MEK1 (IC50 = 14 nM); MEK1 (Kd = 99 nM); MEK2 (Kd = 530 nM)
MEK1/2 (IC50 = 14 nmol/L against purified MEK1 [3])
MEK2 (similar inhibition as MEK1, data not shown [3])

NVP-BHG712 dose-dependently inhibited the autophosphorylation of EphRs in Hek293 cells transfected with various EphRs. When compared to EphB2, EphA2, EphB3, and NVP-BHG712, EphB4 exhibited a stronger inhibitory preference. AZD6244 inhibits the phosphorylation of ERK1/2 at IC50 concentrations below 40 nM and is not ATP-competitive. Inhibiting ERK1/2 and p90RSK phosphorylation, as well as elevating caspase-3 and caspase-7 cleavage and cleaved poly(ADP)ribose polymerase, all contribute to AZD6244's ability to stop the growth of primary HCC cells. Phosphatidylinositol 3-kinase, MEK5/ERK5 and p38 pathways are not significantly affected by AZD6244. Ras mutations and Raf mutations both affect AZD6244's sensitivity to breast cancer cell lines and NSCLC cell lines, respectively.

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In primary HCC cells (2-1318, 4-1318, 26-1004), Selumetinib sulfate (AZD6244 sulfate) caused a time- and dose-dependent reduction in DNA synthesis (BrdU incorporation) and cell viability (MTT assay), with 50% reduction in viability at <0.5 μmol/L after 48 h. It dose-dependently elevated phosphorylated MEK1/2 (Ser217/221 and Ser218/222) and B-Raf, while reducing ERK1/2 and p90RSK (Ser359/363) phosphorylation. Cleaved PARP (89 kDa), cleaved caspase-3, and cleaved caspase-7 were detected from 0.5 μmol/L. Apoptosis (TUNEL) increased dose-dependently (P<0.01). At 1 μmol/L, phosphorylated ERK1/2 decreased at 3 h and was barely detectable at 6 h; p90RSK phosphorylation inhibited at 6 h; cleaved caspases and PARP detected at 6 h and max at 12 h. Z-VAD-FMK blocked AZD6244-induced apoptosis and ERK1/2 activation [1].
In 31 breast cancer cell lines, 5 had IC50 <1 μM (sensitive); sensitivity correlated with BRAF mutations (p=0.022) and non-luminal subtypes. In 43 NSCLC cell lines, 15 had IC50 <1 μM; sensitivity correlated with KRAS/NRAS mutations (p=0.045). Western blot of 27 NSCLC lines showed that 1 μM Selumetinib sulfate (AZD6244 sulfate) nearly eliminated pERK in all lines regardless of sensitivity, with no change in pAKT. G0/G1 arrest was seen in sensitive but not resistant cell lines. Baseline gene expression analysis identified PIK3R3 higher in resistant breast cancer lines (p<0.05) and FAM77C, THC1981357, MSRA differentially expressed in both histologies [2].
In Malme-3M, A431, and other cell lines, Selumetinib sulfate (AZD6244 sulfate) inhibited basal and EGF-induced ERK1/2 phosphorylation with IC50 <40 nmol/L; in Malme-3M, IC50 for pERK was 10.3 nmol/L. It inhibited growth of B-Raf mutant (HT-29, Malme-3M, SK-MEL-28) and Ras mutant (MIA PaCa-2, SK-MEL-2) cells with IC50 59-473 nmol/L, but had minimal effect on normal fibroblast Malme-3 or wild-type breast cancer cells (IC50 >50 μmol/L). It did not inhibit ERK5 phosphorylation up to 10 μmol/L. In HT-29 and Malme-3M, 24 h treatment induced G1-S cell cycle arrest; in Malme-3M and SK-MEL-2 (N-Ras mutant), it activated caspase-3/7, but not in HT-29 or SK-MEL-28 [3].

AZD6244 significantly inhibits phosphorylation of ERK1/2 in 2-1318, 5-1318, 26-1004 and 4-1318 xenografts and induces apoptosis in primary 2-1318 cells by activating the caspase pathway. At a dose of 100 mg/kg, AZD6244 could slow the growth of the tumor in the HT-29 xenograft, a colorectal tumor model with a B-Raf mutation; this tumor growth inhibition is superior to that of Gemcitabine. Apoptosis and the down-regulation of cell cycle regulators like cyclin D1, Cdc-2, CDK2 and 4, cyclin B1, and c-Myc are associated with increased apoptosis, which is why AZD6244 could inhibit the growth of HCC xenograft tumors in the absence of these factors.

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In seven HCC xenografts (2-1318, 4-1318, 5-1318, 26-1004, 30-1004, 29-1104, 2006), growth rate positively correlated with phosphorylated MEK1 (Ser218/222 and Thr286) levels. Oral administration of Selumetinib sulfate (AZD6244 sulfate) at 50 mg/kg (BID) or 100 mg/kg for 21 days dose-dependently suppressed tumor growth in 4-1318 (treated/control ratio 0.41 and 0.18 respectively). At 50 mg/kg, growth suppression was also significant in 2-1318 (0.50), 26-1004 (0.31), 5-1318 (0.64), and 29-1104 (0.70), with higher sensitivity in high pMEK lines (P<0.05). AZD6244 inhibited pERK1/2 and MEK kinase activity, elevated B-Raf and pMEK (Ser218/222), reduced p90RSK (Ser380 and Thr359/363), and increased cleaved PARP, caspase-3, and caspase-7 in tumors. Ki-67 labeling index decreased from 14.4% to 4.3%, and cleaved caspase-3 positive cells increased from 0.1% to 4.5% [1].
In HT-29 (B-Raf mutant) xenografts, oral Selumetinib sulfate (AZD6244 sulfate) at 10, 25, 50, 100 mg/kg BID for 21 days inhibited tumor growth dose-dependently (55% at 10 mg/kg, 70% at 100 mg/kg by day 11). Tumor regrowth resumed after dosing cessation; time to 1000 mm³ was 36 days for high doses vs 18 days for vehicle (P<0.01). pERK1/2 was inhibited in treated tumors. In BxPC3 (pancreatic) xenografts, 10, 25, 50 mg/kg BID for 18 days induced tumor regressions (6/10 at 50 mg/kg) with 94% growth inhibition, superior to gemcitabine. pERK was inhibited. In a second BxPC3 study, 21-day treatment (25 or 50 mg/kg BID) followed by 7-day holiday and then another 21-day cycle showed that tumors remained responsive to re-treatment, with regressions seen in both cycles [3].

MEK1. [3]
NH2-terminal hexahistidine tagged, constitutively active MEK1 (S218D, S222D ΔR4F; ref. 18) was expressed in baculovirus-infected Hi5 insect cells and purified by immobilized metal affinity chromatography, ion exchange, and gel filtration. The activity of MEK1 was assessed by measuring the incorporation of [γ-33P]phosphate from [γ-33P]ATP onto ERK2. The assay was carried out in a 96-well polypropylene plate with an incubation mixture (100 μL) composed of 25 mmol/L HEPES (pH 7.4), 10 mmol/L MgCl2, 5 mmol/L β-glycerolphosphate, 100 μmol/L sodium orthovanadate, 5 mmol/L DTT, 5 nmol/L MEK1, 1 μmol/L ERK2, and 0 to 80 nmol/L compound (final concentration of 1% DMSO). The reactions were initiated by the addition of 10 μmol/L ATP (with 0.5 μC k[γ-33P]ATP/well) and incubated at room temperature for 45 min. An equal volume of 25% trichloracetic acid was added to stop the reaction and precipitate the proteins. Precipitated proteins were trapped onto glass fiber B filter plates, excess labeled ATP was washed off with 0.5% phosphoric acid, and radioactivity was counted in a liquid scintillation counter. ATP dependence was determined by varying the amount of ATP in the reaction mixture. The data were globally fitted using SigmaPlot. Values were calculated using the following equation for noncompetitive inhibition: v = [Vmax × S / (1 + I / Ki)] / (Km + S).[3]

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ERK2. [3]
To measure inhibition of ERK2, the kinase activity of ERK2 was first activated by MEK1. Wild-type (WT) ERK2 containing an NH2-terminal hexahistidine tag was overexpressed in Escherichia coli and purified by immobilized metal affinity chromatography, ion exchange, and gel filtration. To activate WT ERK2, 2 mg WT ERK2 was mixed with 17 μg of constitutively active MEK1 in 4 mL of 25 mmol/L HEPES (pH 7.5) containing 1 mmol/L ATP. The reaction mixture was incubated at room temperature for 40 min, and the addition of two phosphates was confirmed by mass spectrometry. Activated WT ERK2 was further purified by ion exchange. ERK2 activity was assayed as described for constitutively active MEK, using 10 nmol/L activated ERK2. The substrate used was myelin basic protein at a concentration of 1 μmol/L.[3]

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MEK1 molecules are immunoprecipitated using an anti-MEK1 antibody. When recombinant ERK1 is activated by immuno-isolated MEK1 in a coupled assay with MBP as the end point, MEK kinase activity is calculated. Before being exposed to X-ray film, phosphorylated MBP is resolved on a 14% SDS-PAGE gel and vacuum-dried.

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MEK1 enzymatic activity assay: Constitutively active MEK1 (S218D, S222D) was incubated with Selumetinib sulfate (AZD6244 sulfate) (0-80 nmol/L), ERK2, and [γ-33P]ATP in buffer (25 mmol/L HEPES pH 7.4, 10 mmol/L MgCl2, 5 mmol/L β-glycerolphosphate, 100 μmol/L sodium orthovanadate, 5 mmol/L DTT). Reaction initiated by 10 μmol/L ATP (0.5 μCi/well), incubated 45 min at room temperature, stopped with 25% trichloroacetic acid, precipitated proteins trapped on glass fiber B filter, washed with 0.5% phosphoric acid, and counted. IC50 = 14.1 ± 0.79 nmol/L. ATP dependence: varying ATP concentrations showed noncompetitive inhibition (Km ATP = 14.9 μmol/L, Ki = 22.6 nmol/L) [3].
MEK kinase activity assay (from HCC xenografts): Anti-MEK1 antibody immunoprecipitated MEK1; kinase activity measured by ability to activate recombinant ERK1 in a coupled assay using myelin basic protein (MBP) as end point. Phosphorylated MBP resolved on 14% SDS-PAGE and visualized by autoradiography [1].
Other kinase assays: p38α, MKK6, EGFR, ErbB2, B-Raf (V600E), and over 40 other kinases were tested using standard protocols (radioactive, TR-FRET, or ELISA based). No inhibition was observed at 10 μmol/L Selumetinib sulfate (AZD6244 sulfate) against any of these kinases, including p38α (88% control), MKK6 (100%), EGFR (90%), ErbB2 (100%), ERK2 (100%), B-Raf (100%), CDK2/cyclinA (93%), c-Raf (98%), c-Src (95%), insulin receptor kinase (103%), JNK2α2 (100%), MAPKAP-K2 (101%), PDGFRα (99%), PKBα (97%), PKCα (93%) [3].

Cell Viability and Cell Proliferation[1]
Primary HCC cells were plated at a density of 2.0 × 104 per well in growth medium. After 48 h in growth medium, the cell monolayer was rinsed twice with MEM. Cells were treated with various concentrations of AZD6244 (0, 0.5, 1.0, 2.0, 3.0, and 4.0 μmol/L) for 24 or 48 h. Cell viability was determined by the 3-(4,5-dimethylthiazol-2y1)-2,5-diphenyltetrazolium bromide (MTT) assay (32). Cell proliferation was assayed using a bromodeoxyuridine kit (Roche) as described by the manufacturer. Experiments were repeated at least thrice, and the data were expressed as mean ± SE.[1]

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Detection of Apoptosis[1]
Primary HCC cells were grown in eight-chamber slides and treated with 0, 0.5, 1.0, 2.0, 3.0, and 4.0 μmol/L of AZD6244 in SRF medium for 24 h. Cells were fixed with PBS containing 4% formalin solution for 1 h at room temperature and washed with PBS. Apoptosis was detected by the terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling (TUNEL) assay using the In situ Cell Death Detection kit (Roche) as described by the manufacturer. Apoptotic cells were then visualized under a fluorescent microscope equipped with a FITC filter. The labeling index was obtained by counting the number of positive cells among 500 cells per region. They were expressed as percentage values.[1]

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At a density of2.0 × 104, cells are seeded. The cells undergo two culture media rinses after 48 hours of incubation. AZD6244 is used to treat cells for 24 or 48 hours at various concentrations. The MTT assay uses 3-(4,5-dimethylthiazol-2y1)-2,5-diphenyltetrazolium bromide to measure the viability of cells. With the help of a bromodeoxyuridine kit, cell proliferation is measured.

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Isolation of primary HCC cells: Tumors minced, washed with MEM, incubated with MEM containing 5% FBS and 5 mg/mL collagenase at 37°C for 12 h, centrifuged (800×g, 10 min), washed, and grown in MEM+10% FBS. Cell viability: MTT assay; cells plated at 2×10⁴/well, treated with Selumetinib sulfate (AZD6244 sulfate) (0-4 μmol/L) for 24 or 48 h. Cell proliferation: BrdU ELISA. Apoptosis: TUNEL assay using In situ Cell Death Detection kit; cells grown in chamber slides, treated with compound for 24 h, fixed in 4% formalin, stained, and visualized under fluorescence microscope. Western blot: Cells/tissues lysed, blotted with antibodies against cleaved caspase-3/7, pMEK, pERK, p90RSK, cleaved PARP, etc. [1].
Breast and NSCLC cell line proliferation assay: Cells plated in 24-well plates at 5×10⁴ to 1×10⁵ per well, treated with Selumetinib sulfate (AZD6244 sulfate) (10 μmol/L down to 1 nmol/L) for 6 days, harvested by trypsinization, counted using Coulter counter. Percent inhibition = 1 - (treated/control). IC50 calculated by linear regression. Cell cycle analysis: Cells treated with 1 μmol/L selumetinib for 48 h, stained with Nim-Dapi, analyzed by flow cytometry (UV). Western blot: Cells grown to log phase, treated with 1 μmol/L compound for 30 min, lysed, proteins resolved by SDS-PAGE, transferred to nitrocellulose, probed with antibodies against total ERK, pERK (Thr202/Tyr204), total AKT, pAKT (Ser308 and Ser473), and tubulin [2].
Cellular ERK1/2 phosphorylation assay: Cells grown in 24-well plates, treated with Selumetinib sulfate (AZD6244 sulfate) for 1 h, stimulated with 100 ng/mL EGF for 5 min where indicated, lysed in RIPA buffer, Western blotted with anti-pERK, stripped and reprobed with anti-ERK1/2. For 96-well format: Malme-3M cells treated, fixed with 3.7% formaldehyde, permeabilized with methanol, stained with anti-pERK and anti-total ERK, followed by fluorescent secondary antibodies, quantified by LI-COR imager (IC50 = 10.3 nmol/L). Whole blood assay: Human whole blood treated with compound for 1 h at 37°C, then 400 nmol/L TPA for 10 min, fixed with 2% formaldehyde, PBMCs isolated by Ficoll, stained with anti-pERK and FITC-secondary, analyzed by flow cytometry. At 100 nmol/L compound, % cells with pERK reduced from 74% to 34%; at 1 μmol/L nearly blocked [3].

HCC xenografts in mice homozygous for the SCID (severe combined immunodeficiency) mutation
50 or 100mg/kg
Administered via p.o.

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o investigate the effects of AZD6244 on HCC xenografts, AZD6244 was suspended in water at an appropriate concentration. Mice bearing HCC xenografts were p.o. given, twice a day, with either 100 μL of water (n = 12) or 50 mg (n = 12) or 100 mg (n = 12) of AZD6244 per kilogram of body weight for 21 days, starting from day 7 after tumor implantation. Growth of established tumor xenografts was monitored at least twice weekly by Vernier caliper measurement of the length (a) and width (b) of the tumor. Tumor volume was calculated as (a × b2)/2. Animals were sacrificed 3 h after the last dose of ADZ6244, and body and tumor weights were recorded, with the tumors harvested for analysis.[1]

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To study the effects of AZD6244 on caspase-3 activation and MEK1/2 phosphorylation, mice bearing HCC tumors (∼800 mm3) were treated with vehicle (n = 4) or 50 mg of AZD6244 per kilogram of body weight (n = 4) for 3 days as described above. Animals were sacrificed 3 h after the last dose, and tumors were harvested and frozen in liquid nitrogen for later analysis. Part of the tumor harvest was fixed in neutral buffer containing 10% formalin for immunohistochemistry.[1]

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HT-29 human colon carcinoma or BxPC3 human pancreatic tumor fragments were implanted s.c. in the flank of nude mice and allowed to grow to 100 to 150 mg. Mice (n = 10 per group) were randomized to treatment groups to receive vehicle (10 mL/kg and 10% ethanol/10% cremophor EL/80% D5W) or AZD6244/ARRY-142886 (10, 25, 50, or 100 mg/kg, oral, BID) on days 1 to 21. Tumors [(W2 × L) / L] were measured twice weekly. [3]

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HCC xenograft model: Male SCID mice (homozygous for SCID mutation) implanted subcutaneously with minced tumor fragments (passed through 18-gauge needle) mixed with RPMI 1640. Treatment started on day 7 post-implantation. Selumetinib sulfate (AZD6244 sulfate) suspended in water; mice given orally (p.o.) twice daily (BID) with either water (vehicle), 50 mg/kg, or 100 mg/kg for 21 days. Tumor volume measured twice weekly by caliper (length a, width b; volume = a×b²/2). Animals sacrificed 3 h after last dose; tumors harvested for analysis [1].
HT-29 and BxPC3 xenograft models: Nude mice implanted subcutaneously with tumor fragments (~100-150 mg). Randomized to treatment groups (n=10). Vehicle: 10% ethanol / 10% cremophor EL / 80% D5W, 10 mL/kg. Selumetinib sulfate (AZD6244 sulfate) given orally BID at 10, 25, 50, or 100 mg/kg for 21 days (or 18 days for BxPC3 due to control tumor growth). Tumor measurements twice weekly. For HT-29, tumor regrowth monitored until 1000 mm³. For BxPC3, gemcitabine control: 160 mg/kg i.p. every 3rd day for 4 doses. In a second BxPC3 study, mice treated for 21 days, then 7-day dosing holiday, then another 21-day cycle. Four hours after last dose, tumors excised for pERK analysis [3].

At 100 mg/kg dose of Selumetinib sulfate (AZD6244 sulfate) in HCC xenograft-bearing mice, a ~10% decrease in body weight was observed, suggesting significant toxicity. At 50 mg/kg, no overt toxicity as measured by weight loss or morbidity was observed, indicating good safety [1].
In HT-29 and BxPC3 xenograft studies, no significant toxicity was reported; the compound was tolerated at doses up to 100 mg/kg BID [3].

[1]. Targeted inhibition of the extracellular signal-regulated kinase kinase pathway with AZD6244 (ARRY-142886) in the treatment of hepatocellular carcinoma. Mol Cancer Therapy, 2007, 6 (1), 138-146 .

[2]. Identification of common predictive markers of in vitro response to the Mek inhibitor selumetinib (AZD6244; ARRY-142886) in human breast cancer and non-small cell lung cancer cell lines. Mol Cancer Thera, 2010, 9 (7), 1985-1994.

[3]. Biological characterization of ARRY-142886 (AZD6244), a potent, highly selective mitogen-activated protein kinase kinase 1/2 inhibitor. Clin Cancer Res, 2007, 13 (5), 1576-1583.

Cerumetinib sulfate is the sulfate salt of cerumetinib, an orally effective small molecule drug with potential antitumor activity. Cerumetinib is an ATP-independent inhibitor of mitogen-activated protein kinase kinases (MEK or MAPK/ERK kinases) 1 and 2. MEK1 and 2 are bispecific kinases and key mediators of RAS/RAF/MEK/ERK pathway activation, often highly expressed in various cancer cells, and drive various cellular responses, including proliferation. Cerumetinib inhibits the proliferation of cells in various cancers by inhibiting MEK1 and MEK2, preventing the activation of MEK1/2-dependent effector proteins and transcription factors.
See also: Cerumetinib (containing the active ingredient).
Drug Indications
Koselugo monotherapy is indicated for the treatment of symptomatic, unresectable plexiform neurofibromas (PN) in children aged 3 years and older with neurofibromatosis type 1 (NF1).

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The Ras-Raf-MEK-ERK pathway is constitutively active in many human cancers, including HCC, colorectal, pancreatic, lung, breast, and melanoma. Selumetinib sulfate (AZD6244 sulfate) targets MEK1/2, blocking ERK1/2 phosphorylation and downstream signaling, leading to growth arrest and apoptosis. In HCC, MEK1/2 overexpression and phosphorylation are common; AZD6244-induced growth suppression correlates with inhibition of ERK and p90RSK, and activation of caspases. In breast and NSCLC, sensitivity is associated with BRAF mutations (breast) and KRAS/NRAS mutations (NSCLC). AZD6244 has shown better efficacy than the first-generation MEK inhibitor CI-1040 in preclinical models and is currently in phase I/II clinical trials for various solid tumors [1][2][3].

C₁₇H₁₇BRCLFN₄O₇S

555.76

553.967

943332-08-9

Selumetinib;606143-52-6

16214875

Light yellow to yellow solid

4.601

5

10

6

32

604

0

O=C(C1C(NC2C(Cl)=CC(Br)=CC=2)=C(F)C2N=CN(C=2C=1)C)NOCCO.O=S(O)(O)=O

GRKFGZYYYYISDX-UHFFFAOYSA-N

InChI=1S/C17H15BrClFN4O3.H2O4S/c1-24-8-21-16-13(24)7-10(17(26)23-27-5-4-25)15(14(16)20)22-12-3-2-9(18)6-11(12)19;1-5(2,3)4/h2-3,6-8,22,25H,4-5H2,1H3,(H,23,26);(H2,1,2,3,4)

6-(4-bromo-2-chloroanilino)-7-fluoro-N-(2-hydroxyethoxy)-3-methylbenzimidazole-5-carboxamide;sulfuric acid

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.50 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (4.50 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 3: ≥ 2.5 mg/mL (4.50 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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Solubility in Formulation 4: 4% DMSO+30% PEG 300+5% Tween 80+ddH2O: 5mg/mL

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