (Also known as GS-4997) is a highly selective and potent once-daily orally available ASK1 (apoptosis signal-regulating kinase 1) inhibitor that is highly potent and selective, with a pIC50 of 8.3–0.07. A once-daily oral ASK1 inhibitor with high selectivity and potency, selonsertib (GS-4997), competes with ATP in the catalytic kinase domain of ASK1.

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1. Structure-based drug design assay for ASK1 inhibitors: Known ASK1 inhibitors were deconstructed and reoptimized using structural biology techniques (X-ray crystallography for ASK1-inhibitor binding mode analysis); key interactions between ASK1 protein and small molecule inhibitors were characterized, and crystal structures of lead compounds bound to ASK1 kinase domain were resolved to guide the design of potent ASK1 inhibitors (including Selonsertib) [3]
2. Kinase selectivity assay for ASK1 inhibitors: The overall kinase selectivity of lead ASK1 inhibitor compounds (analogs of Selonsertib) was evaluated to confirm specific inhibition of ASK1/MAP3K pathway [3]

ASK1-HEK293 P-JNK Cellular Assay. [3]
Human ASK1/HEK-293 cells were engineered to express human ASK1 utilizing a tetracycline-inducible gene expression (Tet-on) system, in which the gene of interest is activated by adding tetracycline (Tet) to the culture medium. Cells were maintained in DMEM high glucose medium containing 10% Fetal Bovine Serum, 100units/ml Penicillin G/100ug/mL Streptomycin Sulfate, 500ug/ml Geneticin and 5ug/ml Blasticidin. Cells were seeded at 300,000 cell/ml, 145ul/well, in 96 well black clear plates coated with PolyD Lysine and incubated at 37°C under 5% CO2 for 5 hours before ASK1 expression was induced by adding 15ul of tetracycline (11ug/ml) to all wells (except for no Tet/No H2O2 negative controls). Cells were incubated overnight at 37°C under 5% CO2. Five-fold concentrated serial diluted compound plates were prepared the next morning and 0.040 mL from each dilution was transferred to the cell plates. Plates were then incubated for 30 minutes at 37°C under 5% CO2 before activating ASK-1 by adding 20ul of 10mM H2O2 for 30 more minutes to all wells (except for negative controls). At the end of incubation with H2O2, cell supernatants were gently aspirated, and cells were lysed by adding 70ul/well of complete MSD lysis buffer. Phospho- and total JNK levels were determined by MESO Scale ELISA. [3]

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The mouse macrophage cell line RAW264.7 was cultured in 1640 medium containing 10% foetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin and maintained at 37 °C in a humidified atmosphere containing 5% CO2. The cells were preincubated with Selonsertib (GS-4997) (5 µM) or mdivi (10 µM) for 6 h and then incubated with LPS (500 ng/ml) for 4 h. The supernatant and cell samples were then collected for further analysis[4].

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An oral bioavailable inhibitor of apoptosis signal-regulating kinase 1 (ASK1) that may have anti-inflammatory, anti-tumor, and anti-fibrotic properties. The ATP-competitive ASK1 inhibitor GS-4997 targets and binds to the catalytic kinase domain of ASK1 after oral administration, preventing its phosphorylation and activation. This stops downstream kinases, including p38 MAPK and c-Jun N-terminal kinases (JNKs), from becoming phosphorylated. GS-4997 suppresses excessive apoptosis, inhibits cellular proliferation, down-regulates the expression of fibrosis-related genes, and prevents the production of inflammatory cytokines by preventing the activation of ASK1-dependent signal transduction pathways.

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1. RAW264.7 macrophage cell assay: RAW264.7 cells were primed with LPS in the presence/absence of Selonsertib (5 µM); mitochondrial oxidative stress was detected by MitoSox staining, mitochondrial membrane potential (Δψm) was measured by JC-1 staining, mitochondrial permeability transition pore (mPTP) opening was assessed by calcein staining; western blot was used to analyze the levels of p-JNK, JNK, p-DRP1 and DRP1 in whole cells and mitochondrial fractions; ELISA was performed to quantify TNF-α and IL-1α secretion levels [4]
2. Primary hepatic macrophage assay: Primary hepatic macrophages were isolated from LPS/GalN-injected mice treated with/without Selonsertib (30 mg/kg); western blot was used to detect p-JNK, JNK, p-DRP1, DRP1 and mitochondrial dynamics-related protein levels; MitoSox staining was used to determine mitochondrial oxidative stress [4]

ALF mouse model and treatment[4]
\n\\nC57BL/6J mice were intraperitoneally injected with LPS (10 μg/kg, Sigma-Aldrich, St Louis, MO, USA) and D-GalN (400 mg/kg, Sigma-Aldrich) to establish a mouse model of LPS/GalN-induced ALF. Selonsertib (GS-4997) (15, 30, and 60 mg/kg, MCE) was administered via i.p. injection 30 min prior to LPS/GalN injection or at 0.5, 1, 2, and 4 h after LPS/GalN injection. A JNK activator, anisomycin (20 mg/kg, MCE), was administered via i.p. injection combined with Selonsertib (GS-4997) to further investigate the role of the JNK pathway in mediating the protective effect of Selonsertib (GS-4997). For another therapy, mdivi (30 mg/kg, MCE) was administered via i.p. injection 30 min prior to LPS/GalN injection. The control group was administered vehicle (n = 6). At 0.5, 1, 2, 4, and 6 h after LPS/GalN injection, the mice were sacrificed, and serum and liver samples were collected to assess the extent of liver injury. Serum was evaluated for biochemical parameters. The liver samples were evaluated for histochemistry and Western blot analysis.\\n\\nRat Pharmacokinetics: [3]
\n\\nCannulated male Sprague-Dawley (SD) rats were fasted overnight then treated with 19 (analog of Selonsertib (GS-4997)) formulated in 20% (2-hydroxypropyl)-β-cyclodextrin in 0.05M methanesulfonic acid at 5 mg/kg by oral gavage. Dose volume was 5 ml/kg and 19 was dosed as a solution at a concentration of 1 mg/ml. Residual dose form was saved and analyzed for exact dose concentration. Following administration of test article, 200 ul of blood was collected through the jugular vein catheter from conscious animals at 0.25, 0.5, 1, 2, 4, 7 and 24 hours. Blood samples were kept on ice until processed for plasma. Plasma was prepared by centrifugation at 5C, frozen and stored at -70C until analyzed. Analysis of 19 plasma concentrations was performed by LC-MS/MS analysis. Briefly, 25 ul of plasma was mixed with 100 ul acetonitrile containing an appropriate internal standard. Samples were vortexed for 1 minute then centrifuged at 300 rpm for 5 min at 2-8C. Forty ul of supernatant was then diluted 1:2 with water and vortexed for 5 min. Samples were analyzed with a AB Sciex API-4000 triple quadropole mass spectrometer equipped with a Shimadzu LC System and a LEAP autosampler. A reverse-phase gradient method running at a flow rate of 0.500 mL/min on an Phenomenex, Kinetic C18 column (2.1 mm ID  50 mm; particle size 5.0 m) was used for the test article separation. The mobile phase used was water (A) and acetonitrile (B), and both were supplemented with formic acid (0.04%, volume-to-volume ratio [v:v]). Samples were ionized and detected in multiple reactions monitoring (MRM) mode by monitoring the transition m/z 394.051→ 336.100. Samples were quantitated by use of analyte standards prepared in pooled rat plasma with internal standard. The lower limit of quantitation in this assay was 1.00 ng/mL and linearity was achieved in the concentration range of 1.00 ng/mL to 2500 ng/mL. \\n\\nLangendorff Perfused Heart Model: [3]
\n\\nMale Sprague Dawley rats were obtained from Harlan laboratories and were allowed to acclimate at least 48 hours before being used in the study. Rats were housed 2-3 animals/cage and had free access to food and water throughout the study. Rats were dosed with vehicle or compound 1, 4, 6, or 8 hours prior to ex vivo ischemia/reperfusion (I/R). Global no-flow ex vivo I/R was performed on a constant pressure recirculating Langendorff apparatus. Rats were treated with 500 U/kg heparin 10 min prior to administration of ketamine (60 mg/kg), xylazine (7.5 mg/kg) via intraperitoneal injection. Analgesics (buprenorphine hydrochloride, 0.05 mg/kg) were administered prior to surgery via subcutaneous injection. Once appropriate depth of anesthesia and analgesia were confirmed, animals were sacrificed and the hearts were quickly removed and placed in ice-cold modified Krebs-Henseleit buffer. The aorta was then cannulated and the heart was mounted on a Langendorff apparatus and perfused with oxygenated Krebs-Henseleit buffer at a constant pressure of 80 mmHg. Hearts were submerged in buffer warmed to 37°C at all times. Following a 30 minutes equilibration period, hearts were subjected to 30 minutes of no flow ischemia followed by 90 minutes of reperfusion. At the completion of I/R, hearts were flash frozen on dry ice and sectioned coronally into 3mm pieces (6 in total) using a rat heart matrix and razor blades. Hearts were then stained with 1% 2,3,5-triphenyltetrazolium chloride (TTC) for 10 minutes to visualize viable tissue and then fixed overnight in 10% formalin.

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\n1. Murine ALF model protocol: LPS/GalN was used to induce ALF in mice; Selonsertib was administered at doses of 15 mg/kg, 30 mg/kg, 60 mg/kg via unspecified route (pretreatment or treatment at 0.5 h/1 h/2 h/4 h after LPS/GalN injection); anisomycin (20 mg/kg) and mdivi (30 mg/kg, DRP1 inhibitor) were used as positive/control agents; serum levels of ALT, AST, TBIL and inflammatory cytokines were measured by CBA cytokine analysis; liver tissue pathological changes were evaluated by H&E staining; western blot was used to analyze p-JNK, JNK, p-DRP1, DRP1 and mitochondrial dynamics-related proteins in whole liver and mitochondrial fractions [4]
\n2. Isolated perfused heart model protocol: Isolated hearts were used to establish cardiac ischemia/reperfusion injury model; lead ASK1 inhibitor (analog of Selonsertib) was administered (dose unspecified) to evaluate its effect on infarct size reduction [3]
\n3. Phase 2 clinical trial protocol for DKD: ~300 patients with T2DM and stage 3/4 DKD receiving standard care were randomized in a stratified manner (based on eGFR and urine albumin to creatinine ratio) to one of four arms (dose-ranging); Selonsertib was administered orally once daily for 48 weeks; primary endpoint was change in eGFR at 48 weeks, key secondary endpoint was change in albuminuria [1]

1. The pharmacokinetics of a lead ASK1 inhibitor (an analogue of Selonsertib) at a simulated rat dose of 2.25 mpk were evaluated, and simulated ASK1 inhibition data were reported [3]

[1]. Nephron . 2015;129(1):29-33.

[2]. NCI Drug Dictionary. ASK1 inhibitor GS-4997

[3]. ACS Med Chem Lett . 2017 Feb 8;8(3):316-320.

[4]. Cell Biosci . 2021 Jan 7;11(1):9.

Selonsertib is being investigated in the clinical trial NCT03053050 (Safety and efficacy study of selonsertib in adults with non-alcoholic steatohepatitis (NASH) and bridging (F3) fibrosis). Selonsertib is an orally bioavailable inhibitor of apoptosis signal-regulated kinase 1 (ASK1) with potential anti-inflammatory, antitumor, and antifibrotic activities. After oral administration, selonsertib targets and binds to the catalytic kinase domain of ASK1 in an ATP-competitive manner, thereby preventing its phosphorylation and activation. This prevents the phosphorylation of downstream kinases such as c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38 MAPK). GS-4997 inhibits the production of inflammatory cytokines, downregulates the expression of fibrosis-related genes, inhibits excessive apoptosis, and suppresses cell proliferation by inhibiting the activation of ASK1-dependent signaling pathways. ASK1, also known as mitogen-activated protein kinase kinase 5 (MAP3K5), is activated under conditions such as oxidative stress, endoplasmic reticulum (ER) stress, calcium ion influx and infection. It plays a key role in the development of certain cardiovascular diseases, neurodegenerative diseases, diabetes and certain types of cancer.

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Drug indications
For the treatment of non-alcoholic steatohepatitis (NASH).

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Apoptosis signal-regulated kinase 1 (ASK1/MAP3K) is a member of the mitogen-activated protein kinase family and has been shown to be involved in acute ischemia/reperfusion injury. Using structure-based drug design methods, known ASK1 inhibitors were deconstructed and re-optimized to identify a lead compound. This compound showed a strong inhibitory effect on the MAP3K pathway in an in vitro perfusion heart injury model and could reduce the infarct area. [3]

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Background: Acute liver failure (ALF) has a high mortality rate, and there are currently no other effective treatments besides liver transplantation and artificial liver therapy. This study aimed to investigate the efficacy, therapeutic window, and mechanism of the selective ASK1 inhibitor selonsertib in treating alanine fibrosis (ALF). Results: ALF was simulated using a lipopolysaccharide/D-galactosamine (LPS/GalN) model. We found that selonsertib pretreatment significantly improved ALF, manifested by a reduction in liver necrosis area and decreased serum alanine aminotransferase, aspartate aminotransferase, and inflammatory cytokine levels. However, selonsertib was only effective in the early stages after LPS/GalN administration, and its limited therapeutic window was associated with the activation of JNK and DRP1 and their mitochondrial translocation. Further experiments showed that selonsertib could alleviate LPS-induced macrophage mitochondrial damage by assessing macrophage mitochondrial membrane potential and the opening of the mitochondrial permeability transition pore. Furthermore, selonsertib also inhibited the release of inflammatory cytokines from macrophages by reducing DRP1-mediated mitochondrial dysfunction, a result confirmed by the use of the specific DRP1 inhibitor mdivi. Conclusion: selonsertib protects the body from LPS/GalN-induced acute liver failure (ALF) by attenuating JNK-mediated DRP1 mitochondrial translocation, thereby rescuing mitochondrial damage in macrophages, and may have therapeutic potential for patients with early-stage ALF. [4] Selonsertib (GS-4997) is a once-daily oral selective ASK1 inhibitor that directly reduces the pathological consequences of oxidative stress (ASK1 is a key mediator of harmful effects induced by oxidative stress) [1]
2. Selonsertib protects the body from LPS/GalN-induced acute liver failure (ALF) by inhibiting the ASK1-JNK-DRP1 pathway in macrophages, restoring mitochondrial fusion-fission balance, repairing mitochondrial damage, and reducing the release of inflammatory cytokines; its limited therapeutic window is related to the activation of JNK and DRP1/mitochondrial translocation [4]
3. ASK1 is involved in acute myocardial ischemia/reperfusion injury, and structure-based ASK1 inhibitors (including Selonsertib analogues) are designed to develop potential drugs for the treatment of heart failure [3]
4. Most patients with diabetic nephropathy (DKD) experience disease progression despite standard treatment; Selonsertib is undergoing a second phase II clinical trial for DKD. A placebo-controlled clinical trial was conducted, and the design of the trial was optimized for the specific challenges of DKD (study population, efficacy indicators, treatment period, statistical methods) [1]
5. Selonsertib has potential therapeutic value for patients with early acute liver failure (ALF). Currently, apart from liver transplantation and artificial liver therapy, there are no other effective treatments for ALF [4]

C24H24FN7O

445.49

445.202

C, 64.71; H, 5.43; F, 4.26; N, 22.01; O, 3.59

1448428-04-3

1448428-04-3;1448428-05-4 (HCl);

71245288

White to off-white

1.4±0.1 g/cm3

1.704

3.17

1

6

6

33

692

0

FC1C([H])=C(C([H])([H])[H])C(=C([H])C=1C(N([H])C1=C([H])C([H])=C([H])C(C2=NN=C([H])N2C([H])(C([H])([H])[H])C([H])([H])[H])=N1)=O)N1C([H])=NC(=C1[H])C1([H])C([H])([H])C1([H])[H]

YIDDLAAKOYYGJG-UHFFFAOYSA-N

InChI=1S/C24H24FN7O/c1-14(2)32-13-27-30-23(32)19-5-4-6-22(28-19)29-24(33)17-10-21(15(3)9-18(17)25)31-11-20(26-12-31)16-7-8-16/h4-6,9-14,16H,7-8H2,1-3H3,(H,28,29,33)

5-(4-cyclopropylimidazol-1-yl)-2-fluoro-4-methyl-N-[6-(4-propan-2-yl-1,2,4-triazol-3-yl)pyridin-2-yl]benzamide

Selonsertib free base; GS-4997; GS4997; 5-(4-cyclopropyl-1H-imidazol-1-yl)-2-fluoro-N-(6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-4-methylbenzamide; Selonsertib [INN]; Selonsertib(GS-4997); 5-(4-cyclopropylimidazol-1-yl)-2-fluoro-4-methyl-N-[6-(4-propan-2-yl-1,2,4-triazol-3-yl)pyridin-2-yl]benzamide; GS 4997; Selonsertib

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: ≥ 2.62 mg/mL (5.88 mM) (saturation unknown) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), 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.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (4.67 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 20.8 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 3: ≥ 2.08 mg/mL (4.67 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 20.8 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 4: (saturation unknown) in (add these co-solvents sequentially from left to right, and one by one),

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