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Purity: ≥98%
Pamufetinib (TAS-115) mesylate, the mesylate salt of Pamufetinib (TAS115; TAS 115), is an inhibitor of VEGFR and c-MET with anticancer activity. It inhibits rVEGFR2 and rMET with IC50s of 30 and 32 nM, respectively, and has an enhanced safety profile. Tumor angiogenesis is largely dependent on VEGF receptor (VEGFR) signaling. Associated toxicities or resistance to such therapy limit the usefulness of some VEGFR signal-targeted medications, despite their approval for clinical use. In order to get around these restrictions, TAS-115 was created to impede both VEGFR2 and MET's kinase activity as well as their signal-dependent cell growth to the same extent as other known inhibitors of VEGFR or MET. TAS-115 produced relatively weak growth inhibition (GI50 > 10 μmol/L) in cells that were either MET or VEGFR signal-independent, and its kinase selectivity was more specific than sunitinib's. Moreover, TAS-115 caused less harm to different types of normal cells than other VEGFR inhibitors. According to these findings, TAS-115 is remarkably specific and selective, at least when used in vitro. Even at a serum-saturating dose of TAS-115, daily administration for six weeks completely suppressed the growth of MET-inactivated tumors by blocking angiogenesis without causing toxicity in in vivo studies. Improved tolerability and the capacity to continue treatment without dosage reduction or a washout period were linked to TAS-115's notable selectivity for kinases and targeted cells. Additionally, in MET-amplified human cancer-bearing mice, TAS-115 produced a significant reduction in tumor size and extended survival. Based on these data, TAS-115 appears to be a novel inhibitor that targets VEGFR/MET, better able to suppress tumor growth while posing less side effects. The triple inhibition of EGFR, HGF/Met, and VEGF/VEGF receptor 2 by either a triplet of clinical drugs or TAS-115 in combination with erlotinib has also been reported to be helpful in reversing EGFR-TKI resistance and preventing angiogenesis, which may help control the progression of EGFR-mutant lung cancer. These findings are noteworthy.
| Targets |
VEGFR2 (IC50 = 30 nM); c-Met (IC50 = 32 nM)
Pamufetinib mesylate significantly inhibits the proliferation of MET-amplified cancer cells (GI50=0.032-0.362 μM) and strongly suppresses the VEGF-dependent proliferation of HUVECs (IC50=0.019 μM) as a MET-targeted inhibitor. In comparison to other VEGFR-targeted kinase inhibitors, pamufetinib mesylate is significantly less toxic in a variety of normal cell lines[1]. In PC-9 and HCC827 cells, erlotinib resistance, Met phosphorylation, and VEGF production induced by HGF are all inhibited by crizotinib and Pamufetinib mesylate[2]. |
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| ln Vitro |
Pamufetinib mesylate significantly inhibits the proliferation of MET-amplified cancer cells (GI50=0.032-0.362 μM) and strongly suppresses the VEGF-dependent proliferation of HUVECs (IC50=0.019 μM) as a MET-targeted inhibitor. In comparison to other VEGFR-targeted kinase inhibitors, pamufetinib mesylate is significantly less toxic in a variety of normal cell lines[1]. In PC-9 and HCC827 cells, erlotinib resistance, Met phosphorylation, and VEGF production induced by HGF are all inhibited by crizotinib and Pamufetinib mesylate[2].
TAS-115 inhibits MET phosphorylation in PC-9/HGF and HCC827 lung cancer cells. At concentrations less than 10 μmol/L, TAS-115 does not affect the growth of PC-9 or HCC827 cells. However, when combined with erlotinib (0.3 μmol/L), it reverses hepatocyte growth factor (HGF)-induced resistance to erlotinib in these cell lines in a concentration-dependent manner. [2] TAS-115 inhibits VEGF production stimulated by exogenous or endogenous HGF in PC-9 cancer cells. [2] TAS-115 inhibits VEGF-stimulated viability of human dermal microvascular endothelial cells (HMVECs) in a dose-dependent manner, similar to bevacizumab. Crizotinib does not show this effect. [2] Western blot analysis indicates that VEGFR-2 phosphorylation induced by VEGF in HMVECs is inhibited by TAS-115 and bevacizumab. [2] |
| ln Vivo |
Pamufetinib mesylate completely suppresses the progression of MET-inactivated tumors by blocking angiogenesis without causing toxicity when administered daily for six weeks, even at a serum-saturating dose of Pamufetinib mesylate. In MET-amplified human cancer-bearing mice, pamufetinib mesylate significantly reduces tumor size and increases survival[1].
In nude mice bearing PC-9/HGF tumors (which overexpress HGF), oral administration of TAS-115 (75 mg/kg daily) alone modestly inhibits tumor growth (76.6% inhibition on day 39). [2] The combination of TAS-115 (75 mg/kg daily, orally) and erlotinib (50 mg/kg daily, orally) potently inhibits the growth of PC-9/HGF tumors, showing 93.7% inhibition on day 39. This inhibitory effect is greater than that achieved with the triplet combination of erlotinib, crizotinib, and bevacizumab. [2] Treatment with TAS-115 (75 mg/kg daily, orally for 4 days) inhibits angiogenesis (measured by CD31+ vessel density) in PC-9/HGF tumors in vivo. The anti-angiogenic effect of TAS-115 is more potent than that of bevacizumab. [2] The combination of erlotinib and TAS-115 significantly delays tumor regrowth after cessation of a 39-day treatment course. Ten days after stopping treatment, tumors in the erlotinib+TAS-115 group regrew to only 1.7 times their initial size at cessation, compared to 4.5 and 3.3 times for tumors treated with erlotinib+crizotinib and erlotinib+crizotinib+bevacizumab, respectively. [2] This delayed regrowth is associated with sustained inhibition of angiogenesis (low vessel density) and a high number of apoptotic cells in the tumors even 10 days after treatment cessation. [2] |
| Enzyme Assay |
Mobilization shift assays were used in studies of enzyme inhibition. Briefly, 0.3 μg/mL of recombinant MET (rMET, N-terminal glutathione S-transferase (GST) Tag; Carna Biosciences) and 1.5 μmol/L of FL-Peptide 2 (Caliper Life Sciences) or 2 μg/mL of recombinant VEGFR2 (rVEGFR2, amino acid 790-end, N-terminal 6His Tagged; Upstate) and 1.5 μmol/L of FL-Peptide 22 (Caliper Life Sciences) were added to a 25 μL mixture containing 1/2 the Michaelis constant (Km) level of ATP, 100 mmol/L of HEPES (pH 7.2), 0.003% (w/v) Brij35, 0.04% (v/v) Tween 20, 10 mmol/L of MgCl2, 1 mmol/L of dithiothreitol, a Complete Mini EDTA-free Protease Inhibitor Cocktail Tablet (Roche Diagnostics, K.K.), and a PhosSTOP Phosphatase Inhibitor Cocktail Tablet (Roche Diagnostics, K.K.), with the addition of 0.05% (w/v) CHAPSO only in the case of rVEGFR2. The reaction mixture was stopped by adding 15 mmol/L of EDTA after it had been incubated for 90 minutes at 28°C. Utilizing a LabChip EZ Reader, Version 2.1.82.0 (UCC Version: 1.96, CCD Version: 102), the phosphorylated peptide was computed. Utilizing a logistic regression analysis, the 50% inhibitory concentration (IC50) was determined based on the quantity of phosphorylated peptide generated in the drug-treated well and the control well. The ProfilerPro Kit 1-8 (Caliper Life Sciences) was used to conduct 192 kinase panel assays, which were then analyzed using a mobility shift assay.
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| Cell Assay |
For 72 hours, MRC-5 (1000 cells/300 μL) and tumor cells (8000 cells/800 mL) in the upper chamber of Transwell collagen-coated chambers are cocultured with or without TAS-115 (1.0 μM) or erlotinib (0.3 μM). Next comes the removal of the upper chamber. Utilizing the MTT assay, cell viability is determined[2].
Cell Viability Assay (MTT Assay): Tumor cells (PC-9, HCC827, PC-9/Vec, PC-9/HGF) are plated in 96-well plates. After 24 hours, reagents including TAS-115, erlotinib, HGF, or combinations are added. Cells are incubated for 72 hours. MTT solution is then added to each well and incubated for 2 hours. The medium is removed, formazan crystals are dissolved in dimethyl sulfoxide, and absorbance is measured at 550 nm with a reference at 630 nm. Growth percentage is calculated relative to untreated controls. [2] Coculture Assay: Tumor cells with or without TAS-115 or erlotinib are placed in the lower chamber of a Transwell system. Fibroblasts (MRC-5 cells) are placed in the upper chamber. After 72 hours of coculture, the upper chamber is removed, and MTT assay is performed on the lower chamber to assess tumor cell viability. [2] Western Blotting: Cell lysates are prepared from treated cells. Proteins are separated by SDS-PAGE, transferred to membranes, blocked, and incubated with primary antibodies (e.g., against phospho-Met, total Met, phospho-Akt, total Akt, phospho-Erk, total Erk, phospho-VEGFR-2, total VEGFR-2). After washing, membranes are incubated with horseradish peroxidase-conjugated secondary antibodies. Immunoreactive bands are visualized using enhanced chemiluminescence substrates. [2] VEGF/HGF Production Measurement: Cells are cultured in medium with serum. Supernatants are collected after 48 hours, centrifuged, and stored. Concentrations of HGF and VEGF are determined using commercial enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer's protocols. Color intensity is measured at 450 nm. [2] |
| Animal Protocol |
Mice: The dose ranges for TAS-115 are 12.5, 50, and 200 mg/kg/d. The recommended daily dose of sunitinib is 40 mg/kg. When using oral medication for chronic dosing in the SC-9 xenograft model, the regimen is continued for either 14 or 42 days in a row. Two weekly measurements of TV and body weight are made during the course of treatment. At the conclusion of every study period, the antitumor efficacy is evaluated[1].
Subcutaneous Xenograft Model for Efficacy: Cultured PC-9/Vec or PC-9/HGF tumor cells are implanted subcutaneously into the flanks of male nude mice. When tumor volumes reach 100-200 mm³, mice are randomized into treatment groups. TAS-115 is administered orally at 75 mg/kg, once daily. Erlotinib is administered orally at 50 mg/kg, once daily. Bevacizumab is administered intraperitoneally at 100 µg per mouse, once a week. Treatment continues for 39 days. Tumor dimensions are measured three times a week, and volume is calculated. After 39 days, treatment is ceased in some groups to monitor tumor regrowth for an additional 10 days. [2] Short-term Treatment Model for Angiogenesis Assessment: Nude mice bearing established subcutaneous tumors (≈100 mm³) are treated with drugs (e.g., erlotinib, TAS-115) for 4 days. Mice are euthanized on day 4, tumors are harvested, and vascularization is assessed via CD31 immunohistochemical staining. [2] |
| Toxicity/Toxicokinetics |
In nude mice, no significant adverse reactions, including weight loss, were observed after daily oral administration of TAS-115 (75 mg/kg) in combination with daily oral administration of erlotinib (50 mg/kg) for 39 days. [2]
The manuscript notes a potential concern based on previous studies: anti-VEGF therapy (part of the mechanism of action of TAS-115) may induce malignant progression of tumors, such as increased local invasion and distant metastasis. Therefore, biomarkers may be crucial for optimizing the use of dual MET/VEGFR inhibitors. [2] |
| References |
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| Additional Infomation |
TAS-115 is described as a novel oral inhibitor active against both VEGFR-2 and MET. [2]
This study suggests that using erlotinib in combination with TAS-115 to triple-inhibit EGFR, MET, and angiogenesis (via VEGFR-2) may be an effective strategy for controlling the progression of EGFR-mutant lung cancer that has developed resistance to EGFR tyrosine kinase inhibitors (TKIs) due to HGF/MET activation. [2] Under the experimental conditions used, the two-drug regimen (erlotinib + TAS-115) was significantly more effective than the clinically available three-drug regimen (erlotinib + crizotinib + bevacizumab) in delaying tumor recurrence after treatment cessation. [2] |
| Molecular Formula |
C28H27FN4O7S2
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|---|---|
| Molecular Weight |
614.664987802505
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| Exact Mass |
614.13
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| Elemental Analysis |
C, 54.71; H, 4.43; F, 3.09; N, 9.12; O, 18.22; S, 10.43
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| CAS # |
1688673-09-7
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| Related CAS # |
Pamufetinib;1190836-34-0
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| PubChem CID |
118130255
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| Appearance |
White to off-white solid powder
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| Hydrogen Bond Donor Count |
4
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| Hydrogen Bond Acceptor Count |
10
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| Rotatable Bond Count |
7
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| Heavy Atom Count |
42
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| Complexity |
891
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| Defined Atom Stereocenter Count |
0
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| InChi Key |
NUYQWFCUOAVQAL-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C27H23FN4O4S.CH4O3S/c1-29-26(34)19-14-18-21(15-24(19)35-2)30-11-10-22(18)36-23-9-8-17(13-20(23)28)31-27(37)32-25(33)12-16-6-4-3-5-7-16;1-5(2,3)4/h3-11,13-15H,12H2,1-2H3,(H,29,34)(H2,31,32,33,37);1H3,(H,2,3,4)
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| Chemical Name |
4-[2-fluoro-4-[(2-phenylacetyl)carbamothioylamino]phenoxy]-7-methoxy-N-methylquinoline-6-carboxamide;methanesulfonic acid
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| Synonyms |
TAS115 mesylate; TAS115; Pamufetinib; TAS-115; TAS 115; Pamufetinib mesylate
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| HS Tariff Code |
2934.99.9001
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| Storage |
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. |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
DMSO : ~75 mg/mL (~122.02 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (4.07 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. Solubility in Formulation 2: ≥ 2.5 mg/mL (4.07 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. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.6269 mL | 8.1344 mL | 16.2689 mL | |
| 5 mM | 0.3254 mL | 1.6269 mL | 3.2538 mL | |
| 10 mM | 0.1627 mL | 0.8134 mL | 1.6269 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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