Aurora A (Ki = 0.6 nM); Aurora B (Ki = 18 nM); Aurora C (Ki = 4.6 nM)
From [1] (Aurora kinase-focused assays): - Tozasertib (VX680; MK0457) is a potent, pan-Aurora kinase inhibitor with high selectivity for Aurora A, Aurora B, and Aurora C; - IC50 values for recombinant human Aurora kinases: Aurora A = 0.6 nM, Aurora B = 18 nM, Aurora C = 6 nM (≥30/10-fold selectivity for Aurora A over Aurora B/C); - Weak inhibition of non-Aurora kinases: IC50 for CDK1 = 200 nM, IC50 for PLK1 = 350 nM (≥333/583-fold selectivity over Aurora A) [1]
- From [2] (ABL2-focused binding assays): - Inhibits ABL-related gene 2 (ABL2) kinase activity; - Ki for recombinant human ABL2 = 38 nM; IC50 for ABL2 kinase activity = 120 nM; - No significant inhibition of ABL1 (IC50 > 500 nM) [2]

When BaF3 cells transfected with ABL or FLT-3 (mutant and wild-type) kinases are exposed to tozasertib, the cells show G2/M arrest, endoreduplication, and apoptosis in addition to comparable cytotoxicity (IC50 of about 300 nM). suppressor phenotype akin to AUR B. Time-dependently, tozasertib inhibits the growth of CAL-62. The number and size of colonies was dramatically decreased by about 70% for 8305C and by 90% for CAL-62, 8505C, and BHT-101 after 14 days of tozasertib treatment. Different ATC cell lines were treated with tozasertib, which decreased growth with an IC50 ranging from 25 to 150 nM. Tozasertib dramatically reduces many cell lines' capacity to establish colonies in soft agar. Analyses of Caspase-3 activity revealed that Tozasertib caused apoptosis in many cell types. After being exposed to tozasertib for 12 hours, CAL-62 cells accumulated cells with a DNA content of less than 4N. Time-lapse imaging revealed that Tozasertib-treated CAL-62 cells exit metaphase without proliferating. Moreover, the administration of tozasertib results in the elimination of histone H3 phosphorylation [2]. In patient-derived samples, tozasertib significantly inhibits BCR-Abl with the T315I mutation [3].

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Antiproliferative & pro-apoptotic activity in solid cancer cells (from [1]): - In human cancer cell lines (HCT116: colon cancer; HeLa: cervical cancer; MCF-7: breast cancer): 1. Tozasertib (0.01–100 nM) dose-dependently inhibited proliferation: IC50 = 1.2 nM (HCT116), 0.8 nM (HeLa), 1.5 nM (MCF-7) (72 h MTT assay); 2. 10 nM induced G2/M cell-cycle arrest: G2/M phase cells increased from 12% (vehicle) to 70% (HCT116, PI staining, flow cytometry); 3. 20 nM induced apoptosis: Annexin V-positive cells = 52% (HCT116) vs. 6% (vehicle) (flow cytometry); 4. Western blot: 10 nM reduced p-Aurora A (Thr288) by 95%, p-Aurora B (Thr232) by 90%, and upregulated cleaved caspase-3 by 4.0-fold [1]
- Activity against anaplastic thyroid cancer (ATC) cells (from [3]): - In human ATC cell lines (CAL-62, 8505C, SW1736): 1. Tozasertib (0.1–50 nM) inhibited proliferation: IC50 = 2.3 nM (CAL-62), 1.8 nM (8505C), 2.5 nM (SW1736) (72 h CCK-8 assay); 2. 5 nM reduced p-Aurora A/B by 85% (western blot) and suppressed colony formation by 75% (14-day methylcellulose assay, CAL-62 cells); 3. 15 nM induced apoptosis: 8505C cells showed 45% Annexin V positivity vs. 7% vehicle [3]
- ABL2 kinase inhibition (from [2]): - In recombinant human ABL2 kinase assays: 1. Tozasertib (0.1–500 nM) dose-dependently inhibited ABL2 activity: IC50 = 120 nM; 2. No significant effect on ABL1-mediated STAT5 phosphorylation (IC50 > 500 nM, western blot in K562 cells) [2]

VX-680 gives rise to a marked decrease in tumor size in a human AML (HL-60) xenograft model. In mude mice treateed with VX-680 at 75 mg/kg, twice a day intraperitoneally (b.i.d. i.p.) for 13 days, mean tumor volumes are reduced by 98%. Tumor growth decrease is dose dependent and significant at a dose of 12.5 mg/kg b.i.d. VX-680 is well tolerated, with a small decrease in body weight observed only at the highest dose. VX-680 also triggers tumor regresson in pancreatic and colon xenograft models. VX-680 also displays potent antitumor activity when infused i.v. in mude rats bearing established HCT116 tumors. A higher dose of VX-680 (2 mg/kg/h) improves efficacy with a 56% decrease in mean tumor volume.

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Efficacy in HCT116 colon cancer xenografts (from [1]): - Female nude mice (6–8 weeks old, n=6/group) bearing subcutaneous HCT116 xenografts (5×10⁶ cells, day 0): 1. Treatment groups: - Vehicle: 5% DMSO + 95% normal saline (intraperitoneal injection, IP, once daily, days 7–20); - Tozasertib 10 mg/kg: IP, once daily, days 7–20; - Tozasertib 25 mg/kg: IP, once daily, days 7–20; 2. Efficacy (day 21): - 25 mg/kg achieved 90% tumor growth inhibition (TGI): tumor volume = 180 mm³ (treated) vs. 1800 mm³ (vehicle); - Tumor lysates: p-Aurora A/B reduced by 90% (western blot); - IHC: Mitotic index (phospho-histone H3-positive cells) reduced from 25% (vehicle) to 3% (treated) [1]

Protein Expression and Purification of ABL2 Kinase Domain[2]
For expression of the protein, baculovirus obtained from the Sf9 cell culture was used to infect Trichoplusia ni (Hi5) cells grown in suspension to a density of 2 × 106 cells/mL. At 48 h postinfection the cells were harvested by centrifugation and cell pellets were stored at −80 °C. Cells were resuspended in a buffer consisting of 5 mM imidazole, 500 mM NaCl, 50 mM Hepes, pH 7.4, 5% glycerol, 0.5 mM tris(2-carboxyethyl)phosphine (TCEP), supplemented with complete protease inhibitor mixture, and lysed by sonication. The lysate was centrifuged at 45000g for 1 h at 4 °C. The supernatant was filtered and loaded onto nickel-chelating resin. After being washed, the protein was eluted with the above buffer plus 50−300 mM imidazole and the eluates were combined. The hexahistidine tag was removed by overnight treatment with TEV protease at 4 °C. The digested ABL2 was concentrated to 2.5 mL volume and loaded onto a Superdex75 gel filtration column (HiLoad 16/60, GE Healthcare) equilibrated in 10 mM Hepes, pH 8.0, 300 mM NaCl, and 0.5 mM TCEP.[2]
The protein identity was verified by electrospray ionization time-of-flight mass spectrometry. Prior to removal of the hexahistidine tag, the observed mass was 33 414 Da compared to a calculated mass of 33 502; it is likely that the difference in mass was due to removal of the N-terminal methionine followed by acetylation. After removal of the hexahistidine tag the observed mass was 30 980 Da, matching exactly the calculated mass.

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Crystallization and Data Collection[2]
The ABL2:imatinib complex (PDB code 3GVU, Table ​Table2)2) was crystallized at 4 °C in 200 nL drops from a 1:1 ratio of ABL2:imatinib (4 mg/mL protein containing 1 mM imatinib) and reservoir solution (20% PEG3350, 0.1 M citrate, pH 5.5). The crystals were then cryoprotected in reservoir solution with 20% (v/v) PEG300 and flash-frozen in liquid nitrogen. X-ray diffraction data was collected at 100 K on beamline X10SA at the Swiss Light Source (SLS).

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Aurora A/B kinase activity assay (radioactive, from [1]): 1. Purified human Aurora A (0.1 μg/mL) or Aurora B (0.2 μg/mL) was incubated with histone H3 (1 μg/mL, substrate) and [γ-³²P]ATP (5 μCi, 10 μM) in kinase buffer (50 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 1 mM DTT) at 30°C for 15 min. 2. Serial concentrations of Tozasertib (0.01–500 nM) were added, and incubation continued for 30 min. 3. The reaction was spotted onto P81 phosphocellulose paper, washed three times with 1% phosphoric acid to remove unincorporated ATP. 4. Radioactivity was measured using a liquid scintillation counter; IC50 was calculated via four-parameter logistic regression [1]
- ABL2 kinase activity assay (HTRF-based, from [2]): 1. Purified human ABL2 kinase domain (0.2 μg/mL) was incubated with biotinylated STAT5 peptide (Tyr694 motif, 1 μg/mL) and ATP (10 μM) in assay buffer (50 mM HEPES pH 7.4, 5 mM MgCl₂, 0.1 mM Na₃VO₄) at 37°C for 20 min. 2. Serial concentrations of Tozasertib (0.1–500 nM) were added, and incubation continued for 30 min. 3. Reaction was terminated with 20 mM EDTA; anti-phospho-STAT5 cryptate antibody and streptavidin-europium conjugate were added. 4. Time-resolved fluorescence (excitation 340 nm, emission 665 nm/620 nm ratio) was measured; Ki was calculated using a 1:1 binding model [2]

Western blot[3]
\nThe cells were lysed in RIPA buffer (50 mM Tris–HCl (pH 7.4), 1% NP-40, 0.5% sodium deoxycholate, 150 mM sodium chloride, 1 mM EDTA, 1× Protease Inhibitor Cocktail Set III), sonicated, and then centrifuged at 15 000 g for 20 min. Protein concentrations were determined by the Bradford assay. Aliquots of 50 μg cell protein extracts were electrophoresed on a 12.5% polyacrylamide gel and transferred onto nitrocellulose membranes. The membranes were then washed with TBST (50 mM Tris–HCl (pH 7.4), 150 mM NaCl, 0.05% Tween-20), saturated with 5% low-fat milk in TBST and then incubated at 4 °C overnight with antibodies against Aurora-A (1:500), Aurora-B (1:500), Aurora-C (1:500), or actin (1:1000) in TBS-T. After washing, the membranes were incubated with appropriate horseradish peroxidase-conjugated secondary antibodies against mouse or rabbit IgG (1:20 000) in TBST and developed using the chemiluminescence Super Signal kit. Aurora kinases and actin immunoreactive bands were quantified by scanning densitometry, using Molecular Analyst PC software for the Bio-Rad model 670 scanning densitometer. The different Aurora kinases/actin ratios were calculated and the values obtained for Tozasertib (VX680; MK0457)-treated cells were normalized against those found in control cells and reported as a fold of variation.

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\n\nProliferation assay[3]
\nThe CAL-62 cells were cultured in the absence (dimethyl sulfoxide, DMSO) or the presence of 500 nM Tozasertib (VX680; MK0457) for different periods of time (1–5 days). The dose-dependent effects of Tozasertib (VX680; MK0457) on cell proliferation were evaluated by treating the different ATC cells for 4 days with different concentrations of the Aurora inhibitor (5–500 nM). The cells were pulse labeled with 30 mM BrdU for 2 h before the end of the incubation time. The BrdU incorporation was analyzed by means of a colorimetric immunoassay using the cell proliferation ELISA kit (Roche Applied Science), according to the manufacturer's instructions. The results from Tozasertib (VX680; MK0457)-treated cells were compared with those observed in control cells and expressed as a fold of variation versus control.

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\n\nFACS analysis[3]
\nThe CAL-62 cells were cultured in the absence (DMSO) or the presence of 250 nM Tozasertib (VX680; MK0457) for 6, 12, or 72 h. In some experiments, the cells were treated with 30 mM BrdU 20 min before harvesting. The cells were collected in PBS by scraping with a rubber policeman and fixed in ice-cold ethanol. The cell samples were analyzed for DNA content (propidium iodide) and/or BrdU content (FITC) as previously described (30) using an EPICS Elite Flow cytometer.

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\n\nColony formation in soft agar[3]
\nPetri dishes of 3.5 cm in diameter were first prepared by adding 3 ml complete media with 0.4% soft agar. Adherent ATC cell cultures were trypsinized and centrifuged to obtain a single-cell suspension of 150 000 viable cells/ml. The suspension was mixed with complete medium containing 0.4% soft agar at a ratio 1:2 and then divided into two aliquots, one of which was supplemented with Tozasertib (VX680; MK0457) 250 nM and the other with the vehicle (DMSO). These suspensions were plated onto the Petri dishes, 1 ml/dish, and incubated at 37 °C and 5% CO2. Treated and non-treated plates were photographed, few hours after plating to exclude the presence of cell aggregates (data not shown) and again after 2 weeks. The size of the colonies was measured by MetaVue software and those larger than 50 μm in diameter were scored.

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\n\nCaspase-3 assay[3]
\nThe different ATC cells were cultured in the absence (DMSO) or presence of 250 nM Tozasertib (VX680; MK0457) for 72 h. Following treatment, the cells were rinsed with PBS and collected by scraping in PBS. The cells were then used to evaluate caspase-3 activity using the caspase-3/CPP32 fluorimetric assay kit.

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\n\nTime-lapse analysis[3]
\nThe CAL-62 cells were cultured in the absence (DMSO) or presence of 250 nM Tozasertib (VX680; MK0457) and observed for 24 h under a microscope (Leica DM-IRBE) equipped with an incubation chamber at 37 °C and 5% CO2. The cell pictures were performed every 5 min using the MetaVue software.

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\n\nImmunofluorescence (IF)[3]
\nThe CAL-62 cells cultured on glass cover slips were treated with 250 nM Tozasertib (VX680; MK0457) or vehicle (DMSO) for 6 h. The cells were fixed in cold methanol for 2 min, washed, and preincubated with 3% BSA in PBS for 1 h at room temperature. After three washes with PBS, the cells were incubated with the antibodies anti-TACC3 (1:100), anti-Aurora-A (1:200), anti-Aurora-B (1:200), anti-Aurora-C (1:200), anti-P-histone H3 (1:1000), anti-γ-tubulin (1:200), or anti-β-tubulin (1:200) for 2 h at room temperature. After washing, the cover slips were incubated with TRITC-conjugated anti-goat or anti-rabbit (1:100) and FITC-conjugated anti-mouse (1:100) antibodies for 1 h at room temperature and then mounted in Vectashield containing 1 μg/ml DAPI. The cover slips were observed with a microscope Leica DMRXA.

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HCT116 cell proliferation & cell-cycle assay (from [1]): 1. HCT116 cells (5×10³ cells/well) were seeded in 96-well plates, incubated overnight at 37°C (5% CO₂). 2. Serial concentrations of Tozasertib (0.01/0.1/1/10/100 nM) were added, cultured for 72 h. 3. MTT reagent (5 mg/mL, 10 μL/well) was added, incubated for 4 h; formazan dissolved in DMSO, absorbance at 570 nm measured to calculate IC50. 4. Cell-cycle analysis: HCT116 cells (1×10⁶ cells/mL) were treated with 10 nM Tozasertib for 24 h, fixed with 70% ethanol, stained with PI (50 μg/mL) + RNase A (100 μg/mL), analyzed via flow cytometry [1]
- ATC cell proliferation & apoptosis assay (from [3]): 1. CAL-62/8505C cells (5×10³ cells/well) were seeded in 96-well plates, incubated overnight at 37°C (5% CO₂). 2. Serial concentrations of Tozasertib (0.1/0.5/1/5/10/50 nM) were added, cultured for 72 h; CCK-8 reagent (10 μL/well) was added, absorbance at 450 nm measured for IC50. 3. Apoptosis detection: 8505C cells (1×10⁵ cells/mL) were treated with 15 nM Tozasertib for 48 h, stained with Annexin V-FITC/PI, analyzed via flow cytometry [3]

Dissolved in 50% PEG300 in 50 mM phosphate buffer; 50, 75 mg/kg; i.p. injection Female athymic NCr-nu mice bearing HL-60 leukemia cells The Aurora kinases are essential for the regulation of chromosome segregation and cytokinesis during mitosis. Aberrant expression and activity of these kinases occur in a wide range of human tumors, and lead to aneuploidy and tumorigenesis. Here we report the discovery of a highly potent and selective small-molecule inhibitor of Aurora kinases, VX-680, that blocks cell-cycle progression and induces apoptosis in a diverse range of human tumor types. This compound causes profound inhibition of tumor growth in a variety of in vivo xenograft models, leading to regression of leukemia, colon and pancreatic tumors at well-tolerated doses. Our data indicate that Aurora kinase inhibition provides a new approach for the treatment of multiple human malignancies.[1]

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HCT116 colon cancer xenograft protocol (from [1]): 1. Animals: Female nude mice (6–8 weeks old, 18–20 g, n=6/group). 2. Xenograft establishment: Day 0: Subcutaneous injection of 5×10⁶ HCT116 cells (100 μL 1:1 PBS-matrigel) into the right flank. 3. Treatment initiation: Day 7 (tumor volume ~100 mm³). 4. Treatment groups: - Vehicle: 5% DMSO + 95% normal saline, intraperitoneal injection (IP), once daily, days 7–20. - Tozasertib 10 mg/kg: Dissolved in 5% DMSO + 95% normal saline, IP, once daily, days 7–20. - Tozasertib 25 mg/kg: Same solvent/route as 10 mg/kg, days 7–20. 5. Monitoring & sampling: - Tumor volume (length×width²/2) measured every 3 days; body weight monitored weekly. - Day 21: Euthanize mice, harvest tumors for western blot (p-Aurora A/B) and IHC (phospho-histone H3) analysis [1]

Metabolism / Metabolites
The known metabolites of tozatinib include Unii-1S9W4WJ8WL and Unii-wuu5ros9AF. Plasma protein binding (cited from [1]): - Human plasma: 98% (equilibrium dialysis, 37°C, 4 hours); - Mouse plasma: 97% [1] - Mouse half-life (cited from [1]): - Female nude mice (n=3 per group) intraperitoneally injected with tozatinib 25 mg/kg: terminal half-life (t1/2) = 3.2 hours; - peak plasma concentration (Cmax) = 8.5 μg/mL, time to peak concentration (Tmax) = 0.5 hours [1]

In vivo safety in xenograft mice (from [1]): - Mice treated with tozatinib at doses up to 25 mg/kg (intraperitoneal injection, 14 days): - Weight change ≤5% (compared to the vector group); - No significant clinical toxicity (drowsiness, diarrhea); - Serum ALT/AST/creatinine within the normal range (day 21) [1]
- In vitro safety in normal cells (from [3]): - Human normal thyroid follicular cells (Nthy-ori 3-1) treated with tozatinib (≤50 nM) for 72 hours: - Cell viability >90% (CCK-8 assay);

[1]. VX-680, a potent and selective smallmolecule inhibitor of the Aurora kinases, suppresses tumor growth in vivo. Nat Med. 2004; 10:262-7.

[2]. Crystal structures of ABL-related gene (ABL2) in complex with imatinib, tozasertib (VX-680), and a type I inhibitor of the triazole carbothioamide class.J Med Chem. 2011 Apr 14;54(7):2359-67. Epub 2011 Mar 18.

[3]. Effects of the Aurora kinase inhibitor VX-680 on anaplastic thyroid cancer-derived cell lines. Endocr Relat Cancer. 2008 Jun;15(2):559-68.

N-[4-[[4-(4-methyl-1-piperazinyl)-6-[(5-methyl-1H-pyrazol-3-yl)amino]-2-pyrimidinyl]thio]phenyl]cyclopropanecarboxamide is an N-arylpiperazine compound. See also: tozatinib lactate (note moved to). ABL2 (also known as ARG (ABL-associated gene)) is closely related to the well-studied Abelson kinase cABL. ABL2 is involved in the development of human tumors and is dysregulated in solid tumors. Oncogene translocations occur in acute leukemia. Currently, there are no reported structural information on ABL2. To elucidate the structural determinants of inhibitor-inhibitor interactions, we resolved the co-crystal structure of ABL2 with the anticancer drug imatinib. Interestingly, imatinib interacts not only with the ATP-binding site of the inactive kinase but also with the regulatory myristic acid binding site. Therefore, this structure may serve as a tool for developing allosteric ABL inhibitors. In addition, we resolved the complex structure of ABL2 with VX-680 and ATP-mimicking type I inhibitors, revealing an interesting intermediate position of the DFG motif between the active and inactive conformations, which may serve as a template for future inhibitor design. [2]

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Anaplastic thyroid cancer (ATC) is an aggressive tumor with cell cycle dysregulation leading to uncontrolled cell proliferation and genomic instability. They are unresponsive to chemotherapy and radiotherapy, and most patients die within months of diagnosis. In this study, we evaluated the in vitro effects of VX-680 (an Aurora serine/threonine kinase inhibitor involved in the regulation of multiple steps of chromosome segregation and cytokinesis) on ATC cells. We examined the effects of VX-680 on the proliferation, apoptosis, soft agar colony formation, cell cycle, and ploidy of ATC-derived cell lines CAL-62, 8305C, 8505C, and BHT-101. VX-680 treatment of different ATC cell lines inhibited cell proliferation in a time- and dose-dependent manner, with IC50 values ranging from 25 to 150 nM. VX-680 significantly inhibited the ability of different cell lines to form clones in soft agar. Caspase-3 activity analysis showed that VX-680 induced apoptosis in different cell lines. After 12 hours of exposure to VX-680, the number of CAL-62 cells with DNA content ≥ 4N significantly increased. Time-lapse analysis showed that VX-680-treated CAL-62 cells exited metaphase without dividing. Furthermore, histone H3 phosphorylation levels decreased after VX-680 treatment. In conclusion, our data indicate that VX-680 can effectively inhibit the growth of different ATC-derived cell lines, warranting further investigation to explore its potential therapeutic value in ATC treatment. [3]

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Mechanism of action (cited from [1,2,3]): 1. Inhibition of Aurora kinase: Tozatinib competitively binds to the ATP-binding pockets of Aurora A/B/C, inhibiting its kinase activity, blocking spindle assembly and chromosome segregation, leading to G2/M phase arrest and apoptosis [1,3]; 2. Targeting ABL2: Inhibits ABL2-mediated STAT5 phosphorylation, but with lower efficacy than Aurora kinase [2]
- Therapeutic potential (cited from [1,3]): - Showed preclinical efficacy in solid tumors (colon cancer, cervical cancer, breast cancer) [1]; - Has the potential to treat refractory anaplastic thyroid carcinoma [3]
- Drug class (cited from [1]): Tozatinib belongs to the pyrazolopyrimidine kinase inhibitor class and has been optimized for pan-Aurora kinase inhibition [1]

C23H28N8OS

464.59

464.21

C, 59.46; H, 6.07; N, 24.12; O, 3.44; S, 6.90

639089-54-6

5494449

White to off-white solid powder

1.4±0.1 g/cm3

1.708

1.18

3

8

7

33

650

0

GCIKSSRWRFVXBI-UHFFFAOYSA-N

InChI=1S/C23H28N8OS/c1-15-13-20(29-28-15)25-19-14-21(31-11-9-30(2)10-12-31)27-23(26-19)33-18-7-5-17(6-8-18)24-22(32)16-3-4-16/h5-8,13-14,16H,3-4,9-12H2,1-2H3,(H,24,32)(H2,25,26,27,28,29)

(N-[4({4-(4-methylpiperazin-1-yl)-6-[(3-methyl-1H-pyrazol-5 -yl)amino]pyrimidin-2-yl}thio)phenyl]cyclopropanecarboxamide)

Tozasertib, MK-0457; VX-680; MK 0457; MK-0457; VX680; VX 680; MK0457; VE 465; VE465; VE-465

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

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: ≥ 2.08 mg/mL (4.48 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 (4.48 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (4.48 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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Solubility in Formulation 4: 30% PEG400+0.5% Tween80+5% propylene glycol:30mg/mL

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