SETD2[1]

A panel of MM and DLBCL cell lines are inhibited by EZM0414, with an IC50 of 0.24 μM for t(4;14) cells and 0.023 μM->10 μM for DLBCL cell lines [2].
Inhibition of SETD2 by EZM0414 results in potent anti-proliferative effects in a panel of MM and DLBCL cell lines. EZM0414 inhibited proliferation in both t(4;14) and non-t(4;14) MM cell lines, with higher anti-proliferative activity generally observed in the t(4;14) subset of MM cell lines. The median IC 50value for EZM0414 in t(4;14) cell lines was 0.24 μM as compared to 1.2 μM for non-t(4;14) MM cell lines. Additionally, inhibitory growth effects on DLBCL cell lines demonstrated a wide range of sensitivity with IC 50 values from 0.023 μM to >10 μM. [2]

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In vitro testing of EZM0414 in a safety panel consisting of 47 targets and a diversity panel of 72 kinases showed IC50 > 25 μM for all targets except D2 (IC50 = 13.0 μM, antagonist) and 5-HT1B (IC50 = 3.2 μM, agonist).[3]

In a NOD SCID mouse xenograft model implanted with human KMS-11 cells, EZM0414 (15 and 30 mg/kg, po, BID, daily) suppresses tumor growth and is well tolerated [3]. In rats and mice, EZM0414 (50 mg/kg, oral) has nearly 100% oral bioavailability, with t1/2 values of 1.8 hours for mice and 3.8 hours for rats [3].

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EZM0414 resulted in statistically significant potent antitumor activity compared to the vehicle control in three MM and four DLBCL cell line-derived xenograft models. In the t(4;14) MM cell line-derived xenograft model, KMS-11, robust tumor growth regressions were observed at the top two doses with maximal TGI of 95%. In addition, two non-t(4;14) MM (RPMI-8226, MM.1S) and two DLBCL xenograft models (TMD8, KARPAS422) demonstrated > 75% TGI; with two additional DLBCL models (WSU-DLCL2, SU-DHL-10) exhibiting > 50% TGI in response to EZM0414. In all models tested, the antitumor effects observed correlated with reductions in intratumoral H3K36me3 levels demonstrating on-target inhibition of SETD2 methyltransferase activity in vivo. [2]

SETD2 (1434-1711) Assay [3]
The biochemical assay monitored the incorporation of the tritiated methyl group from S-adenosyl-methionine (SAM) into a biotinylated histone 3 peptide corresponding to residues 26-40. The sequence of the substrate peptide is biotin-Ahx-RKSAPATGGVKKPHR-NH2 and 3H-SAM was purchased from American Radiolabeled Chemicals, Inc. For the assay, 40 L of enzyme was incubated with 1 L of compound or DMSO for 30 minutes before initiating the reaction with 10 L of substrate solution in a 384- well assay plate. The assay was performed at room temperature in assay buffer composed of 25 mM bicine, pH 8.0, 7.5 mM -mercaptoethanol, 0.002 % Tween-20, and 0.01 % bovine skin gelatin (BSG). The reaction was quenched during the linear portion of product formation with 10 L of 1 mM S-adenosyl-homocysteine (SAH) and 1 mM SAM. From the quenched reaction, 50 L was transferred to a streptavidin-coated Flashplate (Perkin Elmer) and incubated for at least 2h before washing once with 0.1 % Tween-20. Signal from the 3H-labeled peptide captured by the streptavidin-coated plates was counted by a Topcount plate reader. Percent inhibition (%I) and IC50 values were calculated using equations 1 and 2 respectively. %𝐼 = (1 − ( 𝑆−𝑚𝑖𝑛 𝑚𝑎𝑥−𝑚𝑖𝑛)) (eq 1) %𝐼 = (100 − 𝑏𝑜𝑡𝑡𝑜𝑚) ( 1 1+( 𝐼𝐶50 𝐼 ) 𝑛)+ 𝑏𝑜𝑡𝑡𝑜𝑚 (eq 2) Min is the signal from fully inhibited SETD2 from wells with a final concentration of 20 uM SAH and max is the signal from wells with DMSO instead of compound. For IC50 calculation, bottom is the theoretical minimum %I, I is the concentration of inhibitor, and n is the Hill slope. Compound IC50 determination was performed by testing 10 concentrations of compound diluted 3-fold in duplicate at final concentrations of 4 nM enzyme and substrate concentrations equal to their KM values of 0.7 M peptide and 2 M SAM.

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Screening Method: [3]
EZM0414 (10 nL) from 384-well source plates was added directly to Poly-D-Lysine (PDL) coated 384-well culture plates. A549 cells were seeded in assay medium at a concentration of 80 000 cells per mL and added to PDL coated plates with a volume of 50 µL per well. Plates were left on the bench top for ~20 minutes to allow cells to settle on the bottom of the well. Plates were incubated at 37°C, 5% CO2 for 3 days. After three days of incubation, plates were removed from the incubator and allowed to come to room temperature. Media was blotted out of the plate and 50 µL per well of ice cold 100% methanol was added to each plate and incubated for 30 minutes. Methanol was removed by aspiration and plates were washed 3 times with 115 µL per well of wash buffer (1X PBS with 0.05% Tween-20 (v/v)). Next, 50 µL per well of Odyssey blocking buffer + 0.1% Tween-20 was added to each plate and incubated for 1 hour at room temperature. Blocking buffer was removed, plates were washed 3 times, and 20 µL per well of primary antibody was added (anti-histone H3 tri-methyl K36 diluted 1:1000 in Odyssey buffer with 0.1% Tween-20 (v/v)) and plates were incubated overnight (16 hours) at 4°C. Plates were washed 5 times with 115 µL per well of wash buffer (1X PBS with 0.05% Tween-20 (v/v)). Next 20 µL per well of secondary antibody was added (1:500 800CW goat anti-rabbit IgG (H+L) antibody, 1:1000 DRAQ5 in Odyssey buffer with 0.1% Tween-20 (v/v)) and incubated for 1 hour at room temperature. The plates were washed 5 times with 115 µL per well wash buffer (1X PBS with 0.05% Tween-20 (v/v)) then 3 times with 115 µL per well of water. After rinsing, plates were centrifuged upside down on a thin bed of paper towels up to 1000 rpm for 1 minute to remove excess reagent then allowed to dry at room temperature, out of direct exposure to light. Plates were imaged on the Licor Odyssey system which measures integrated intensity at 700 nm and 800 nm wavelengths. Both 700 and 800 channels were scanned.

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For CYP1A2 inhibition, 1 μL of specific drug substrate (Phenacetin: 8 mM) was added at the final concentration of 40 μM to the above solution. For CYP2B6 inhibition, 1 μL of specific drug substrate (Bupropion: 10 mM) was added at the final concentration of 50 μM to the above solution. For CYP2C8 inhibition, 1 μL of specific drug substrate (Paclitaxel: 1 mM) was added at the final concentration of 5 μM to the above solution. For CYP2C9 inhibition, 1 μL of specific drug substrate (Tolbutamide: 40 mM) was added at the final concentration of 200 μM to the above solution. For CYP2C19 inhibition, 1 μL of specific drug substrate ((s)-Mephenytoin: 10 mM) was added at the final concentration of 50 μM to the above solution. For CYP2D6 inhibition, 1 μL of specific drug substrate (Dextromethorphan: 2 mM) was added at the final concentration of 10 μM to the above solution. For CYP3A4 inhibition, 1 μL of specific drug substrate (Midazolam: 1 mM) was added at the final concentration of 5 μM to the above solution. The mixture was pre-warmed at 37°C for 5 min. The reaction was started by the addition of 20 μL of 10 mM NADPH solution at the final concentration of 1 mM and carried out at 37°C. The reaction was stopped by addition of 400 μL of cold quench solution (methanol containing internal standards [IS: 100 nM alprazolam, 500 nM labetalol and 2 μM ketoprofen]) at the designated time points (Phenacetin: 20 min; Bupropion: 20 min: Paclitaxel: 10 min; Tolbutamide: 20 min; (s)-Mephenytoin: 20 min; Dextromethorphan: 20 min; Midazolam: 5 min). Samples were vortexed for 5 minutes and centrifuged at 3220 g for 40 minutes at 4 °C. And then 100 μL of the supernatant was transferred to a new 96-well plate with 100 μL water (depends on the LC-MS signal response and peak shape) for LC-MS/MS analysis. All experiments were performed in duplicate. [3]

Cellular proliferation assays determined IC 50 values of EZM0414 in MM and DLBCL cell line panels. Cell line-derived xenograft preclinical models of MM and DLBCL were evaluated for tumor growth inhibition (TGI) in response to EZM0414. H3K36me3 levels were determined by western blot analysis to evaluate target engagement. Combinatorial potential of SETD2 inhibition with MM and DLBCL standard of care (SOC) agents was evaluated in 7-day cotreatment in vitro cellular assays.[2]

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Cell lines in log phase growth were treated with EZM0414 in a concentration-dependent manner ranging from 0.5x10-3 to 10 μmol/L, in a final DMSO concentration of 0.2% volume/volume (v/v). Cells were plated at the determined (1.25 x 105 cells/mL) plating densities, in triplicate, in 96-well plates. Assay plates were incubated in a humidified atmosphere of 5% CO2 at 37°C. Every 3-4 days, viable cell numbers were determined using Calcein AM and analyzed on the Acumen high content imager. After cell counts, cells were split back to the original plating density, and growth media and compound replaced. The final split-adjusted number of viable cells/mL from day 14 were used to calculated percent inhibition. Averages of percent inhibition in technical triplicates were used to plot concentration response curves and calculate absolute half-maximal inhibitory concentration (IC50) values at each time point. To calculate growth for days 4,7,11, and 14: 1. Calculate the split factor for day 4 to 7, day 7 to 11, and day 11-14 The split factor is the viable cells/mL on Day X (either 4, 7, or 11) divided by the density the cells are being split back to 2. For growth of cells from day 4 to 7, multiply the day 7 viable cells/mL density by the split factor from day 4. 2. For growth of cells from day 7 to 11, multiply the day 11 viable cells/mL density by the days 4, and 7 split factors. 3. For growth of cells from Day 11 to 14, multiply the Day 14 viable cells/mL density by the days 4, 7, and 11 split factors. 5. Plot growth on semi-log chart (viable cells/mL on Y axis, in log, and days on X axis). [3]

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Cell lines in log phase growth were treated with EZM0414 in a concentration-dependent manner ranging from 0.5x10-3 to 10 μmol/L, in a final DMSO concentration of 0.2% v/v. Cells were plated at the determined plating densities in either 96-well plates (1.25 x 105 cells/mL) in triplicate (for the day 0–7-time course) or 6-well plates (3.75 x 105 cells/mL) (for re-plating on day 7 for the remainder of the time course). Assay plates were incubated in a humidified atmosphere of 5% CO2 at 37°C. Plates were read on day 0, 4, and 7, with compound/media being replenished on day 4. On day 7, the 6-well plates were trypsinized, centrifuged, and re-suspended in fresh media for counting by Vi-CELL. Cells from each treatment were re-plated at the original density in 96-well plates in triplicate and re-treated with the same compound concentration. Plates were read on days 7 (another baseline reading for day 7 seeding), 11, and 14, with compound/media being replenished on day 11. Quantification of proliferation through measurement of cellular adenosine-5’-triphosphate (ATP) was performed via a luminescent cell viability assay using CellTiter-Glo® and luminescence was detected using an EnSpire multimode microplate reader. The final raw luminescence values from day 14 were used to calculate percent inhibition. Averages of percent inhibition in technical triplicates were used to plot concentration response curves and calculate absolute IC50 values at each time point.[3]

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The Caco-2 plate was removed from the incubator and washed twice with pre-warmed HBSS (10 mM HEPES, pH 7.4), and then incubated at 37 °C for 30 minutes. The stock solutions of control compounds were diluted in DMSO to get 1 mM solutions and then diluted with HBSS (10 mM HEPES, pH 7.4) to get 5 μM working solutions. The stock solutions of the test compounds were diluted in DMSO to get 1 mM solutions, then diluted with SUPPORTING INFORMATION Conformational-Design-Driven Discovery of EZM0414: A Selective, Potent SETD2 Inhibitor for Clinical Studies SI-47 HBSS (10 mM HEPES, pH 7.4) to get 5 μM working solutions. The final concentration of DMSO in the incubation system was 0.5%. To determine the rate of drug transport in the apical to basolateral direction. 75 μL of working solution of test compound and control compounds was added to the Transwell insert (apical compartment) and the wells in the receiver plate (basolateral compartment) were filled with 235 μL of HBSS (10 mM HEPES, pH 7.4). To determine the rate of drug transport in the basolateral to apical direction, 235 μL of working solution of test compound and control compounds was to the receiver plate wells (basolateral compartment) and then the Transwell inserts (apical compartment) were filled with 75 μL of HBSS (10 mM HEPES, pH 7.4). Time 0 samples were prepared by transferring 10 μL of working solution to 40 μL HBSS (10 mM HEPES, pH 7.4) in a new 96-well plate, followed by the addition of 200 μL cold acetonitrile or methanol containing appropriate internal standards (IS). The plates were incubated at 37 °C for 2 hours. At the end of the incubation, 10 μL samples from donor sides (apical compartment for A→B flux, and basolateral compartment for B→A) to 40 μL HBSS (10 mM HEPES, pH 7.4) and 50 μL samples from receiver sides (basolateral compartment for A→B flux, and apical compartment for B→A) were transferred to wells of a new 96-well plate, followed by the addition of 4 volume of cold acetonitrile or methanol containing appropriate internal standards (IS). Samples were vortexed for 5 minutes and then centrifuged at 3,220 g for 40 minutes. An aliquot of 100 µL of the supernatant was mixed with an appropriate volume of ultrapure water before LC-MS/MS analysis. [3]

Pharmacokinetics [3]
Study Design: A single intravenous (IV) dose was administered to the animal. Fasted animals were also dosed orally (PO). At the designated time points, blood was collected via the dorsal metatarsal vein in mice. Blood was transferred into collection tubes containing K2-EDTA. For plasma analysis, blood was immediately processed for plasma by centrifugation and stored in a freezer set to be maintained at approximately -80C until analysis.

Sample Preparation: The desired serial concentrations of working solutions were achieved by diluting stock solution (1 mg/mL in DMSO) of analyte with 50% acetonitrile in water. Ten microliters of working solutions were added to 10 μL of the blank animal plasma to achieve calibration standards of 0.5-1000 ng/mL in a total volume of 20 μL. The resulting 20 µL standard samples were added to 200 μL of acetonitrile for protein precipitation. All samples were then vortexed for 30 seconds. After centrifugation at 4°C and 4000 rpm (ca. 3740 x g) for 15 minutes, the supernatant was diluted with water.

Analytical Method: Concentrations in extracted samples were determined by liquid chromatography–tandem mass spectrometry (LC-MS/MS) using reversed-phase liquid chromatography. Analytes were monitored using Electron Spray Ionization (ESI) with multiple reaction monitoring in positive ion mode. Peak areas were integrated by Analyst® where concentrations were determined by a weighted (1/x2 ) linear or quadratic regression of peak area ratios (peak area of analyte/peak area of IS) versus the theoretical concentrations of the plasma calibration standards.

Pharmacokinetic Analysis: Individual plasma concentration-time data of mice were analyzed by non-compartmental methods using the Linear/Log trapezoidal method (IV) or the Linear-up/Log-down trapezoidal method (PO) (Phoenix WinNonlin 6.1, Certara, Princeton, NJ). After IV dosing, clearance (CL), steady-state volume of distribution (Vss), terminal elimination half-life (t½), area under the curve from time zero to infinity (AUCINF), mean resonance time from time zero to infinity (MRTINF), and terminal phase volume of distribution (Vz) were calculated. After PO dosing, maximum observed concentration (Cmax), time of Cmax (tmax), terminal elimination half-life (t½), area under the curve from time zero to infinity (AUCINF), mean resonance time from time zero to infinity (MRTINF), apparent total clearance (CL/F), bioavailability (%F), and estimated fraction absorbed (Fa) were calculated. [3]

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In vivo efficacy analysis in mice Animals were quarantined for 7 days before the study. The treatments were started for the efficacy study when the mean tumor volume reached about 119 mm3 . Based on the tumor volume, mice were randomly assigned to respective groups such that the mean starting tumor volume was the same between groups. EZM0414 dissolved in methyl cellulose/Tween-80 in water (0.5% CMC/0.1% Tween-80 (Sigma-Aldrich)), or vehicle alone was administered p.o. to NOD SCID mice (n = 10 per dose group). [3]
All study animals were monitored not only for tumor growth but also behavior such as mobility, food and water consumption, body weight, eye/hair matting, and any other abnormal effects. The measurement of tumor volume was conducted twice a week throughout the efficacy study with a caliper and the tumor volume (mm3 ) was estimated using the formula, Tumor Volume = a x b2 /2, where “a” and “b” are the long and short diameters of a tumor. The tumor volumes were used for calculating the tumor growth inhibition (TGI, an indicator of antitumor effectiveness) value using the formula: TGI = (1-T/C) × 100%, where “T” and “C” is the mean relative volumes (tumor growth) of the tumors in the treated (T) and the control (C) groups, respectively, on a given day after tumor inoculation.

While both EZM0414 and analogue 7 showed favorable PK profiles following 50 mg/kg po administration in mice, EZM0414 had ∼2-fold higher exposure (AUC) with better oral bioavailability (F) than 7, a trend that was observed in rats as well. Table 3 presents a comparison of the PK parameters for EZM0414, 7, and the starting parent compound 3. Improving the lipophilic efficiency of the earlier series vastly enhanced the overall PK profile, with a 17-fold increase in overall exposure levels (AUC0–∞) achieved in CD-1 mice at 50 mg/kg following oral administration with EZM0414. With regard to the potential for drug–drug interactions as perpetrator of CYP enzymes, EZM0414 exhibited only weak inhibition of CYP isoform 2C8 (4.8 μM), and no inhibition of other tested isoforms was seen (IC50 > 30 μM for isoforms 1A2, 2B6, 2C9, 2C19, 2D6, and 3A4). Analogue 7 showed modest inhibition of isoforms 2B6 (3.2 μM) and 2D6 (10.8 μM), with IC50 > 25 μM for the remaining isoforms tested. Additional characterization of EZM0414 indicated a favorable safety pharmacology profile. In vitro testing of EZM0414 in a safety panel consisting of 47 targets and a diversity panel of 72 kinases showed IC50 > 25 μM for all targets except D2 (IC50 = 13.0 μM, antagonist) and 5-HT1B (IC50 = 3.2 μM, agonist).[3]

[1]. Substituted indoles and methods of use thereof. WO2020037079A1.

SETD2 Inhibitor EZM0414 is an orally bioavailable selective inhibitor of the histone methyltransferase (HMT) SETD2 (SET domain containing 2, histone lysine methyltransferase), with potential antineoplastic activity. Upon oral administration, SETD2 inhibitor EZM0414 binds to SETD2 and inhibits its activity. This prevents several key biological processes that are mediated by SETD2, including the methylation of histones and non-histone proteins, transcriptional regulation, RNA splicing, DNA damage repair and B cell development and maturation. The inhibition of SETD2 by EZM0414 may inhibit tumor cell proliferation. SETD2 plays multiple important roles in oncogenesis.

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Introduction: SETD2 is the only known histone methyltransferase (HMT) capable of catalyzing H3K36 trimethylation (H3K36me3) in vivo. It plays an important role in several biological processes including B cell development and maturation, leading to the hypothesis that SETD2 inhibition in these settings could provide anti-tumor effects. The normal process of B cell development/maturation renders B cells susceptible to genetic vulnerabilities that can result in a dysregulated epigenome and tumorigenesis, including in multiple myeloma (MM) and diffuse large B-cell lymphoma (DLBCL). For example, 15%-20% of MM harbors the high risk (4;14) chromosomal translocation, resulting in high expression of the multiple myeloma SET domain (MMSET) gene. MMSET is an HMT that catalyzes H3K36me1 and H3K36me2 formation and extensive scientific work has established overexpressed MMSET as a key factor in t(4;14) myeloma pathogenesis. To the best of our knowledge MMSET has eluded drug discovery efforts, however, since t(4;14) results in high levels of the H3K36me2 substrate for SETD2, inhibiting SETD2 offers promise for targeting the underlying oncogenic mechanism driven by MMSET overexpression in t(4;14) MM patients. In addition, SETD2 loss of function mutations described to date in leukemia and DLBCL are always heterozygous, suggesting a haploinsufficient tumor suppressor role for SETD2. This observation points to a key role for SETD2 in leukemia and lymphoma biology and suggests that therapeutic potential of SETD2 inhibition may also exist in these or similar settings. EZM0414 is a first-in-class, potent, selective, orally bioavailable small molecule inhibitor of the enzymatic activity of SETD2. We explored the anti-tumor effects of SETD2 inhibition with EZM0414 in MM and DLBCL preclinical studies to validate its potential as a therapy in these tumor types.[2]

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Targeting SETD2 with a small molecule inhibitor results in significantly reduced growth of t(4;14) MM, as well as non-t(4;14) MM and DLBCL cell lines, in both in vitro and in vivo preclinical studies. In addition, in vitro synergy was observed with EZM0414 and certain SOC agents commonly used in MM and DLBCL, supporting the combination of SETD2 inhibition with current MM and DLBCL therapies. This work provides the rationale for targeting SETD2 in B cell malignancies such as MM, especially t(4;14) MM, as well as DLBCL, and forms the basis for conducting Phase 1/1b clinical studies to evaluate the safety and activity of EZM0414 in patients with R/R MM and DLBCL.[2]

C24H30F4N4O4

514.513020038605

514.22

2411759-92-5

EZM0414;2411748-50-8

155971202

White to light yellow solid powder

3

9

3

36

693

2

FC1=CC=C(C)C2=C1C=C(C(N[C@@H]1CCC[C@@H](C1)N1CCN(C(C)=O)CC1)=O)N2.FC(C(=O)O)(F)F

HASNOYOGMISGTP-PPPUBMIESA-N

InChI=1S/C22H29FN4O2.C2HF3O2/c1-14-6-7-19(23)18-13-20(25-21(14)18)22(29)24-16-4-3-5-17(12-16)27-10-8-26(9-11-27)15(2)28;3-2(4,5)1(6)7/h6-7,13,16-17,25H,3-5,8-12H2,1-2H3,(H,24,29);(H,6,7)/t16-,17+;/m1./s1

N-[(1R,3S)-3-(4-acetylpiperazin-1-yl)cyclohexyl]-4-fluoro-7-methyl-1H-indole-2-carboxamide;2,2,2-trifluoroacetic acid

SETD2-IN-1 TFA; 2411759-92-5; SETD2-IN-1 (TFA); EZM0414 (TFA); N-[(1R,3S)-3-(4-acetylpiperazin-1-yl)cyclohexyl]-4-fluoro-7-methyl-1H-indole-2-carboxamide;2,2,2-trifluoroacetic acid; EZM0414 TFA; CHEMBL5303391;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

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

DMSO : 200 mg/mL (388.72 mM)
H2O : 6.67 mg/mL (12.96 mM)

Solubility in Formulation 1: ≥ 5 mg/mL (9.72 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 50.0 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: ≥ 5 mg/mL (9.72 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 50.0 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: ≥ 5 mg/mL (9.72 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 50.0 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.)