USP25(IC50= 0.7 μM);USP28(IC50= 0.6 μM)
USP28 (IC50 = 0.7 μM in Ub-Rh110 assay; 1.0 μM in Ub-TMR assay; 0.8 μM in tetra-Ub assay) & USP25 (IC50 = 0.6 μM in Ub-Rh110 assay) [1]
Also demonstrates equipotent activity against USP25, which is the closest homologue [1].

The in vitro profiles of AZ1, AZ2, and AZ4 show them to be active against the USP28 enzyme and to bind to USP28 by iso-thermal calorimetry (ITC) and microscale thermophoresis (MST). They were also shown to be selective over USP2a. A fourth analogue (AZ3) was shown to be significantly less potent at inhibiting USP28 but retained selectivity over USP2a. The data shown in the table were generated from one experimental replicate, unless stated otherwise[1].

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Enzyme Inhibition: AZ1 potently inhibits USP28 with IC50 values of 0.7 μM (Ub-Rh110), 1.0 μM (Ub-TMR), and 0.8 μM (tetra-Ub). It inhibits USP25 with an IC50 of 0.6 μM (Ub-Rh110). It shows high selectivity, with no significant inhibition (IC50 > 100 μM) against USP2a and less than 10% inhibition against a panel of 20 other DUBs (including USPs 1, 2, 4, 5, 7, 11, 15, 19, 20, 36, 45, CYL; UCHs L1, L3, L5, BAP1; OTUs OTUB2, OTUD6B, Cezanne; and JAMM AMSHLP) at 10 μM. At 30 μM, it showed <25% inhibition against caspases 1, 2, 4, 5, 8 and cathepsins H, L, S [1].
- Binding Affinity: Binds to USP28 with a Kd of 0.2 μM as determined by Isothermal Titration Calorimetry (ITC), and with Kd values of 3.7 μM by Microscale Thermophoresis (MST) using intrinsic protein fluorescence [1].
- Mechanism of Action: Kinetic analysis revealed AZ1 acts as a non-competitive inhibitor of USP28, with a Ki of 1.5 μM. The compound binds reversibly to the USP28 target [1].
- Cellular Target Engagement: In HCT116 cells, AZ1 engages USP28 and USP25 in a concentration-dependent manner, as shown by the reduction of Ub-VS covalent labeling, with EC50 values of 5.3 μM (USP28) and 3.3 μM (USP25). No engagement was observed for USP7 or USP4 up to 60 μM [1].
- Modulation of c-Myc: In HCT116 cells, AZ1 treatment for 3 hours led to a rapid, concentration-dependent decrease in total c-Myc protein levels, as shown by Western blot. Complete reduction was observed at the highest concentrations tested. This effect was not due to general protein degradation as β-actin and USP28 levels remained steady [1].
- Apoptosis Induction: AZ1 induced apoptosis in HCT116 cells, evidenced by the cleavage of full-length PARP (116 kDa) to the 85 kDa cleaved fragment. The increase in PARP cleavage mirrored the decrease in c-Myc levels [1].
- c-Myc Half-life: In HCT116 cells treated with cycloheximide to block protein synthesis, AZ1 (60 μM) reduced the half-life of c-Myc from 72 minutes (control) to 40 minutes. This effect was blocked by the proteasome inhibitor MG132, indicating the reduction is mediated by proteasomal degradation [1].
- Cell Viability/Anti-proliferative Effect: AZ1 reduced cell viability in a dose-dependent manner across multiple cancer cell lines. In HCT116 cells, the EC50 for loss of viability after 72 hours was 18.0 - 20.0 μM. Similar anti-proliferative effects were observed in SW480 and HT29 cells. Across a panel of 22 cancer cell lines, EC50 values were typically clustered around 20 μM [1].

The USP25/28 inhibitor AZ1 (AZ1; 40 mg/kg; gavage; daily; for 7 days) prevents diarrhea and weight loss brought on by dextran sulfate sodium (DSS) as well as impaired colon shortening[1].
Colon tumor numbers are significantly decreased by USP25/28 inhibitor AZ1 (20 mg/kg/day; gavage; 6 times a week in the 1, 3, and 6 weeks) treatment. Tumors exhibit elevated SOCS3 levels, decreased pSTAT3 levels, and Wnt-related gene expression. AZ1 gavage is not effective in treating spontaneous colitis in Il10-/-mice or DSS-induced colitis in Usp25-/-mice[1].
The USP25/28 inhibitor AZ1 (20 mg/kg/day; gavage; every 3 days from 13–20 weeks) prolongs the survival of AOM/Vil-Cre;Trp53fl/fl (VP) mice and significantly inhibits colon tumorigenesis. Treatment with AZ1 has little effect on tumorigenesis in the background deficient in USP25[1].

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For these binding studies, two independent biophysical techniques were utilized, the first of which was isothermal titration calorimetry (ITC). This label free methodology directly measures the heat of binding for the interaction that occurs when a ligand binds to a target protein and can be used to confirm ligand binding and calculate the equilibrium dissociation constant (Kd) and stoichiometry of the interaction. These parameters are additionally useful in characterizing the binding interaction and demonstrating the functional integrity of the enzyme. Under the conditions of our experiments, Kd values of 0.2, 0.9, and 2.7 μM were derived for AZ1, AZ2, and AZ4, respectively (Figure 1a–c; Table 1). These values are consistent with the biochemical activity data described previously. In addition, corresponding stoichiometry values of 0.6, 0.7, and 0.8 were derived for AZ1, AZ2, and AZ4, respectively (Figure 1a–c) in line with a nonspurious and specific mode of binding, hence supporting our initial observations from the “ratio test” experiments. For comparison, two compounds determined by the “ratio test” as potentially acting via alternative mechanisms were also tested and produced noisy and difficult to interpret data (data not shown). The second approach taken to confirm binding of the compounds to USP28 utilized the NanoTemper Microscale Thermophoresis (MST) methodology, which produced comparable data. In this instance, Kd values of 3.7 and 10.3 μM were determined for AZ1 and AZ2, respectively (Supporting Information Figure S2a,b and Table 1). In agreement with the ITC data, AZ3 failed to produce a determinable result. The binding constants for the compounds were determined using the protein’s intrinsic fluorescence. We believe this resulted in a less sensitive detection system than the fluorescently labeled version of the approach. This may in part account for the apparent discrepancy in Kd estimates between MST and ITC. In contrast, AZ4 generated a Kd using the labeled protein, which was in much closer agreement with the ITC result (3.6 vs 2.7 μM; Table 1). Despite Kd values being higher than the IC50 values obtained in the in vitro enzyme assay and the Kd values from our ITC experiments, these data further confirm target-ligand engagement. Taken together, these data derived from two independent methodologies demonstrate that AZ1, AZ2, and AZ4 interact with and bind to USP28 in a nonspurious and specific manner[1].\n

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\nBinding Assays—Isothermal Titration Calorimetry [3]
\nThe USP28 protein and test compounds were dialyzed in 40 mM HEPES (pH 7.5) and 150 mM NaCl, in order to minimize heat effects due to buffer mismatch or ionization. ITC experiments were carried out with 20 μM USP28 protein, contained in the cell of a Microcal iTC200 instrument, titrated with 200 μM test compound, contained in the instrument injection syringe. The interaction of USP28 with the test compound was quantified using a Microcal ITC 200. The titration data were recorded at 25 °C in 40 mM HEPES at pH 7.5, 150 mM NaCl, and 2% DMSO. Aliquots of 200 μM ligand stocks were added to 20 μM USP28 in multiple 2 μL intervals. Data were analyzed using nonlinear least-squares regression using Microcal Origin software.\n

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\n\n\nUSP Selectivity Assessment [3]
\nThe selectivity of compounds across the DUB family was analyzed through testing in the DUBProfiler panel at Ubiquigent (www.ubiquigent.com). This involved inclusion of test compounds at a concentration of 10 μM in a range of Ub-Rho110 in vitro enzyme assays. Enzyme assays were generated and run for the following DUB-family members: 14 USPs (USPs 1, 2, 4, 5, 7, 11, 15, 19, 20, 25, 28, 36, 45, and CYLD), four UCHs (UCHL1, UCHL3, UCHL5, and BAP1), three OTUs (OTUB2, OTUD6B), and one JAMM (AMSH-LP). Data generated are displayed as a percentage inhibition of total enzyme activity for each enzyme. Dose response testing against USP28 and USP25 was performed in the same way, testing compounds in the range of 100–0.03 μM. Selectivity of AZ1 against cysteine proteases including caspases 1/2/4/5/8 and cathepsins H/L/S was tested at a fixed screening concentration of 30 μM and data reported as % of enzyme activity relative to DMSO control.

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USP28/25 Inhibition Assay (Ub-Rh110, Ub-TMR, tetra-Ub): The deubiquitinating activity of purified USP28 and USP25 was assessed using various substrates, including Ubiquitin-Rhodamine110 (Ub-Rh110), Ubiquitin-tetramethylrhodamine (Ub-TMR), and tetra-ubiquitin (tetra-Ub) chains. The assay measured the cleavage of the substrate, which resulted in a fluorescence signal. Inhibitors were tested at various concentrations to determine the half-maximal inhibitory concentration (IC50). The data were generated from multiple replicates (n=3-24) [1].
- Isothermal Titration Calorimetry (ITC): To confirm and characterize binding, ITC experiments were performed using a Microcal iTC200 instrument. The USP28 protein (20 μM) was placed in the sample cell, and the test compound (AZ1, 200 μM) was titrated into the cell via the injection syringe in multiple 2 μL aliquots. The experiments were conducted at 25°C in a buffer containing 40 mM HEPES (pH 7.5), 150 mM NaCl, and 2% DMSO. The resulting heat of binding was measured, and data were analyzed using non-linear least squares regression to determine the equilibrium dissociation constant (Kd) and stoichiometry [1].
- Selectivity Profiling: The selectivity of AZ1 across the DUB family was assessed using the DUBProfiler panel. This involved screening the compound at a fixed concentration (10 μM) in a range of purified in vitro enzyme assays using Ubiquitin-Rhodamine110 as a substrate. The panel included 14 USPs, 4 UCHs, 3 OTUs, and 1 JAMM family member. Activity was reported as the percentage inhibition of total enzyme activity relative to a DMSO control [1].
- Reversibility Assay (High-Dilution): To test for reversible binding, USP28 was pre-incubated with AZ1 at a concentration 10 times its IC50. This mixture was then diluted 100-fold. Following dilution, USP28 activity was re-assessed. Full restoration of enzyme activity upon re-equilibration indicated reversible binding [1].
- Kinetic Analysis (Mode of Inhibition): To determine the mode of inhibition, USP28 activity was assayed across a range of tetra-Ub substrate concentrations in the presence of increasing concentrations of AZ1. The data were analyzed using a Lineweaver-Burk plot to determine the mechanism and calculate the inhibitory constant (Ki) [1].

HCT116 cells were pretreated with cycloheximide (100 μg/mL) and the proteasome inhibitor MG132 (20 μM) and subsequently exposed to AZ1 (60 μM) or vehicle control (DMSO). Cells were lysed at the indicated time points (from 0 to 180 min) and samples analyzed by Western blotting probing for c-Myc. The half-life values of c-Myc were determined by densitometry analysis based on these blots. β-Actin was included as a loading control. These data are representative data from at least three independent experiments[1].

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Cell Assays [3]
In order to assess cellular activity of the compounds, HCT116 cells were treated with USP28 inhibitor compounds for a period of 3 h. Following this incubation, cells were harvested, lysed, and subjected to Western Blot analysis. Samples were probed for c-Myc protein levels and also probed for β-actin levels, as a loading control. In order to determine the half-life of c-Myc in cells, HCT116 cells were treated for 3 h with USP28 inhibitors in the presence of cycloheximide (100 μg/mL) to block nascent protein synthesis. Thus any alteration in c-Myc levels would be indicative of changes in degradation as opposed to protein synthesis. Following compound treatment, cells were harvested and lysed prior to Western blot analysis

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Reagents for Cellular Characterization [3]
The USP28 inhibitors (AZ1, AZ2, AZ3, and AZ4) were prepared as 100 mM DMSO stocks for cell culture experiments. Cycloheximide was used at a final concentration of 100 μg/mL. MG132 and propidium iodide (PI) were used at a final concentration of 20 μM and 10 μg/mL respectively. RNaseA was used at a final concentration of 250 μg/mL. CellTiter-Glo (cell viability assay) was purchased from Promega. Ubiquitin-Vinyl Sulfone (Ub-VS) was used at a final concentration of 32 μg/mL.

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Target Engagement Assay [3]
HCT116 cells were treated with USP28 inhibitors for 2 h. Following incubation, cells were harvested in TE lysis buffer containing 50 mM Tris (pH7.4), 150 mM NaCl, 5 mM MgCl2, 0.5 mM EDTA, 0.5% NP40, 10% glycerol, and 2 mM DTT and clarified cell lysates (40 μg) incubated with ubiquitin-vinyl sulfone in assay buffer containing 50 mM Tris (pH7.6), 5 mM MgCl2, 250 mM sucrose, 0.5 mM EDTA, and 2 mM DTT for 1 h on ice. The reaction was terminated by the addition of LDS sample buffer and heated to 70 °C. Samples were then analyzed by Western blotting.

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Proliferation Assays [3]
Cells were typically seeded in 96 well plate format (typically 4000–6000 cells/well) and treated after 24 h with increasing concentration of compound from 0 to 100 μM in 1/2 log unit increments. Cell viability was assessed after 72 h by CellTiter-Glo as recommended by the manufacturer’s instructions. Analysis and EC50 values were derived using GraphPadPrism.

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Cell Culture: HCT116, SW480, HT29, and other cancer and normal cell lines were cultured in their respective media (e.g., McCoys 5A, RPMI, DMEM) supplemented with fetal bovine serum (FBS), penicillin/streptomycin, and L-glutamine at 37°C with 5% CO2. All cell lines were authenticated and tested for mycoplasma [1].
- Target Engagement Assay (Ub-VS): HCT116 cells were treated with AZ1 for 2 hours. Following incubation, cells were harvested and lysed in TE lysis buffer. Clarified cell lysates (40 μg) were then incubated with Ubiquitin-vinyl sulfone (Ub-VS) for 1 hour on ice. The reaction was terminated by adding LDS sample buffer and heating to 70°C. Samples were then analyzed by Western blotting. The covalent interaction of Ub-VS with the DUB was detected as a mobility shift (higher band). Target engagement was quantified by densitometry to determine EC50 values [1].
- Western Blotting: Cells were lysed in RIPA buffer supplemented with phosphatase and protease inhibitors. Protein lysates were subjected to SDS-PAGE and transferred to membranes. Membranes were probed with primary antibodies against c-Myc, USP28, USP25, PARP, USP7, USP4, and β-actin (as a loading control), followed by HRP-conjugated secondary antibodies [1].
- c-Myc Half-life Determination: HCT116 cells were pre-treated with cycloheximide (CHX, 100 μg/mL) to block protein synthesis, with or without the proteasome inhibitor MG132 (20 μM). They were then exposed to AZ1 (60 μM) or vehicle. Cells were lysed at various time points (0 to 180 minutes), and samples were analyzed by Western blotting for c-Myc. Half-life values were determined by densitometry analysis [1].
- Proliferation/Viability Assay: Cells were seeded in 96-well plates (4,000-6,000 cells/well). After 24 hours, they were treated with increasing concentrations of AZ1 (0 to 100 μM) in 1/2 log unit increments. Cell viability was assessed after 72 hours using the CellTiter-Glo® assay. EC50 values were derived using GraphPad Prism [1].

Animal Model: Male Usp25+/+ and Usp25-/- mice aged 12 weeks[1]
Dosage: 40 mg/kg
Administration: Gavage; daily; for 7 days
Result: In comparison to control mice, the colons of Usp25-/-mice showed increased expression of proinflammatory cytokines and antibacterial peptides, and they were shielded from the weight loss and diarrhea caused by dextran sulfate sodium (DSS). They also showed impaired colon shortening.

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AZ1 is orally bioavailable . It is soluble in DMSO (up to 84 mg/mL or 250 mg/mL according to different sources) . For in vivo administration, a common formulation is 10% DMSO + 40% PEG300 + 5% Tween 80 + 45% saline, which achieves a working concentration of 5 mg/mL (11.84 mM) . Stock solutions in DMSO can be stored at -20°C for up to 1-3 months, and powder form is stable for up to 2-3 years when stored at -20°C .

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Physicochemical Properties: Molecular Weight: 422. LogD7.4: 3.3. Solubility at pH 7.4: 801 μM. Human Plasma Protein Binding: 3.7% free (96.3% bound) [1].

AZ1 is intended for laboratory research use only and is not approved for human or veterinary use . According to safety data sheets, AZ1 contains a pharmaceutically active ingredient and is classified as moderately to severely irritating to the skin and eyes . Handling should be performed by trained personnel wearing appropriate personal protective equipment including chemical-resistant gloves, safety goggles, and protective clothing . The substance is not classified as hazardous for transport under DOT, IMDG, or IATA regulations . It contains no substances known to the State of California to cause cancer, birth defects, or reproductive harm under Proposition 65 . Specific acute toxicity data (LD50) and chronic toxicity profiles have not been thoroughly characterized in available literature.

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Cellular Toxicity/Therapeutic Index: AZ1 reduced cell viability in both cancer and normal cell lines. When profiled against a panel of 7 tissue-matched normal cell lines, AZ1 showed a minimal therapeutic index (narrow window of 3-5-fold) compared to the less active analogue AZ3. No significant differentiation in response was observed between tumour and normal cell types [1].

[1]. ACS Chem Biol. 2017 Dec 15;12(12):3113-3125.

[2]. Nature Cancer volume 1, pages811–825(2020).

[3]. ACS Chem Biol. 2017 Dec 15;12(12):3113-3125. doi: 10.1021/acschembio.7b00334.

The ubiquitin-proteasome system is widely considered an important emerging field in future drug development, with ubiquitin-specific proteases (USPs) being among the most attractive targets. Many USPs are associated with key pathways for therapeutic intervention, and the role of USP28 in maintaining c-Myc stability suggests that inhibiting USP28 may be a novel approach to targeting this currently untapped oncogene. This article reports the discovery of the first USP28 inhibitors that bind to and inhibit USP28 activity. While these inhibitors also exhibit dual activity against USP25 (and its homologs), they show high selectivity for other deubiquitinating enzymes (DUBs). Demonstrating that these compounds can simultaneously target USP25 and USP28 in cells highlights their value as important probes for studying and further exploring cellular DUB biology. Furthermore, we demonstrate that these inhibitors regulate the total level and half-life of intracellular c-Myc oncoproteins and induce apoptosis and loss of cell viability in various cancer cell lines. However, we observed a narrower therapeutic index compared to a range of tissue-matched normal cell lines. Therefore, we hope that the probes and data presented in this paper will further deepen our understanding of the biological characteristics and operability of deubiquitinating enzymes (DUBs) as potential future therapeutic targets. [1]

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Gastrointestinal bacterial infection or abnormal colonization is associated with some inflammatory bowel disease and colorectal cancer. This paper confirms that ubiquitin-specific protease 25 (USP25) plays an important role in experimental colitis, bacterial infection and colorectal cancer. Knockout or pharmacological inhibition of USP25 can enhance the immune response induced by experimental colitis or bacterial infection, promote the clearance of infected bacteria and the resolution of inflammation, and attenuate the Wnt and SOCS3-pSTAT3 signaling pathways, thereby inhibiting the occurrence of colorectal tumors. USP25 levels are positively or negatively correlated with the colonization of Fusobacterium nucleatum and the levels of β-catenin or SOCS3 in human colorectal tumor biopsy tissues, and can predict poor prognosis in patients with gastrointestinal cancer. Our results suggest that USP25 is a promoter of gastrointestinal infection and cancer and a potential drug target. [2]

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The ubiquitin-proteasome system is widely considered an important emerging field in future drug development, with ubiquitin-specific proteases (USPs) being one of the most attractive target classes in this field. Many USP proteins are associated with key pathways for therapeutic intervention, and the discovery that USP28 is essential for c-Myc stability suggests that inhibiting USP28 may be a novel approach to targeting this oncogene for which there is currently no drug target. This paper reports the discovery of the first USP28 inhibitors, which we demonstrate can bind to and inhibit USP28, and while also being active against the USP28 homolog USP25, they exhibit high selectivity for other deubiquitinating enzymes (DUBs). The utility of these compounds as valuable probes for studying and further exploring the biology of cellular DUBs is demonstrated by our demonstration that these inhibitors can simultaneously target USP25 and USP28 in cells. In addition, we also demonstrate that these inhibitors can regulate the total level and half-life of intracellular c-Myc oncoproteins and induce apoptosis and decreased cell viability in various cancer cell lines. However, we observed a narrower therapeutic index compared to a group of tissue-matched normal cell lines. Therefore, we hope that the probes and data presented in this paper will further deepen our understanding of the biological characteristics and operability of DUBs as potential future therapeutic targets. [3]

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Compound Class and Identification: AZ1 (compound identifier in the study) is a benzylaminoethanol derivative. It was identified as a hit from a high-throughput screening (HTS) campaign aimed at discovering USP28 inhibitors. The compound was validated as a specific, reversible, and non-competitive inhibitor of USP28 [1].
- Utility as a Chemical Probe: The authors highlight AZ1 as one of the first reported inhibitors of USP28 and USP25. It is presented as a valuable chemical probe for investigating the cellular biology of these deubiquitinating enzymes, particularly the regulation of c-Myc stability and its downstream effects. Its dual activity against USP25 and USP28 makes it useful for studying the selectivity and redundancy within this closely related DUB subfamily [1].

C17H16BRF4NO2

422.2121

421.0301

C, 48.36; H, 3.82; Br, 18.92; F, 18.00; N, 3.32; O, 7.58

2165322-94-9

2165322-94-9

135397656

White to off-white solid powder

3.7

2

7

7

25

399

0

BrC1C([H])=C([H])C(=C(C=1[H])C([H])([H])N([H])C([H])([H])C([H])([H])O[H])OC([H])([H])C1C([H])=C([H])C(=C(C(F)(F)F)C=1[H])F

ITHSFXDGKQYOED-UHFFFAOYSA-N

InChI=1S/C17H16BrF4NO2/c18-13-2-4-16(12(8-13)9-23-5-6-24)25-10-11-1-3-15(19)14(7-11)17(20,21)22/h1-4,7-8,23-24H,5-6,9-10H2

2-(5-Bromo-2-(4-fluoro-3-(trifluoromethyl)benzyloxy)benzylamino)ethanol

AZ1; AZ-1; USP25/28 inhibitor AZ1; 2165322-94-9; USP25 and 28 inhibitor AZ-1; Ethanol, 2-[[[5-bromo-2-[[4-fluoro-3-(trifluoromethyl)phenyl]methoxy]phenyl]methyl]amino]-; CHEMBL4442615; 2-[[5-bromo-2-[[4-fluoro-3-(trifluoromethyl)phenyl]methoxy]phenyl]methylamino]ethanol; 2-((5-bromo-2-((4-fluoro-3-(trifluoromethyl)benzyl)oxy)benzyl)amino)ethanol; C17H16BrF4NO2; AZ 1; AZ1

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 : 84~250 mg/mL ( 198.95~592.12 mM )
Ethanol : ~84 mg/mL

Solubility in Formulation 1: ≥ 2.08 mg/mL (4.93 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 2: 2.08 mg/mL (4.93 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 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 3: ≥ 2.08 mg/mL (4.93 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 20.8 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.)