Activin receptor-like kinase 5 (ALK5) (IC50 = 12.9 nM)

Vactosertib (10-1000 nM; 30 minutes; 4T1 cells) treatment suppresses TGFβ-induced Smad2 or Smad3 phosphorylation in 4T1 cells in a dose-dependent manner [1]. In 4T1 and MCF10A cells, vactosertib prevents TGFβ-induced Smad2/3 nuclear translocation. In 4T1 cells, Vactosertib's IC50 value against pSmad3 is 10–30 nM[1]. TGFb1-induced tumor cell migration and invasion can be stopped by vactosertib [1]. TGFβ1 upregulates FN1, HMGA2 (high mobility group AT-hook 2), SNAI1 and SNAI2 (Snail family zinc fingers 1 and 2, respectively) and downregulates CDH1 mRNA levels. Furthermore, TGFβ1-induced effects on genes associated to the epithelial-mesenchymal transition (EMT) were eliminated by vactosertib [1].

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Novel ALK5 kinase inhibitor EW‐7197 suppresses growth of CML‐LICs in vitro. EW‐7197 suppresses in vitro colony‐forming capacity of human CML‐LICs [2].

MMTV/c-Neu female mice treated with vabotosertib (40 mg/kg; i.p.; every other day; for 10 weeks) are less likely to exhibit lung metastases, cell migration, invasion, and Smad/TGFβ signaling [1]. Additionally, in 4T1 orthotopically transplanted mice and in breast cancer cells treated with TGFβ, vactosertib suppresses the epithelial-to-mesenchymal transition (EMT). Furthermore, in 4T1 orthotopic transplanted mice, vactosertib can increase the activity of cytotoxic T cells and extend the survival period of 4T1-Luc and 4T1 breast cancer tumor-bearing animals [1].

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Prolonged survival of CML‐affected mice treated with a combination of TKI plus EW‐7197. Eradication of murine CML‐MPPs in vivo by a combination of TKI plus EW‐7197. Suppression of disease recurrence in TKI‐resistant CML‐affected mice by treatment with EW‐7197 plus ponatinib.[2]

Protein kinase assay [1]
Selectivity profiling of Vactosertib (EW7197) against 320 protein kinases was performed at doses of 1 × 10−8, 1 × 10−7, 1 × 10−6, and 1 × 10−5 mol/L with a radiometric protein kinase assay provided by ProQinase. Detailed information is described in the Supplementary Materials and Methods.

Western Blot Analysis[1]
Cell Types: 4T1 cells
Tested Concentrations: 10 nM, 30 µM, 50 nM, 100 µM, 300 nM, 500 nM, 1000 nM
Incubation Duration: 30 minutes
Experimental Results: Blocked the TGFb-induced phosphorylation of Smad2 or Smad3 in a dose-dependent manner.

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Western blot analysis [1]
Mouse tissues or cells were homogenized in RIPA buffer. Lysates containing 4 to 50 μg of total protein were separated by electrophoresis on polyacrylamide gels and then transferred electrophoretically to a polyvinylidene difluoride transfer membrane. The membrane was blocked with 5% bovine serum albumi and incubated overnight at 4°C with the indicated primary antibodies (Supplementary Table S1). The membrane was then incubated with horseradish peroxidase–conjugated secondary antibodies. Bound antibodies were detected with the Western Blotting Luminol Reagent.

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Wound-healing assay [1]
4T1 cells and MDA-MB-231 cells were seeded in 6-well plates. When more than 80% of the area of each well was occupied by cells, 10% HI-FBS medium was changed to 0.2% HI-FBS medium. After 24 hours, a “wound” was made by scraping with a plastic pipette tip (time = 0), and the cells were treated with TGFβ1 (2 ng/mL) with or without ALK5 inhibitors for 24 hours (4T1) or 53 hours (MDA-MB-231). The wound area at zero time or the end point was measured using the ImageJ program according to phase-contrast images of the cells captured with a camera attached to a microscope. The closure of the wound area was calculated as a percentage of the initial wound area.

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Matrigel invasion assay [1]
The upper surface of a Transwell (6.5-mm diameter and 8-μm pore size; Corning) was coated with 20 μL of diluted 33.3% Matrigel. 4T1 cells were seeded at 4 × 104 cells per well in the upper chamber of the Transwell in serum-free medium with or without TGFβ1 (2 ng/mL) in the presence or absence of ALK5 inhibitors. The lower chamber was filled with the same medium as in the upper chamber but with 10% HI-FBS. After incubation for 20 hours, the cells remaining on the upper surface of the membrane were removed with a cotton swab, and the DAPI-stained cells remaining on the bottom surface were observed using fluorescence microscopy. The average cell number per field of view was obtained from five random fields.

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Colony‐forming capacity [2]
To determine colony‐forming capacity after a combination treatment of Vactosertib (EW7197) plus IM, or EW‐7197 plus ponatinib, we cocultured freshly isolated CML‐LICs on OP‐9 stromal cells in the presence of DMSO or EW‐7197 for 24 h.18 Cells were then treated with additional DMSO, 1 μM IM, or 1 μM ponatinib (AP24534) and cultured for another 2 days (total, 3 days). Colonies were counted 7 days later as previously described.

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Colony‐forming capacity of primary human CML‐LICs [2]
Viable BM mononuclear cells from three human patients with CP‐CML were purchased from Allcells. Cells were stained with anti‐CD34(8G12), anti‐CD38(HIT2), anti‐CD3(SK7), anti‐CD16(3G8), anti‐CD19(SJ25C1), anti‐CD20(L27), anti‐CD14(MϕP9), and anti‐CD56(NCAM16.2) antibodies. A mixture of mAbs recognizing CD3, CD16, CD19, CD20, CD14, and CD56 was used to identify Lin− cells, and CD34+CD38−Lin− cells were purified.18 To determine the effects of Vactosertib (EW7197) alone or a combination of EW‐7197 plus dasatinib, CD34+CD38−Lin− cells were cultured on OP‐9 stromal cells under hypoxic conditions (3% O2). After harvesting and washing in PBS, the colony‐forming ability of primitive human CML‐LICs was evaluated by culture in semi‐solid medium.

Animal/Disease Models: Mammary tumor virus (MMTV)/c-Neu female mice (32weeks old)[1]
Doses: 40 mg/kg
Route of Administration: intraperitoneal (ip)injection; every other day; for 10 weeks
Experimental Results: Inhibited Smad/TGFβ signaling , cell migration, invasion, and lung metastasis in MMTV/c-Neu mice.

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Breast cancer model #1 using MMTV/c-Neu mice [1]
When the total mammary tumor volume was 100 mm3, 32-week-old MMTV/c-Neu mice were randomized and treated (i.p.) with saline (Veh; n = 7) or Vactosertib (EW7197)·HCl dissolved in saline (43.7 mg/kg; equivalent to 40 mg/kg of EW-7197, n = 10) three times per week for 10 weeks.

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Breast cancer models #2, #3, and #4 using 4T1 orthotopic–grafted mice [1]
In efficacy experiments (models #2 and #3), 1.2 × 104 4T1 cells were suspended in saline and implanted into the left mammary fat pads (#4) of female BALB/c mice (50 μL/mouse; day 0). Tumor size and body weight were measured weekly. Artificial gastric fluid (Veh), Vactosertib (EW7197) (5 or 20 mg/kg), or LY2157299 (40 or 80 mg/kg) dissolved in Veh was administered orally to mice five times per week starting from day 4 for a total of 28 days (model #2; n = 10/group). Veh or Vactosertib (EW7197) (5, 10, 20, or 40 mg/kg) was administered to mice three times per week from day 4 for a total of 28 days (model #3; n = 6∼8/group). For the TGFβ1 challenge experiment in the model #3, 2 mice from each group were selected and treated with the indicated concentration of EW-7197 on day 28. After 30 minutes, 1 animal per group was injected (i.v.) with TGFβ1 (50 ng/mouse), and the other animal was left untreated. At 90 minutes after TGFβ1 injection, the mice were sacrificed. In survival experiments (model #4), 1.6 × 104 4T1 cells were injected as in the model #2, and Veh or EW-7197 (2.5 or 5 mg/kg) dissolved in Veh was administered orally to the mice five times per week from day 7 until death (n = 11/group).

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The breast cancer model #5 using the 4T1-luc tail vein mouse model [1]
4T1-luc cells [2 × 105; transfected with the plasmid construct pGL4(CMV-Luc)] were suspended in saline and injected into the tail veins of female BALB/c mice (50 μL/mouse; day 0). Artificial gastric fluid (Veh) or Vactosertib (EW7197) (0.625, 1.25, 2.5, or 5 mg/kg) dissolved in Veh was administered orally to mice five times per week from day 0 until death (n = 13/group). On day 15, surviving mice were analyzed using an in vivo imaging system to compare metastases in the lungs. Luciferase-positive 4T1 cells were imaged with the IVIS-200 system. The captured images were quantified using the Living Image Software package.

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Breast cancer model #6 using 4T1-luc orthotopic–grafted mice [1]
4T1-luc cells [3 × 104; transfected with the plasmid construct pGL4(CMV-Luc)] were implanted into the left #4 mammary fat pads of female BALB/c mice as in the model #2 (day 0). Tumor size and body weight were measured weekly. Artificial gastric fluid (Veh) or Vactosertib (EW7197) (2.5, 5, 10, or 20 mg/kg) dissolved in Veh was administered orally to mice five times per week (2.5, 5, or 10 mg/kg) or three times per week (20 mg/kg) starting from day 4 for a total of 28 days (n = 15/group).

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Mouse survival [2]
For “TKI‐insensitive” survival experiments using CML‐affected mice, IM (Novartis, Basel, Switzerland) was given to mice by oral gavage (200 mg/kg/day) on days 8–90 after transplantation of BCR‐ABL1 + CML‐MPPs.18 For “TKI‐resistant” recurrence experiments using T315I CML‐affected mice, ponatinib (AP24534) was given to mice by oral gavage (15 mg/kg/day) on days 8–60 after transplantation of BCR‐ABL1‐T315I+ CML‐MPPs. In both cases, we accessed a stock solution of Vactosertib (EW7197) that was prepared at 2 μg/mL in an artificial gastric fluid solution (900 mL ddH2O containing 2.0 g NaCl, 7 mL conc. HCl, and 3.2 g pepsin). Once every 3 days between days 15 to 90 post‐transplantation with BCR‐ABL1 + CML‐MPPs, or between days 15 to 60 post‐transplantation with BCR‐ABL1‐T315I + CML‐MPPs, a dilution of this mixture in artificial gastric fluid solution was given by oral gavage to CML‐affected mice to achieve a final concentration of 2.5 mg EW‐7197/kg body weight. Mouse survival was monitored for 125 days in “TKI‐insensitive” experiments and for 100 days in “TKI‐resistant” recurrence experiments.

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Pharmacokinetics [2]
To assess the PK of Vactosertib (EW7197), tetracycline‐inducible tg‐CML‐affected mice were fasted overnight and given Vactosertib (EW7197) (10 mg/kg) by oral gavage as described above. Blood samples were collected before treatment and at 30 min, 2 h, 4 h, and 8 h post‐treatment. Plasma concentrations of EW‐7197 were determined by liquid chromatography/tandem mass spectroscopy using an Agilent 1200 series HPLC and an Agilent 6410 triple quadrupole mass spectrometer equipped with an electrospray ionization source.

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Pharmacodynamics using Duolink® in situ PLA technology [2]
The PD of Vactosertib (EW7197) was determined based on phosphorylation of Smad3. Vactosertib (EW7197) (2.5 mg/kg) was given to tetracycline‐inducible tg‐CML‐affected mice by oral gavage. Then we isolated a cell fraction containing primitive LT‐CML stem cells (CD150+CD135−CD48−KLS cells) from tg‐CML‐affected mice by flow cytometry using a FACSAria III instrument (BD Biosciences).27 Phospho‐Smad3 was detected in cells using anti‐Smad3 and rabbit anti‐phospho‐Ser423/425 Smad3 (ab51451) antibodies by the highly sensitive Duolink® in situ PLA technology approach.

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Determination of WBC numbers in PB [2]
The CML‐affected mice received dasatinib (5 mg/kg/day) plus vehicle or Vactosertib (EW7197) (2.5 mg/kg every third day) by oral gavage for 30 days. For blood cell counts, PB from the post‐orbital vein was collected in a heparinized microtube and analyzed on a CellTac

Pharmacokinetics and PD of EW‐7197 in tg‐CML‐affected mice [2]
We next determined the PK of EW‐7197 in the bloodstream of tetracycline‐inducible tg‐CML‐affected mice by assessing its plasma concentration–time profile post‐treatment. We observed that EW‐7197 was rapidly absorbed into the bloodstream from the gastrointestinal tract of tg‐CML‐affected mice, and was then eliminated with a terminal phase half‐life (T1/2) of 3.26 ± 2.47 h (Fig. 2a). The maximum concentration (Cmax) of the drug in plasma was determined to be 625.0 ± 529.7 ng/mL (n = 7) at 30 min post‐administration.

[1]. EW-7197, a novel ALK-5 kinase inhibitor, potently inhibits breast to lung metastasis, 2014 Jul, 13(7):1704-16.

[2]. Novel oral transforming growth factor-β signaling inhibitor EW-7197 eradicates CML-initiating cells. Cancer Sci. 2016 Feb;107(2):140-8.

Vactosertib is under investigation in clinical trial NCT03724851 (Vactosertib in Combination With Pembrolizumab in Metastatic Colorectal or Gastric Cancer).
Vactosertib is an orally bioavailable inhibitor of the serine/threonine kinase, transforming growth factor (TGF)-beta receptor type 1 (TGFBR1), also known as activin receptor-like kinase 5 (ALK5), with potential antineoplastic activity. Upon oral administration, vactosertib inhibits the activity of TGFBR1 and prevents TGF-beta/TGFBR1-mediated signaling. This suppresses tumor growth in TGFBR1-overexpressing tumor cells. TGFBR1, which is overexpressed in a variety of tumor cell types, plays a key role in tumor cell proliferation. Expression of TGF-beta promotes tumor cell proliferation, enhances the migration of tumor cells and suppresses the response of the host immune system to tumor cells.

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Advanced tumors produce an excessive amount of transforming growth factor β (TGFβ), which promotes tumor progression at late stages of malignancy. The purpose of this study was to develop anti-TGFβ therapeutics for cancer. We synthesized a novel small-molecule TGFβ receptor I kinase (activin receptor-like kinase 5) inhibitor termed N-[[4-([1,2,4]triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-1H-imidazol-2-yl]methyl]-2-fluoroaniline (EW-7197), and we investigated its potential antimetastatic efficacy in mouse mammary tumor virus (MMTV)/c-Neu mice and 4T1 orthotopic-grafted mice. EW-7197 inhibited Smad/TGFβ signaling, cell migration, invasion, and lung metastasis in MMTV/c-Neu mice and 4T1 orthotopic-grafted mice. EW-7197 also inhibited the epithelial-to-mesenchymal transition (EMT) in both TGFβ-treated breast cancer cells and 4T1 orthotopic-grafted mice. Furthermore, EW-7197 enhanced cytotoxic T lymphocyte activity in 4T1 orthotopic-grafted mice and increased the survival time of 4T1-Luc and 4T1 breast tumor-bearing mice. In summary, EW-7197 showed potent in vivo antimetastatic activity, indicating its potential for use as an anticancer therapy.[1]

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Recent strategies for treating CML patients have focused on investigating new combinations of tyrosine kinase inhibitors (TKIs) as well as identifying novel translational research agents that can eradicate CML leukemia-initiating cells (CML-LICs). However, little is known about the therapeutic benefits such CML-LIC targeting therapies might bring to CML patients. In this study, we investigated the therapeutic potential of EW-7197, an orally bioavailable transforming growth factor-β signaling inhibitor which has recently been approved as an Investigational New Drug (NIH, USA), to suppress CML-LICs in vivo. Compared to TKI treatment alone, administration of TKI plus EW-7197 to CML-affected mice significantly delayed disease relapse and prolonged survival. Notably, combined treatment with EW-7197 plus TKI was effective in eliminating CML-LICs even if they expressed the TKI-resistant T315I mutant BCR-ABL1 oncogene. Collectively, these results indicate that EW-7197 may be a promising candidate for a new therapeutic that can greatly benefit CML patients by working in combination with TKIs to eradicate CML-LICs.[2]

C22H18FN7

399.42

399.16

C, 66.15; H, 4.54; F, 4.76; N, 24.55

1352608-82-2

Vactosertib Hydrochloride;1352610-25-3; 1352608-82-2

54766013

Light yellow to yellow solid powder

1.4±0.1 g/cm3

1.725

3.19

2

6

5

30

566

0

CC1=NC(=CC=C1)C2=C(N=C(N2)CNC3=CC=CC=C3F)C4=CN5C(=NC=N5)C=C4

FJCDSQATIJKQKA-UHFFFAOYSA-N

InChI=1S/C22H18FN7/c1-14-5-4-8-18(27-14)22-21(15-9-10-20-25-13-26-30(20)12-15)28-19(29-22)11-24-17-7-3-2-6-16(17)23/h2-10,12-13,24H,11H2,1H3,(H,28,29)

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)

Solubility in Formulation 1: 2.5 mg/mL (6.26 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
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 (6.26 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 (6.26 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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