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Purity: ≥98%
Pamufetinib (TAS-115; TAS115) is a novel potent inhibitor of VEGFR and hepatocyte growth factor receptor (MET)-targeted kinase 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 exhibited a higher degree of kinase selectivity in comparison to sunitinib, and it resulted in a comparatively mild growth inhibition (GI50 > 10 μmol/L) in cells that were either MET or VEGFR signal-independent. Moreover, TAS-115 caused less harm than other VEGFR inhibitors in a variety of normal cells. Based on these data, TAS-115 appears to be highly specific and selective, at least when tested in vitro. Given daily for six weeks, even at a serum-saturating dose of TAS-115, TAS-115 completely suppressed the progression of MET-inactivated tumors in in vivo studies by blocking angiogenesis without causing toxicity. TAS-115's remarkable selectivity for kinases and targeted cells was linked to increased tolerability and made it possible to continue treatment without lowering the dosage or requiring a washout period. Moreover, TAS-115 significantly reduced tumor size and increased survival time in mice harboring human cancer that was amplified by MET. Based on these data, TAS-115 appears to be a novel inhibitor that targets VEGFR/MET, offering enhanced antitumor efficacy and reduced toxicity. Furthermore, it has been suggested that EGFR-mutant lung cancer may be controlled in its progression by reversing EGFR-TKI resistance and inhibiting angiogenesis through triple inhibition of EGFR, HGF/Met, and VEGF/VEGF receptor 2, which can be achieved either by a triplet of clinical drugs or by TAS-115 in combination with erlotinib.
| Targets |
VEGFR2 (IC50 = 30 nM); c-Met (IC50 = 32 nM)
Pamufetinib is ATP antagonistic, having inhibition constants (Ki) of 12 and 39 nM against rVEGFR2 and rMET, respectively. Similar to other known inhibitors of VEGFR or MET, pamufetinib also strongly suppresses the kinase activity of both VEGFR2 and MET, as well as their signal-dependent cell growth. In comparison to other VEGFR inhibitors, parmatetinib causes less damage to a variety of normal cells[1]. At concentrations below 10 μM, Pamufetinib has no effect on PC-9 or HCC827 cell growth; however, when combined with Erlotinib, it reverses HGF-induced resistance in the cell lines in a concentration-dependent manner. Pamufetinib inhibits the proliferation of endothelial cells and the production of VEGF by cancer cells[2]. |
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| ln Vitro |
Pamufetinib is ATP antagonistic, having inhibition constants (Ki) of 12 and 39 nM against rVEGFR2 and rMET, respectively. Similar to other known inhibitors of VEGFR or MET, pamufetinib also strongly suppresses the kinase activity of both VEGFR2 and MET, as well as their signal-dependent cell growth. In comparison to other VEGFR inhibitors, parmatetinib causes less damage to a variety of normal cells[1]. At concentrations below 10 μM, Pamufetinib has no effect on PC-9 or HCC827 cell growth; however, when combined with Erlotinib, it reverses HGF-induced resistance in the cell lines in a concentration-dependent manner. Pamufetinib inhibits the proliferation of endothelial cells and the production of VEGF by cancer cells[2].
Pamufetinib (TAS-115) potently inhibited VEGF-induced VEGFR2 tyrosine phosphorylation in HUVECs and MS-1 cells at concentrations ≥ 0.01 µM, and inhibited HGF-induced MET tyrosine phosphorylation and downstream signaling (p-ERK1/2, p-AKT, p-FAK, p-S6, p-STAT3) in MKN45 cells (a MET-amplified gastric cancer line) at concentrations ≥ 0.03 µM. [1] It strongly inhibited VEGF-dependent proliferation of HUVECs with an IC₅₀ of 0.019 µM, but was much less potent (IC₅₀ = 19.3 µM) under VEGF-independent (10% FBS) conditions. [1] Pamufetinib (TAS-115) selectively inhibited the proliferation of MET-amplified cancer cell lines (MKN45, Hs746T, NUGC-4) with GI₅₀ values of 0.032, 0.035, and 0.362 µM, respectively. It showed minimal growth inhibition (GI₅₀ > 10 µM) against cancer cell lines without MET amplification (HCT-116, MCF-7, SK-OV-3, DU145). [1] In contrast to sunitinib, sorafenib, and crizotinib, Pamufetinib (TAS-115) (at 10 µM) induced minimal caspase 3/7 activation (an indicator of apoptosis) and cell damage in various normal cell lines, including rat cardiomyocytes, human coronary artery smooth muscle cells (hCASMC), normal lung fibroblasts (MRC-5), and human alveolar epithelial cells (AEPiC). [1] |
| ln Vivo |
Pamufetinib (50 mg/kg/d) totally stops tumor growth while the patient is receiving treatment. MET-amplified human cancer transplanted models exhibit a 48% regression from the initial tumor volume upon administration of parofendinib (200 mg/kg/d). In this model, 8 mg/kg/d is the estimated 50% effective dose (ED50) of Pamufetinib. These mice's survival is greatly extended by pamufetinib when doses of 50 or 200 mg/kg/d are given[1]. In vivo angiogenesis in PC-9/HGF tumors is inhibited by parofenib. Furthermore, even after the treatment is stopped, the doublet of erlotinib and Pamufetinib effectively inhibits the growth of PC-9/HGF tumors and delays tumor regrowth linked to prolonged inhibition of the tumor vasculature[2].
In the MET-low SC-9 gastric cancer xenograft model, oral administration of Pamufetinib (TAS-115) at 200 mg/kg/day for 42 consecutive days completely suppressed tumor growth by inhibiting angiogenesis, with no body weight loss observed. In contrast, sunitinib (40 mg/kg/day, 2-weeks-on/1-week-off regimen) initially inhibited growth but tumors rapidly regrew during the 1-week washout period. [1] In the MET-amplified MKN45 gastric cancer xenograft model, Pamufetinib (TAS-115) administered orally for 14 consecutive days dose-dependently inhibited tumor growth (TGI=76% at 12.5 mg/kg/day; complete growth inhibition at 50 mg/kg/day) and induced 48% tumor regression at 200 mg/kg/day. The estimated ED₅₀ was 8 mg/kg/day. Sunitinib (40 mg/kg/day) showed significant growth inhibition (TGI=84%) but did not cause regression. [1] In a MET-amplified NUGC-4 gastric cancer peritoneal dissemination model, Pamufetinib (TAS-115) administered orally at 50 and 200 mg/kg/day (5 days/week) significantly prolonged survival (median survival time >60 days at 200 mg/kg/day vs. 29 days for control), while sunitinib (40 mg/kg/day) did not show a significant survival benefit. [1] Pharmacodynamic analysis in AZ-521 tumor-bearing mice showed that a single oral dose of Pamufetinib (TAS-115) inhibited VEGF-induced VEGFR2 phosphorylation in tumors. In MKN45 tumor-bearing mice, it inhibited phosphorylation of MET and its downstream effectors ERK and AKT in tumors at doses as low as 3.1 mg/kg. [1] |
| Enzyme Assay |
The mobility shift assay is used in studies of enzyme inhibition. In summary, a 25 μL mixture containing 1/2 the Michaelis constant (Km) level of ATP, 100 mM of HEPES (pH 7.2), 0.003% (w/v) Brij35, 0.04% (v/v) Tween 20, 10 mM of MgCl2, 1 mM of dithiothreitol, a Complete Mini EDTA-free Protease Inhibitor Cocktail Tablet, and a PhosSTOP Phosphatase Inhibitor Cocktail Tablet are combined with 1.5 μM of FL-Peptide 2 or 2 μg/mL of recombinant VEGFR2 (rVEGFR2, amino acid 790-end, N-terminal 6His Tagged) and 1.5 μM of FL-Peptide 22. The addition of 15 mM EDTA ends the 90-minute incubation period of the reaction mixture at 28°C. Using a LabChip EZ Reader, Version 2.1.82.0 (UCC Version: 1.96, CCD Version: 102), phosphorylated peptide is calculated. Utilizing a logistic regression analysis, the 50% inhibitory concentration (IC50) is determined based on the quantity of phosphorylated peptide generated in the drug-treated well and the control well. Using the ProfilerPro Kit 1-8, 192 kinase panel assays are conducted and analyzed via a mobility shift assay.
Kinase inhibitory activity was assessed using a mobility shift assay. Briefly, recombinant MET or VEGFR2 kinase, a fluorophore-labeled peptide substrate, ATP at the Km concentration, and test compounds were incubated in a reaction buffer containing HEPES, MgCl₂, DTT, and protease/phosphatase inhibitors. The reaction proceeded for 90 minutes at 28°C and was stopped with EDTA. The phosphorylated and non-phosphorylated peptides were separated and quantified using a microfluidic chip reader. The IC₅₀ values were calculated based on the reduction of phosphorylated peptide formation in drug-treated wells compared to control wells. [1] A broad kinase selectivity profile (192 kinases) was determined using a similar mobility shift assay with pre-configured kinase panels. [1] |
| Cell Assay |
The MTT dye reduction method is used to measure the growth of cells. Using 10% foetal bovine serum (FBS) per well, tumor cells are seeded into 96-well plates at a density of 2×103 cells/100 mL RPMI-1640 medium. Following a 24-hour incubation period, different reagents are added to each well, and the cells are incubated for an additional 72 hours.Next, 50 μL of MTT solution (2 mg/mL) is added to each well, and the cells are incubated for a further two hours. The dark blue crystals are dissolved by adding 100 mL of dimethyl sulfoxide after the media containing the MTT solution is removed. At test and reference wavelengths of 550 and 630 nm, respectively, the absorbance of every well is measured using a microplate reader. In comparison to untreated controls, the growth percentage is displayed. Every experiment involves testing each reagent concentration at least three times, and in triplicate if possible.
For cellular phosphorylation assays, HUVECs were serum-starved overnight, treated with Pamufetinib (TAS-115), then stimulated with VEGF for 5 minutes before cell lysis and immunoblotting for p-VEGFR2. [1] MKN45 cells were treated with Pamufetinib (TAS-115) for 30 minutes before lysis and immunoblotting for p-MET and downstream signaling proteins. [1] Proliferation assays: Cancer cells were plated, allowed to adhere, treated with Pamufetinib (TAS-115) for 72 hours, and cell viability was assessed using a luminescent ATP detection assay. [1] VEGF-dependent HUVEC proliferation was assessed by seeding starved HUVECs, adding VEGF and Pamufetinib (TAS-115) simultaneously, and measuring DNA synthesis after 24 hours using a bromodeoxyuridine uptake method. [1] Normal cell damage assessment: Normal cells (e.g., rat cardiomyocytes) were treated with compounds for 72 or 96 hours. Cell viability was measured by crystal violet staining or a multiplex assay, and apoptosis was assessed by measuring caspase-3/7 activity using a luminescent assay. Caspase activity was normalized to cell viability. [1] |
| Animal Protocol |
Each mouse has a subcutaneous SC-9 fragment inserted via a trocar into its right abdomen. Subcutaneous implants of MKN45 cell suspensions are made and inserted into each naked mouse's right abdomen. There is a calculation for the tumor volume (TV, mm3). Doses of 12.5, 50, and 200 mg/kg/d are the established TAS-115. Sunitinib's maximum tolerated dose (MTD) is 40 mg/kg/d, the dose level that has been established. In the SC-9 xenograft model, oral medication therapy is administered for either 14 or 42 days in a row. TV and body weight are measured twice a week for the duration of the therapy.
For subcutaneous xenograft models (SC-9, MKN45), tumor fragments or cell suspensions were implanted into the right abdomen of nude mice. When tumor volumes reached approximately 250 mm³, mice were allocated into groups and treatment began (Day 1). Pamufetinib (TAS-115) was suspended in an appropriate vehicle and administered orally (by gavage) once daily at specified doses (e.g., 12.5, 50, 200 mg/kg/day) for 14 or 42 consecutive days. Tumor volumes and body weights were measured twice weekly. [1] For the NUGC-4 peritoneal dissemination model, NUGC-4 cells in Matrigel were inoculated intraperitoneally into nude mice. Treatment with Pamufetinib (TAS-115) (50 or 200 mg/kg/day) or sunitinib (40 mg/kg/day) began on Day 1 and was administered orally once daily, 5 days per week. Mice were monitored for survival until becoming moribund. [1] For pharmacodynamic studies, tumor-bearing mice received a single oral dose of Pamufetinib (TAS-115). Three hours later, for VEGFR2 analysis, VEGF was injected intravenously, and tumors were harvested 5 minutes post-injection for immunoprecipitation and immunoblotting. For MET analysis, tumors were harvested 3 hours after dosing for direct immunoblotting. [1] |
| ADME/Pharmacokinetics |
The safety margin (the ratio of the maximum tolerated dose to the estimated effective dose) was reported to be greater than 25 times (MTD > 200 mg/kg/day in the MET amplification model, while ED₅₀ was 2-8 mg/kg/day). [1]
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| Toxicity/Toxicokinetics |
Compared with sunitinib, sorafenib and crizotinib, permofiltinib (TAS-115) (10 µM) significantly reduced caspase activation and cell damage in a variety of normal cell lines (rat cardiomyocytes, hCASMC, MRC-5, AEPIC). [1] In chronic in vivo studies, SC-9 tumor-bearing mice were well-tolerated by oral administration of permofiltinib (TAS-115) at a dose of 200 mg/kg/day for 6 weeks, and no weight loss was observed. [1] Unlike sunitinib, permofiltinib (TAS-115) did not inhibit AMP-activated protein kinase (AMPK) activity in vitro or in rat cardiomyocytes, while sunitinib inhibited AMPK (IC₅₀ = 0.061 µM) and induced cardiomyocyte damage. [1]
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| References |
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| Additional Infomation |
Permofiltinib (TAS-115) is a novel oral dual VEGFR and MET tyrosine kinase inhibitor designed to overcome the limitations of existing VEGFR inhibitors, such as toxicity and resistance. Its high selectivity and good tolerability allow for continuous daily administration without the need for a break or dose adjustment, which is crucial for maintaining anti-angiogenic and antitumor efficacy. Simultaneous inhibition of VEGFR and MET may produce synergistic antitumor activity by simultaneously targeting tumor angiogenesis and MET-driven tumor growth, invasion, and resistance mechanisms. [1]
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| Molecular Formula |
C27H23FN4O4S
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|---|---|---|
| Molecular Weight |
518.56
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| Exact Mass |
518.142
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| Elemental Analysis |
C, 62.54; H, 4.47; F, 3.66; N, 10.80; O, 12.34; S, 6.18
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| CAS # |
1190836-34-0
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| Related CAS # |
Pamufetinib mesylate;1688673-09-7
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| PubChem CID |
44247727
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| Appearance |
Solid powder
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| Density |
1.4±0.1 g/cm3
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| Index of Refraction |
1.678
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| LogP |
4.38
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| Hydrogen Bond Donor Count |
3
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| Hydrogen Bond Acceptor Count |
7
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| Rotatable Bond Count |
7
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| Heavy Atom Count |
37
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| Complexity |
798
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| Defined Atom Stereocenter Count |
0
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| SMILES |
S=C(N([H])C(C([H])([H])C1C([H])=C([H])C([H])=C([H])C=1[H])=O)N([H])C1C([H])=C([H])C(=C(C=1[H])F)OC1C([H])=C([H])N=C2C([H])=C(C(C(N([H])C([H])([H])[H])=O)=C([H])C2=1)OC([H])([H])[H]
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| InChi Key |
ORRNXRYWGDUDOG-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C27H23FN4O4S/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/h3-11,13-15H,12H2,1-2H3,(H,29,34)(H2,31,32,33,37)
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| Chemical Name |
4-[2-fluoro-4-[(2-phenylacetyl)carbamothioylamino]phenoxy]-7-methoxy-N-methylquinoline-6-carboxamide
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| Synonyms |
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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 |
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| 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) |
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| Solubility (In Vivo) |
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). View More
Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] Oral Formulations
Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.9284 mL | 9.6421 mL | 19.2842 mL | |
| 5 mM | 0.3857 mL | 1.9284 mL | 3.8568 mL | |
| 10 mM | 0.1928 mL | 0.9642 mL | 1.9284 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.
 Cellular inhibition of the phosphorylation of VEGFR2, MET, and signaling factors downstream from MET by TAS-115.Mol Cancer Ther.2013 Dec;12(12):2685-96. |
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 TAS-115–induced highly potent and selective inhibition of MET and VEGFR signal-dependent cell growth.Mol Cancer Ther.2013 Dec;12(12):2685-96. |
 Kinase inhibitor-induced cell damage in normal cells.Mol Cancer Ther.2013 Dec;12(12):2685-96. |
 Chronic treatment with TAS-115 produced potent antitumor effects by inhibiting angiogenesis.Mol Cancer Ther.2013 Dec;12(12):2685-96. |
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 Antitumor efficacy of TAS-115 against MET-amplified human gastric cancer MKN45 xenograft models.Mol Cancer Ther.2013 Dec;12(12):2685-96. |
 Treatment with erlotinib plus TAS-115 inhibits angiogenesis in PC-9/HGF tumors in vivo.J Thorac Oncol.2014 Jun;9(6):775-83. |
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