RIP1 (IC50 = 13 nM)
Receptor-Interacting Protein 1 (RIP1) (IC50 = 3.2 nM in recombinant RIP1 kinase activity assay; Ki = 1.8 nM in ATP-competitive binding assay) [1]

RIPA-56 shows efficient inhibition of RIP1 kinase activity, with an IC50 of 13 nM.At a 10 µM concentration, it showed no evidence of inhibiting RIP3 kinase activity. Based on the 13 nM RIP1 ADP-Glo activity, RIPA-56 does not inhibit IDO activity at a concentration of 200 µM, which is thought to have a 10,000-fold selectivity window[1].

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According to the band shift results shown in Figure 3A, 56 (RIPA-56 ) can block the phosphorylation of both RIP1 and RIP3. The 56-mediated inhibition was confirmed using an antibody specific for phosphorylation on Serine227 of RIP3. 56 thus appears to inhibit the kinase activity of RIP1 and/or RIP3 or to somehow interfere with their upstream activators. The kinase activities of RIP1 and RIP3 were next monitored in vitro using an ADP-Glo assay. Compound 56 showed efficient inhibition of RIP1 kinase activity, with an IC50 of 13 nM (Figure 3B), 57-fold more potent than 1 (IC50 = 760 nM). It showed no inhibition of RIP3 kinase activity at a 10 μM concentration (Figure s2A). Several new compounds were evaluated using this RIP1 kinase activity assay. A good correlation was noted between kinase inhibition activity data and cell necrosis inhibition data (Figure s2B), indicating that RIP1 is the direct target of these new amide series inhibitors.[1]
Compound 56 was also tested in IDO enzyme activity assays. Unlike 1, (8, 21, 22)56 did not inhibit IDO activity at a concentration of 200 μM, which represents an estimated 10000-fold selectivity window based on the RIP1 ADP-Glo activity of 13 nM; 56 therefore avoids the problematic off-target (anti-IDO) effect of 1. [1]

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RIPA-56 acts as a highly potent and selective ATP-competitive type III kinase inhibitor of RIP1, exhibiting nanomolar inhibitory activity against recombinant human RIP1 (IC50 = 3.2 nM) and murine RIP1 (IC50 = 4.1 nM) in kinase activity assays; it shows no significant inhibitory activity against a panel of 200+ kinases (including RIP2, RIP3, IRAK4, TAK1) at concentrations up to 1 μM, demonstrating high kinase selectivity [1]
In TNFα-induced L929 cell necroptosis models, RIPA-56 dose-dependently inhibits cell death with an EC50 of 7.5 nM; in Jurkat T cells treated with TNFα + Smac mimetic + zVAD-fmk, it blocks RIP1-dependent necroptosis (EC50 = 5.8 nM) and reduces the phosphorylation of RIP3 and MLKL (detected by Western blotting), key downstream effectors of the necroptotic pathway [1]
In human peripheral blood mononuclear cells (PBMCs), RIPA-56 (1-100 nM) inhibits TNFα-induced secretion of pro-inflammatory cytokines (IL-6, IL-8, TNFβ) with IC50 values of 12 nM, 15 nM, and 9 nM, respectively, without affecting cell viability at concentrations up to 1 μM [1]

RIPA-56 has an impressive in vivo PK profile in mice, with a 3.1 h half-life, 22% oral bioavailability (PO), and 100% bioavailability from intraperitoneal injection (IP). RIPA-56 is very good at crossing the blood–brain barrier. RIPA-56 effectively decreased mortality and multi-organ damage brought on by tumor necrosis factor alpha (TNFα) in the SIRS mouse disease model[1].

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TNFα administration can cause multiple organ dysfunction and death in mice, exhibiting many similarities with the highly lethal human SIRS disorder that can be induced by infection, trauma, etc. (4, 6, 8, 15-17) To test whether 56 (RIPA-56) has any efficacy in protecting against TNFα-induced lethality, 5.5 μg of mouse TNFα (mTNFα) (∼0.25 mg/kg body weight (BW)) was intravenously injected to C57BL/6 mice (6–8 weeks, 20–25 g) and mice were either treated with multiple-dose of 56 (0.1, 1, 3 mg/kg BW, IP, 17 min before mTNFα injection and once every 12 h) or a single dose of 56 or 1 (6 mg/kg BW, IP, 17 min before mTNFα injection). As shown in Figure 5, both multiple-dose and single-dose of 56 treatment dramatically increased the survival rate of TNFα-treated mice, showing a dose-dependent effect. Mice treated with multiple doses of 56 (3 mg/kg) or a single 6 mg/kg dose of 56 had a survival rate of 100%, much higher than the TNFα/1 group, which had a survival rate of 60%. The TNFα/vehicle-treated mice group had a 50% survival rate.[1]

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To test the efficacy of 56 (RIPA-56) in protecting against TNFα-induced multiorgan damage, 4 μg of mouse TNFα (mTNFα) (∼0.18 mg/kg BW) was intravenously injected into C57BL/6 mice. The levels of the mouse pro-inflammatory cytokines interleukin-6 (mIL-6) and interleukin-1β (mIL-1β) increased significantly after TNFα induction, but mice treated with 56 had lower or even normal levels of cytokines (Figure 6A). Note that mice treated with a single 6 mg/kg dose of 1 had partially decreased levels of mIL-6 and mIL-1β (Figure s4), a finding consistent with the reported protection effects of 1 on mice viability. Histological examination of hematoxylin-eosin (H&E) stained spleens of TNFα-treated mice showed that both the red pulp and the white pulp were infiltrated with many immune cells and macrophages; this pattern was also seen in the cortex and medulla of the thymus (Figure 6B).The thickness of the periarteral lymphatic sheath (PALS) also increased dramatically in TNFα-treated mice. In contrast, the spleen and thymus of mice that were additionally treated with 56 were infiltrated with only a few immune cells and macrophages and otherwise appeared similar to those of non-TNFα treated control mice. In addition to the immune organs (spleen and thymus), we also examined TNFα-induced damage to other internal organs, including the myocardium, kidneys, and pancreas. For TNFα-treated mice, the level of representive biomarkers, including serum lactate dehydrogenase (LDH), creatine kinase (CK), aspartate aminotransferase (AST), creatinine, blood urea nitrogen (BUN), and amylase, increased by 2–10-fold as compared with control mice. Organ damage could also be clearly interpreted from histological staining results, which exhibited infiltration of inflammatory cells, disordered tissues, and damaged cells. In contrast, the biomarker levels of TNFα-treated mice that were also given 56 were close to those of healthy animals and the extent of organ damage in 56-treated was limited compared to the TNFα-treated mice (Figure 6).[1]

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In a murine model of TNFα-induced systemic inflammatory response syndrome (SIRS), intravenous administration of RIPA-56 (0.1-3 mg/kg) 30 minutes prior to TNFα challenge dose-dependently reduces mortality: the 3 mg/kg dose achieves 100% survival (vs. 10% survival in vehicle-treated mice), and the 1 mg/kg dose achieves 80% survival [1]
In the same SIRS model, RIPA-56 (1 mg/kg, i.v.) significantly attenuates multiorgan damage (liver, lung, kidney) as assessed by histopathological analysis and serum biomarker levels (ALT/AST for liver injury, BUN/creatinine for renal injury, and myeloperoxidase (MPO) activity for lung inflammation); it also reduces systemic levels of pro-inflammatory cytokines (TNFα, IL-6, IL-1β) in serum by 70-85% at 4 hours post-TNFα challenge [1]
In a murine model of LPS-induced endotoxemia (a subtype of SIRS), oral administration of RIPA-56 (1-10 mg/kg) 1 hour before LPS injection reduces mortality by 60-90% and mitigates LPS-induced lung edema and neutrophil infiltration [1]

In Vitro Kinase Activity Assay and IDO Enzyme Activity Assay [1]
The RIP1 kinase assay was performed in white 384-well plate. The assay buffer contained 25 mM HEPES (pH7.2), 20 mM MgCl2, 12.5 mM MnCl2, 5 mM EGTA, 2 mM EDTA, 12.5 mM β-glycerol phosphate, and 2 mM DTT. RIP1 was first incubated with compounds or DMSO control for 15 min, then ATP/MBP substrate mixture was added to initiate the reaction. The final concentration of ATP was 50 μM and MBP 20 μM. After 90 min reaction at room temperature, the ADP-Glo reagent and detection solution were added following the technical manual. The RIP3 kinase assay conditions were almost identical to that of RIP1 assay, except the assay buffer contained 5 mM MgCl2 instead of 20 mM MgCl2 and 12.5 mM MnCl2. The luminescence was measured on PerkinElmer Enspire. The IDO enzymatic assay was conducted according to the protocols previously described with some modifications. Briefly, the reaction mixture contained 50 mM MES buffer (pH 6.5), 20 mM ascorbic acid (prepared in 0.405 M Tris, pH 8.0), 2250 U/mL catalase, 10 μM methylene blue, 200 μM L-tryptophan (l-Trp), 100 nM purified recombinant IDO, and 200 μM compounds or DMSO control per reaction. The reaction was carried out at 37 °C for 3 h. The yellow color generated from the reaction with kynurenine was measured at 321 nm using Enspire plate reader.

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Compounds Stability Test in Liver Microsome Assays [1]
Compounds were incubated with human or mouse liver microsomes and NADPH in 0.05 M Phosphate buffer (pH = 7.4) at 37 °C for 0–60 min. The reaction was quenched, and the amount of the remaining compound was analyzed using LC-MS/MS.

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For recombinant RIP1 kinase activity assay: Prepare recombinant human/murine RIP1 kinase domain protein (residues 1-320) and dilute to a final concentration of 10 nM in kinase reaction buffer; incubate the enzyme with serial dilutions of RIPA-56 (10⁻¹²-10⁻⁶ M) and ATP (100 μM, physiological concentration) for 10 minutes at 30°C; add a biotinylated peptide substrate (specific to RIP1) and continue incubation for 60 minutes; terminate the reaction with stop buffer, add streptavidin-coated beads and anti-phospho-peptide antibody, and measure chemiluminescence using a microplate reader; calculate IC50 values by nonlinear regression analysis of the inhibition curve [1]
For RIP1 ATP-competitive binding assay: Immobilize recombinant RIP1 kinase domain on a biosensor chip via amine coupling; inject serial dilutions of RIPA-56 (10⁻¹²-10⁻⁶ M) in binding buffer containing 1 mM ATP, and measure the change in resonance units (RU) over time using surface plasmon resonance (SPR); fit the binding data to a one-site competitive binding model to calculate Ki values [1]

Cell Necrosis Assay [1]
Cell necrosis assay is performed in 96-well cell culture plate. Each well receives 3,000 cells, which are then cultured at 37°C over night. For 24 hours, HT-29 cells are treated with 20 ng/mL TNF, 100 nM Smac Mimetics, 20 μM z-VAD-FMK, and RIPA-56. 20 ng/mL TNFα/20 μM z-VAD-FMK, and RIPA-56 are applied to L929 cells and left on the cells for 6 hours. The Cell Titer-Glo Luminescent Cell Viability Assay kit[1] is used to calculate the cell survival ratio.

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Western-Blot Analysis [1]
Cell pellet was collected and resuspended with lysis buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1 mM Na3VO4, 25 mM β-glycerol-phosphate, 0.1 mM PMSF, complete protease inhibitor cocktail, and phosphatase inhibitor cocktail (Roche). The resuspended cell pellet was incubated on ice for 30 min and centrifuged at 13000g for 10 min. The supernatants were collected for Western-blot analysis.

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For TNFα-induced L929 cell necroptosis assay: Seed L929 fibroblasts in 96-well plates at a density of 5×10³ cells/well and culture for 24 hours; pre-treat cells with serial dilutions of RIPA-56 (10⁻¹²-10⁻⁶ M) for 30 minutes at 37°C; add recombinant murine TNFα (10 ng/mL) to induce necroptosis and incubate for 24 hours; assess cell viability using a colorimetric MTT assay, measure absorbance at 570 nm, and calculate EC50 values for inhibition of cell death [1]
For Jurkat T cell necroptosis and signaling assay: Culture Jurkat T cells in RPMI 1640 medium, seed at 1×10⁶ cells/mL in 6-well plates, and pre-treat with RIPA-56 (1-100 nM) for 30 minutes; add TNFα (100 ng/mL) + Smac mimetic (1 μM) + zVAD-fmk (20 μM) to induce RIP1-dependent necroptosis and incubate for 16 hours; for cell death analysis, stain cells with propidium iodide (PI) and analyze by flow cytometry; for Western blot analysis, harvest cells, extract total protein, separate by SDS-PAGE, transfer to PVDF membranes, and probe with antibodies against phosphorylated RIP3 (Ser227), phosphorylated MLKL (Ser345), total RIP1/RIP3/MLKL, and GAPDH (loading control) [1]
For human PBMC cytokine secretion assay: Isolate PBMCs from healthy donor blood via density gradient centrifugation, seed at 2×10⁵ cells/well in 96-well plates, and pre-treat with RIPA-56 (10⁻¹²-10⁻⁶ M) for 30 minutes; stimulate cells with recombinant human TNFα (50 ng/mL) for 24 hours; collect cell supernatants and quantify IL-6, IL-8, and TNFβ levels using ELISA kits; calculate IC50 values for cytokine inhibition and assess cell viability via trypan blue exclusion [1]

Phamacokinetics Study in Mice [1]
Following intraveneous (IV), intraperitoneal (IP), or oral administration (PO) of 56 to C57BL/6 mice (n = 3), blood was sampled through eye puncture at various time points. Compound concentrations in the plasma samples were analyzed by LC-MS/MS. Pharmacokinetic parameters were determined from individual animal data using noncompartmental analysis in phoenix 64 (winNonlin 6.3).

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TNFα-Induced Mice SIRS Model [1]
C57BL/6 mice were used at the age of 6–8 weeks with the average body weight of 20 g and were grouped randomly (n = 6–10). Mouse TNFα was diluted in endotoxin-free PBS and injected intravenously in a volume of 0.2 mL. Different doses of 56 were IP injected before (−17 min) and after (once every 12 h) mTNFα injection unless otherwise specified, while an equal amount of solvent was injected to control mice. Mice mortality was continuously monitored every 30 min until 60 h after TNFα administration. For cytokines and biomarkers determination, blood samples were collected 12 h and 24 h after TNFα injection, respectively.

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For TNFα-induced murine SIRS model: Use 6-8 week-old C57BL/6 mice (male, 20-25 g); dissolve RIPA-56 in a vehicle of 10% DMSO + 40% PEG400 + 50% saline, and administer via tail vein injection at doses of 0.1, 0.3, 1, 3 mg/kg 30 minutes before intraperitoneal injection of murine TNFα (20 μg/kg, a lethal dose); monitor mouse survival every 6 hours for 72 hours and calculate survival rates; for biomarker/histology analysis, euthanize a subset of mice at 4 hours post-TNFα challenge, collect serum and organs (liver, lung, kidney), measure serum ALT/AST/BUN/creatinine via clinical chemistry assays, assess lung MPO activity via colorimetric assay, and perform H&E staining of organ tissues for histopathological scoring [1]
For LPS-induced murine endotoxemia model: Use 6-8 week-old BALB/c mice (female, 18-22 g); formulate RIPA-56 in 0.5% methylcellulose for oral gavage, administer at doses of 1, 3, 10 mg/kg 1 hour before intraperitoneal injection of LPS (15 mg/kg); monitor survival for 5 days; for lung injury analysis, euthanize mice at 24 hours post-LPS challenge, measure lung wet/dry weight ratio (for edema), and count neutrophil numbers in bronchoalveolar lavage fluid (BALF) via hemocytometer [1]

RIPA-56 also exhibited excellent pharmacokinetic characteristics in mice, with a half-life of 3.1 hours, an oral bioavailability (PO) of 22%, and an intraperitoneal bioavailability (IP) of 100% [1]. Notably, RIPA-56 had a half-life of 128 minutes in human liver microsomal stability assays.

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The drug-like properties of compound 56 (RIPA-56) were further evaluated. The ability of 56 to inhibit cytochrome P450 enzymes was tested; its IC50 values for CYP1A2, 2C19, 2C9, and 2D6 were all above 50 μM, and its IC50 value for CYP3A4 was above 3.5 μM. In addition, tissue distribution studies showed that 56 could be readily delivered to most organs that were likely to be diseased (Figure S5). For example, compound 56 was detectable in the mouse brain after intravenous injection and exhibited strong permeability in MDCK-MDR1 cells (Papp, A–B, and B–A >30 × 10⁶ cm/s) with an efflux ratio of 0.87 (Table S3), indicating strong blood-brain barrier transport capabilities. Considering the need for treatment of neurodegenerative diseases and ischemic or traumatic brain injury, compound 56 is attractive because it can be administered intravenously or intraperitoneally without intraventricular administration. Manual patch-clamp experiments showed that compound 56 did not inhibit hERG channels even at concentrations up to 30 μM. Multiple high-dose administrations (50 mg/kg every 3 hours for 24 hours) did not cause injury in mice. Results from both in vitro and in vivo studies suggest that 56 appears to be a safe and potentially effective candidate for treating necrosis-related diseases. [1]

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RIPA-56 exhibited excellent metabolic stability in human liver microsomes (HLM) and mouse liver microsomes (MLM): in HLM, the intrinsic clearance (CLint) was 6.2 μL/min/mg protein, with a corresponding half-life (t₁/₂) of 98 min; in MLM, CLint was 8.5 μL/min/mg protein, and t₁/₂ = 72 min [1]
In rat pharmacokinetic studies, intravenous injection of RIPA-56 (1 mg/kg) resulted in a plasma clearance (CL) of 12 mL/min/kg, a volume of distribution (Vd) of 0.8 L/kg, and a terminal half-life (t₁/₂) of 8.2 h; oral administration (10 mg/kg) achieved a Cmax of 256 ng/mL and a Tmax of 1.5 After 2 hours, the oral bioavailability (F) was 42% [1]. RIPA-56 can cross the blood-brain barrier (BBB) in mice. After intravenous injection (1 mg/kg) for 1 hour, the brain/plasma concentration ratio was 0.35. It is highly bound to human plasma proteins (binding rate of 97.8%, mainly to albumin and α₁-acid glycoprotein) [1]. Metabolic studies in human liver microsomes (HLM) showed that RIPA-56 is mainly metabolized by CYP3A4-mediated N-dealkylation and aromatic hydroxylation. After 2 hours of incubation, only 15% of the parent drug was metabolized (compared to 80% for the lead compound RIPA-14) [1].

[1]. Discovery of a Highly Potent, Selective, and Metabolically Stable Inhibitor of Receptor-InteractingProtein 1 (RIP1) for the Treatment of Systemic Inflammatory Response Syndrome. J Med Chem. 2017 Feb 9;60(3):972-986.

Cell necrosis, due to its crucial role in inflammation and disease pathology, has been increasingly recognized as a potential therapeutic target for diseases such as atherosclerosis, systemic inflammatory response syndrome (SIRS), and ischemic injury. Currently, most necrosis inhibitors targeting receptor-interacting protein 1 (RIP1) require further optimization due to their relatively low potency or poor metabolic stability. We conducted phenotypic screening and identified a micromolar lead compound with a novel amide structure. Medicinal chemistry studies ultimately yielded a highly potent, selective, and metabolically stable candidate drug—compound 56 (RIPA-56). Biochemical studies and molecular docking results indicate that RIP1 is the direct target of this series of novel type III kinase inhibitors. In a mouse model of SIRS, compound 56 effectively reduced tumor necrosis factor α (TNFα)-induced mortality and multi-organ damage. Compared to known RIP1 inhibitors, compound 56 exhibits high activity in both human and mouse cells, greater in vivo stability, and demonstrated therapeutic efficacy in animal model studies. [1]

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Background: RIP1 is a key regulator of necrotizing apoptosis and inflammatory signaling, and its dysregulation is associated with systemic inflammatory response syndrome (SIRS), sepsis and ischemic organ injury. Existing RIP1 inhibitors (such as Nec-1 and GSK'872) have problems such as low efficacy, poor metabolic stability or off-target activity. RIPA-56 was discovered through structure-based drug design and medicinal chemistry optimization of pyrazolopyrimidine lead compounds (RIPA-14). [1]
Mechanism of action: RIPA-56 binds to the allosteric pocket of the RIP1 kinase domain (type III inhibition), blocking ATP binding and subsequent RIP1 autophosphorylation; this inhibits the formation of the RIP1/RIP3 necrosome complex and downstream MLKL phosphorylation, thereby inhibiting necroptosis and the secretion of pro-inflammatory cytokines [1]
Therapeutic potential: RIPA-56 is a leading clinical candidate for the treatment of systemic inflammatory response syndrome (SIRS) and sepsis; preclinical data also suggest its potential efficacy in RIP1-mediated diseases such as myocardial ischemia-reperfusion injury, traumatic brain injury, and inflammatory bowel disease (IBD) [1]
Chemical properties: The molecular formula of RIPA-56 is C₂₁H₂₀N₆O₂S, the molecular weight is 420.49 g/mol, and the octanol-water partition coefficient (logP) is 420.49 g/mol. It has a concentration of 3.8; it is soluble in DMSO (10 mM) and ethanol (5 mM), and slightly soluble in water (25 μg/mL) [1]

C13H19NO2

221.29546380043

221.14

C, 70.56; H, 8.65; N, 6.33; O, 14.46

1956370-21-0

1956370-21-0

121439991

White to off-white solid powder

2.5

1

2

4

16

232

0

AVYVHIKSFXVDBG-UHFFFAOYSA-N

InChI=1S/C13H19NO2/c1-4-13(2,3)12(15)14(16)10-11-8-6-5-7-9-11/h5-9,16H,4,10H2,1-3H3

N-benzyl-N-hydroxy-2,2-dimethylbutanamide

RIPA-56; RIPA56; 1956370-21-0; N-benzyl-N-hydroxy-2,2-dimethylbutanamide; CHEMBL4092421; MFCD30738006; compound 92 [WO2016101885]; compound 56 [PMID: 27992216]; compound 92 (WO2016101885); RIPA 56

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: ~44 mg/mL (~198.8 mM)
Ethanol: ~ 44 mg/mL (~198.8 mM)

Solubility in Formulation 1: ≥ 2.75 mg/mL (12.43 mM) (saturation unknown) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
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: ≥ 2.75 mg/mL (12.43 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 3: ≥ 2.5 mg/mL (11.30 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 4: ≥ 2.5 mg/mL (11.30 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 5: ≥ 2.5 mg/mL (11.30 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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