Wnt/β-catenin; c-Met (IC50 = 0.13 nM)

At a dose of around 4 nM and an IC50 value of about 1 nM, capmatinib (INCB28060) inhibits c-MET phosphorylation. Over 90% of c-MET is inhibited by it. This is reversible, and hours after the substance is removed, the effect is greatly diminished. 48 hours later, entirely vanishes [1]. Capmatinib (INCB28060) (0–1000 nM; 72 hours) inhibits SNU-5, S114, H441, and U-87MG from proliferating [1]. The phosphorylation of c-MET and downstream effectors of the c-MET pathway, including ERK1/2, AKT, FAK, GAB1, and STAT3/5, is efficiently inhibited by capmatinib (INCB28060) (0.06-62.25 nM; 2h) [1]. The inhibitor capmatinib (INCB28060) (0.24-63 nM; over night) stops HGF-induced migration of H441 cells [1]. Capmatinib (INCB28060) suppresses EGFR and HER-3 phosphorylation quickly (0.5–50 nM; 20 minutes) [1]. In SNU-5 cells, capmatinib (INCB28060) (0-333 nM; 24 hours) causes apoptosis [1].

Capmatinib (INCB28060) (1-30 mg/kg; orally, twice daily for 2 weeks) displayed dose-dependent reduction of tumor development and was well tolerated at all dosages during treatment , there was no evidence of overt toxicity or body weight loss in the U-87MG tumor mouse model [1]. Capmatinib (INCB28060) (0.03-10 mg/kg; oral, single dosage) suppresses c-MET phosphorylation in the S114 tumor mouse model [1].

The assay buffer has the following contents: pH 7.8, 50 mM Tris-HCl, 10 mM MgCl2, 100 mM NaCl, 0.1 mg/ml BSA, and 5 mM DTT. Spotted on 384-well plates for HTS are 0.8 μL of 5 mM INCB28060 dissolved in DMSO. According to DMSO titration, a solvent concentration of 4% is the highest that can be tolerated. The INCB28060 plate is prepared by serial dilutions at three and eleven points in order to measure IC50s. The assay plate is transferred with 0.8 μL of INCB28060 in DMSO from the INCB28060 plate. DMSO has a final concentration of 2%. In assay buffer, solutions of 0.5 nM phosphorylated c-Met or 8 nM unphosphorylated c-Met are made. In an assay buffer containing 400 μM ATP (unphosphorylated c-Met) or 160 uM ATP (phosphorylated c-Met), a 1 mM stock solution of the peptide substrate Biotin-EQEDEPEGDYFEWLE-amide dissolved in DMSO is diluted to 1 μM. To start the reaction, add 20 μL of substrate solution per well after adding a 20 μL volume of enzyme solution (or assay buffer for the enzyme blank) to the corresponding wells in each plate. For ninety minutes, the plate is incubated at 25 °C with protection from light. To terminate the reaction, introduce 20 μL of a mixture comprising 45 mM EDTA, 50 mM Tris-HCl, 50 mM NaCl, 0.4 mg/ml BSA, 200 nM SA-APC, and 3 nM EUPy20. After incubating the plate at room temperature for 15-30 minutes, the Perkin Elmer Fusion α-FP instrument measures the homogenous time resolved fluorescence (HTRF). The following HTRF program settings are in use: 330/30 primary excitation filter 200 uSec for the primary window, 50 uSec for the primary delay, and 15 flashes total. Time to read well: 2000

Cell Viability Assay[1]
Cell Types: SNU-5, S114, H441 and U-87MG
Tested Concentrations: 0-10000 nM
Incubation Duration: 72 hrs (hours)
Experimental Results: Inhibition of cell viability and colony formation of SNU-5 and S114 H441 and U-87MG The IC50 values are 1.2 nM, 12.4 nM, ~0.5 nM and 2 nM respectively.

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Cell migration assay[1]
Cell Types: H441 (stimulated with 50 ng/mL recombinant human HGF for 24 hrs (hours))
Tested Concentrations: 0.24, 1, 4, 16 and 63 nM
Incubation Duration: Overnight
Experimental Results: Prevents HGF-stimulated H441 cell migration, IC50 Approximately 2 nM, and cell migration is 16 nM.

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Western Blot Analysis[1]
Cell Types: SNU-5
Tested Concentrations: 0.06, 0.24, 0.98, 3.91, 15.63 and 62.25 nM
Incubation Duration: 2 hrs (hours)
Experimental Results: Effectively inhibits c-MET and the phosphorylation of downstream effectors of the c-MET pathway such as ERK1/2, AKT, FAK, GAB1 and STAT3/5.

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Western Blot Analysis[1]
Cell Types: H1993 Cell
Tested Concentrations: 0.5, 5 and 50 nM
Incubation Duration: 20 minutes
Experimental Results: Rapidly inhibits the phosphorylation of EGFR and HER-

Animal/Disease Models: Female Balb/c nu/nu (nude) mice (subcutaneously (sc) (sc) inoculated with 5×106 U-87MG glioblastoma cells) [1]
Doses: 1, 3, 10 and 30 mg/kg
Route of Administration: Orally, daily Two times for 2 weeks.
Experimental Results: 1 mg/kg and 3 mg/kg one time/day had a dose-dependent inhibitory effect on tumor growth, which was 35% and 76% respectively; among 10 U-87MG tumor-bearing mice, Six animals experienced partial regression after taking the 10 mg/kg daily dose; and all doses were well tolerated during treatment, with no evidence of significant toxicity or weight loss.

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Animal/Disease Models: Female Balb/c nu/nu (nude) mice (subcutaneously (sc) (sc) inoculated with 4×106 S114 tumor cells) [1]
Doses: 0.03, 0.1, 0.3, 1, 3 and 10 mg/kg
Doses: po (po (oral gavage)) single dose
Experimental Results: Causes approximately 50% and 90% inhibition of c-MET phosphorylation 30 minutes after administration of 0.03 and 0.3 mg/kg, and more than 90% inhibition of phosphorylation of c-MET after 7 hrs (hrs (hours)).

Absorption
The oral bioavailability of carmatinib is estimated to be >70%. After oral administration, peak plasma drug concentration (Tmax) is reached within 1 to 2 hours. Co-administration with a high-fat meal increases the AUC of carmatinib by 46%, while Cmax remains unchanged (compared to fasting); co-administration with a low-fat meal has no clinically significant effect on drug exposure.
Elimination Pathway
After oral administration of radiolabeled carmatinib, approximately 78% of the radioactive material is excreted in feces, of which approximately 42% is unmetabolized parent drug; 22% is excreted in urine, of which the amount of unmetabolized parent drug is negligible.
Volume of Distribution
The steady-state apparent volume of distribution is 164 L.
Clearance
The steady-state mean apparent clearance of carmatinib is 24 L/h.

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Metabolism/Metabolites
Carmatinib is primarily metabolized via CYP3A4 and aldehyde oxidase. Specific biotransformation pathways and metabolites have not been elucidated.

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Biological half-life
The elimination half-life is 6.5 hours.

Hepatotoxicity
In pre-marketing clinical trials of carmatinib in patients with solid tumors harboring MET mutations, liver function abnormalities were common, but usually self-limiting and mild. 39% of patients treated with carmatinib experienced varying degrees of ALT elevation, with 7% experiencing ALT elevations exceeding 5 times the upper limit of normal (ULN). In these trials involving 373 patients, only 1% discontinued carmatinib prematurely due to elevated AST or ALT. The median time to onset of liver function abnormalities was 2 months after treatment initiation. Although serum transaminases occasionally rose to higher levels (5 to 20 times the ULN), this was not accompanied by elevated serum bilirubin, and no patients developed clinically significant liver injury with jaundice. The carmatinib product information recommends routine liver function tests before treatment, every 2 weeks for the first 3 months of treatment, and monthly as needed thereafter.
Probability Score: E (Unproven but suspected rare cause of clinically significant liver injury).

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Effects during pregnancy and lactation
◉ Overview of use during lactation
There is currently no information regarding the clinical use of carmatinib during lactation. Because carmatinib binds to plasma proteins at a rate of 96%, its concentration in breast milk may be very low. The manufacturer recommends discontinuing breastfeeding during carmatinib treatment and for one week after the last dose.
◉ Effects on breastfed infants
As of the revision date, no relevant published information was found.
◉ Effects on lactation and breast milk
As of the revision date, no relevant published information was found. The plasma protein binding rate is approximately 96% and is independent of serum drug concentration.

[1]. A novel kinase inhibitor, INCB28060, blocks c-MET-dependent signaling, neoplastic activities, and cross-talk with EGFR and HER-3. Clin Cancer Res. 2011 Nov 15;17(22):7127-38.

[2]. Capmatinib (INC280) Is Active Against Models of Non-Small Cell Lung Cancer and Other Cancer Types with Defined Mechanisms of MET Activation. Clin Cancer Res. 2019 May 15;25(10):3164-3175.

[3]. Dhillon S. Capmatinib: First Approval. Drugs. 2020 Jul;80(11):1125-1131.

Objective: c-MET receptor tyrosine kinases play a crucial role in the occurrence, development, and spread of human cancers, making them a highly attractive therapeutic target. This study describes the preclinical characteristics of a novel c-MET kinase inhibitor, INCB28060.
Experimental Design: This study employed a series of in vitro and in vivo biochemical and biological experiments.
Results: INCB28060 exhibits picomolar-level enzyme activity and high specificity for c-MET, with a selectivity exceeding 10,000-fold for various human kinases. This inhibitor effectively blocks c-MET phosphorylation and the activation of key downstream effector molecules in c-MET-dependent tumor cell lines. Therefore, INCB28060 effectively inhibits the proliferation and migration of c-MET-dependent tumor cells and effectively induces apoptosis in vitro. Oral administration of INCB28060 resulted in time- and dose-dependent inhibition of c-MET phosphorylation and tumor growth in a c-MET-driven mouse tumor model, and the inhibitor was well-tolerated at doses that achieved complete tumor inhibition. In further exploring the potential interactions between c-MET and other signaling pathways, we found that activated c-MET positively regulates the activity of epidermal growth factor receptor (EGFR) and HER-3 and the expression of their ligands. INCB28060 treatment reversed these effects. Finally, we confirmed that circulating hepatocyte growth factor levels were significantly elevated in patients with various cancers. Conclusion: Activated c-MET has pleiotropic effects on multiple pro-cancer signaling pathways and may play a key role in driving tumor cell growth and survival. INCB28060 is a potent and selective c-MET kinase inhibitor with potential for cancer treatment. [1] Objective: The selective MET inhibitor capmatinib is being investigated in multiple clinical trials, including monotherapy and combination therapy. This article describes preclinical data of capmatinib that support a rational patient selection clinical strategy based on biomarkers. Experimental Design: The selectivity and cell activity of capmatinib were evaluated in a large-scale cell screening. Antitumor efficacy was quantified in a large number of cell lines or patient-derived xenograft models, and monotherapy or combination therapy was tested based on the genomic characteristics of the respective models. Results: The study found that carmatinib exhibited significantly higher selectivity for MET than other kinases. The drug was effective against cancer models characterized by MET amplification, significant MET overexpression, MET exon 14 skipping mutations, or MET activation via hepatocyte growth factor (HGF) ligand expression. In cancer models where MET is the primary oncogenic driver, combination therapy further enhanced antitumor activity, for example, by adding a BH3 mimicry that induces apoptosis. Combination therapy of carmatinib with other kinase inhibitors enhanced antitumor activity against cancer models where MET activation coexists with other oncogenic drivers, such as EGFR activating mutations. Conclusion: The activity of carmatinib in preclinical models is associated with a few possible genomic characteristics. The low proportion of cancer models responding to carmatinib monotherapy suggests that patient selection strategies based on these biomarkers are crucial for clinical development. Carmatinib is also a reasonable combination therapy partner with other kinase inhibitors to combat MET-driven resistance. [2]

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Carmatinib (Tabrecta™) is an oral small molecule mesenchymal-epithelial transition (MET) inhibitor developed by Novartis Oncology under license from Incyte for the treatment of lung cancer. Carmatinib targets and selectively binds to MET, including mutants that skip exon 14, and inhibits cancer cell growth driven by mutant MET variants. In May 2020, oral carmatinib received the world’s first approval in the United States for the treatment of adult patients with metastatic non-small cell lung cancer (NSCLC) diagnosed by an FDA-approved test for MET exon 14 skipping mutations. Clinical development of carmatinib for the treatment of glioblastoma, hepatocellular carcinoma, malignant melanoma, breast cancer, colorectal cancer, head and neck cancer, and solid tumors is underway in multiple countries. This article summarizes the key milestones in the development of carmatinib that ultimately led to its first approval. [3]

C23H18CLFN6O

448.880026340485

448.121

1029714-89-3

Capmatinib;1029712-80-8;Capmatinib dihydrochloride hydrate;1865733-40-9;Capmatinib dihydrochloride;1197376-85-4

137347172

Solid

2

6

4

32

637

0

JJBXRCLAAKZSNF-UHFFFAOYSA-N

InChI=1S/C23H17FN6O.ClH/c1-25-22(31)18-6-5-16(11-19(18)24)21-13-28-23-27-12-17(30(23)29-21)10-14-4-7-20-15(9-14)3-2-8-26-20;/h2-9,11-13H,10H2,1H3,(H,25,31);1H

2-fluoro-N-methyl-4-[7-(quinolin-6-ylmethyl)imidazo[1,2-b][1,2,4]triazin-2-yl]benzamide;hydrochloride

NVP-INC 280AAA; INCB 028060; Capmatinib hydrochloride; INCB28060; INC280; INC-280; Capmatinib HCl; Capmatinib xHCl; 1029714-89-3; 2-Fluoro-N-methyl-4-(7-(quinolin-6-ylmethyl)imidazo[1,2-b][1,2,4]triazin-2-yl)benzamide hydrochloride; 2126164-56-3; INC 280; INCB028060; INCB-028060; INCB-28060; INCB 28060.

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)

DMSO: > 10 mM

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.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*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).

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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)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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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).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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