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 capmatinib is estimated to be >70%. Following oral administration, maximum plasma concentrations are achieved within 1 to 2 hours (Tmax). Co-administration with a high-fat meal increased capmatinib AUC by 46% with no change in Cmax (as compared to fasted conditions), and co-administration with a low-fat meal had no clinically meaningful effects on exposure.

Route of Elimination
Following oral administration of radiolabeled capmatinib, approximately 78% of the radioactivity is recovered in feces, of which ~42% is unchanged parent drug, and 22% is recovered in the urine, of which a negligible amount remains unchanged parent drug.

Volume of Distribution
The apparent volume of distribution at steady-state is 164 L.

Clearance
The mean apparent clearance of capmatinib at steady-state is 24 L/h.

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Metabolism / Metabolites
Capmatinib undergoes metabolism primarily via CYP3A4 and aldehyde oxidase. Specific biotransformation pathways and metabolic products have yet to be elucidated.

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

Hepatotoxicity
In the prelicensure clinical trials of capmatinib in patients with solid tumors harboring MET mutations, liver test abnormalities were frequent although usually self-limited and mild. Some degree of ALT elevations arose in 39% of capmatinib treated patients and were above 5 times the upper limit of normal (ULN) in 7%. In these trials that enrolled 373 patients, capmatinib was discontinued early due to increased AST or ALT in only 1% of patients. The liver test abnormalities had a median onset of 2 months after initiation of therapy. While serum aminotransferase elevations were occasionally quite high (5 to 20 times upper limit of normal), there were no accompanying elevations in serum bilirubin and no patient developed clinically apparent liver injury with jaundice. The product label for capmatinib recommends monitoring for routine liver tests before, at 2 week intervals during the first 3 months of therapy, and monthly thereafter as clinically indicated.
Likelihood score: E* (unproven but suspected rare cause of clinically apparent liver injury).

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Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
No information is available on the clinical use of capmatinib during breastfeeding. Because capmatinib is 96% bound to plasma proteins, the amount in milk is likely to be low. The manufacturer recommends that breastfeeding be discontinued during capmatinib therapy and for 1 week after the last dose.

◉ Effects in Breastfed Infants
Relevant published information was not found as of the revision date.

◉ Effects on Lactation and Breastmilk
Relevant published information was not found as of the revision date. Plasma protein binding is approximately 96% and is independent of drug serum 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.

Purpose: The c-MET receptor tyrosine kinase plays important roles in the formation, progression, and dissemination of human cancer and presents an attractive therapeutic target. This study describes the preclinical characterization of INCB28060, a novel inhibitor of c-MET kinase.
Experimental design: Studies were conducted using a series of in vitro and in vivo biochemical and biological experiments.
Results: INCB28060 exhibits picomolar enzymatic potency and is highly specific for c-MET with more than 10,000-fold selectivity over a large panel of human kinases. This inhibitor potently blocks c-MET phosphorylation and activation of its key downstream effectors in c-MET-dependent tumor cell lines. As a result, INCB28060 potently inhibits c-MET-dependent tumor cell proliferation and migration and effectively induces apoptosis in vitro. Oral dosing of INCB28060 results in time- and dose-dependent inhibition of c-MET phosphorylation and tumor growth in c-MET-driven mouse tumor models, and the inhibitor is well tolerated at doses that achieve complete tumor inhibition. In a further exploration of potential interactions between c-MET and other signaling pathways, we found that activated c-MET positively regulates the activity of epidermal growth factor receptors (EGFR) and HER-3, as well as expression of their ligands. These effects are reversed with INCB28060 treatment. Finally, we confirmed that circulating hepatocyte growth factor levels are significantly elevated in patients with various cancers.
Conclusions: Activated c-MET has pleiotropic effects on multiple cancer-promoting signaling pathways and may play a critical role in driving tumor cell growth and survival. INCB28060 is a potent and selective c-MET kinase inhibitor that may have therapeutic potential in cancer treatment.[1]

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Purpose: The selective MET inhibitor capmatinib is being investigated in multiple clinical trials, both as a single agent and in combination. Here, we describe the preclinical data of capmatinib, which supported the clinical biomarker strategy for rational patient selection.
Experimental design: The selectivity and cellular activity of capmatinib were assessed in large cellular screening panels. Antitumor efficacy was quantified in a large set of cell line- or patient-derived xenograft models, testing single-agent or combination treatment depending on the genomic profile of the respective models.
Results: Capmatinib was found to be highly selective for MET over other kinases. It was active against cancer models that are characterized by MET amplification, marked MET overexpression, MET exon 14 skipping mutations, or MET activation via expression of the ligand hepatocyte growth factor (HGF). In cancer models where MET is the dominant oncogenic driver, anticancer activity could be further enhanced by combination treatments, for example, by the addition of apoptosis-inducing BH3 mimetics. The combinations of capmatinib and other kinase inhibitors resulted in enhanced anticancer activity against models where MET activation co-occurred with other oncogenic drivers, for example EGFR activating mutations.
Conclusions: Activity of capmatinib in preclinical models is associated with a small number of plausible genomic features. The low fraction of cancer models that respond to capmatinib as a single agent suggests that the implementation of patient selection strategies based on these biomarkers is critical for clinical development. Capmatinib is also a rational combination partner for other kinase inhibitors to combat MET-driven resistance.[2]

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Capmatinib (Tabrecta™) is an oral, small molecule mesenchymal-epithelial transition (MET) inhibitor being developed by Novartis Oncology, under a license from Incyte Corporation, for the treatment of lung cancer. Capmatinib targets and selectively binds to MET, including the mutant variant produced by exon 14 skipping, and inhibits cancer cell growth driven by the mutant MET variant. In May 2020, oral capmatinib received its first global approval in the USA for the treatment of adults with metastatic non-small cell lung cancer (NSCLC) whose tumours have a mutation that leads to MET exon 14 skipping, as detected by an FDA-approved test. Clinical development for the treatment of glioblastoma, liver cancer, malignant melanoma, breast cancer, colorectal cancer, head and neck cancer and solid tumours is ongoing in several countries. This article summarizes the milestones in the development of capmatinib leading 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.)