Wnt/β-catenin; c-Met (IC50 = 0.13 nM)

INCB28060 shows strong antitumor activity in c-MET–dependent mouse tumor models[1]
To assess the in vivo activities of INCB28060, we used the S114 cell–derived mouse tumor model. Because S114 cells express both human c-MET and HGF, tumors from these cells are dependent upon c-MET signaling for their growth. To determine the minimum dose of INCB28060 necessary to control c-MET phosphorylation, we orally administered to mice increasing doses of INCB28060 and measured phospho-c-MET levels in tumors 30 minutes later. As seen in Fig. 4A, 0.03 mg/kg INCB28060, the lowest dose tested, causes approximately 50% inhibition of c-MET phosphorylation. Escalating doses affect phospho-c-MET in a dose-dependent fashion, and single doses of 0.3 mg/kg or more resulted in greater than 90% inhibition. To further characterize the impact of INCB28060 over time, a single dose of 3 mg/kg was selected. Inhibition of phospho-c-MET exceeded 90% through the 7-hour measurement time point (Fig. 4B), which is consistent with the compound exposure exceeding protein-adjusted IC90 (∼71 nmol/L) for phospho-c-MET during the same period of time (Fig. 4B). Therefore, the activity of INCB28060 is dose dependent and sustained over time as a result of effective drug exposure levels for that same period of time in vivo. Similar results were observed with the MKN-45 human gastric cancer cell-derived mouse tumor model that is driven by c-MET activation as a result of c-MET amplification (data not shown).

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INCB28060 demonstrates potent antitumor activity in tumor models in mice dependent on c-MET; oral administration of 0.03 mg/kg INCB28060 results in a 50% reduction in c-MET phosphorylation. In mice exhibiting tumors, there is observed a dose-dependent inhibition of tumor growth.[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

In RPMI-1640 medium with 10% FBS, H441 cells are seeded and grown to full confluence. Using a P200 pipette tip, cells are scraped to create gaps. Next, in the presence of varied INCB28060 concentrations, cells are stimulated with 50 ng/mL recombinant human HGF to induce migration across the gap. Following an overnight incubation period, a semiqualitative evaluation of the inhibition of cell migration is carried out and representative photos are taken.

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Cell viability assay[1]
Optimal cell density used in the viability assay was predetermined for individual cell lines. To determine compound potency, cells were seeded into 96-well microplates at the appropriate density in media containing 1% to 2% FBS and supplemented with serial dilutions of INCB28060 in a final volume of 100 μL per well. After 72-hour incubation, 24 μL of CellTiter 96 AQueous One Solution was added to each well, and the plates were incubated for 2 hours in a 37°C incubator. The optical density was measured in the linear range using a microplate reader at 490 nm with wavelength correction at 650 nm. IC50 values were calculated using the GraphPad Prism Software.

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Soft agar colony formation assay[1]
U-87MG or H441 cells were prepared at adequate densities in 6-well plates mixed with 0.5 mL top layer agar containing 0.3% agarose in appropriate culture medium and supplemented with 1% or 10% FBS, in the presence or absence of 50 ng/mL recombinant human HGF and INCB28060 at various concentrations. Cells were evenly laid over 1 mL solidified base layer agar containing 0.6% agarose in culture medium. The plates were incubated at 37°C in a humidified incubator supplied with 5% CO2. Cells were fed once a week with top agar containing appropriate concentrations of human HGF and INCB28060. The number and size of colonies were evaluated 2 to 3 weeks later when representative photographs were taken.

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Cell migration assay[1]
H441 cells were seeded in RPMI-1640 medium containing 10% FBS and grown to complete confluence. Gaps were introduced by scraping cells with a P200 pipette tip. Cells were then stimulated with 50 ng/mL recombinant human HGF to induce migration across the gap in the presence of various concentrations of INCB28060. After an overnight incubation, representative photographs were taken and a semiqualitative assessment of inhibition of cell migration was conducted.

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Apoptosis assay[1]
Cells were seeded in a 96-well plate and grown overnight in culture medium containing 0.5% FBS. Cells were then treated with INCB28060 at various concentrations for 24 hours. Apoptosis was measured using a DNA fragmentation–based Cell Death Detection ELISAplus kit according to the manufacturer's instructions. To measure PARP cleavage, cells were grown in 10 cm dishes and treated similarly with INCB28060 as described above. Protein extracts were then prepared and subjected to Western blot analysis using a rabbit anti-cleaved PARP (Asp214) antibody.

Eight-week-old female Balb/c nu/nu mice (Charles River) are inoculated subcutaneously with 4 × 10⁶ tumor cells (S114 model) or with 5 × 10⁶ tumor cells (U-87MG glioblastoma model).
3, 10, 30 mg/kg
INCB28060 is orally dosed, twice each day.

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Efficacy studies[1]
Tumor-bearing mice were dosed orally, twice each day with 1, 3, 10, or 30 mg/kg of free base INCB28060 reconstituted in 5% DMAC in 0.5% methylcellulose for up to 2 weeks. Body weights were monitored throughout the study as a gross measure of toxicity/morbidity. Tumor growth inhibition, expressed in percent, was calculated using the formula: (1 − [(volume (treated)/volume (vehicle)]) × 100. Pharmacodynamic analysis[1]
For pharmacodynamic analysis, S114 tumor–bearing mice were monitored for tumor growth and then randomized into groups of 3 with average tumor sizes of approximately 300 to 500 mm3. For time course studies, mice were given a single oral dose of 3 mg/kg INCB28060 reconstituted in 5% DMAC in 0.5% methylcellulose and tumors were harvested at the indicated time points. For dose escalation studies, mice were given a single oral dose of INCB28060 at 0.03, 0.1, 0.3, 1, 3, or 10 mg/kg reconstituted in 5% DMAC in 0.5% methylcellulose and tumors were harvested 30 minutes after dosing. All tumors were processed for the determination of phospho-c-Met levels using the Human Phospho-HGFR/c-Met kit. The plasma concentration of INCB28060 was determined by LC/MS/MS analysis following retro-orbital or cardiac puncture blood collection.

Absorption, Distribution and Excretion
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.
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.
The apparent volume of distribution at steady-state is 164 L.
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.

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Protein Binding
Plasma protein binding is approximately 96% and is independent of drug serum concentration.

[1]. Clin Cancer Res . 2011 Nov 15;17(22):7127-38.

[2]. BMC Res Notes . 2019 Mar 11;12(1):125.

Pharmacodynamics
Capmatinib inhibits the overactivity of c-Met, a receptor tyrosine kinase encoded by the _MET_ proto-oncogene. Mutations in _MET_ are involved in the proliferation of many cancers, including non-small cell lung cancer (NSCLC). Capmatinib may cause photosensitivity reactions in patients following ultraviolet (UV) exposure - patients undergoing therapy with capmatinib should be advised to use sunscreen and protective clothing to limit exposure to UV radiation. Instances of interstitial lung disease/pneumonitis, which can be fatal, occurred in patients being treated with capmatinib. Patients presenting with signs or symptoms of lung disease (e.g. cough, dyspnea, fever) should have capmatinib immediately withheld, and capmatinib should be permanently discontinued if no other feasible causes of the lung-related symptoms are identified.

C23H17FN6O

412.42

412.144

C, 66.98; H, 4.15; F, 4.61; N, 20.38; O, 3.88

1029712-80-8

Capmatinib dihydrochloride hydrate;1865733-40-9;Capmatinib dihydrochloride;1197376-85-4;Capmatinib hydrochloride;1029714-89-3

25145656

Yellow solid powder

1.4±0.1 g/cm3

1.717

-0.12

1

6

4

31

637

0

FC1=C(C(N([H])C([H])([H])[H])=O)C([H])=C([H])C(=C1[H])C1C([H])=NC2=NC([H])=C(C([H])([H])C3C([H])=C([H])C4=C(C([H])=C([H])C([H])=N4)C=3[H])N2N=1

LIOLIMKSCNQPLV-UHFFFAOYSA-N

InChI=1S/C23H17FN6O/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)

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

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: ≥ 2.08 mg/mL (5.04 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% 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 20.8 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: ≥ 2.08 mg/mL (5.04 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 20.8 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: 5%DMSO+40%PEG300+5%Tween80+50%ddH2O: 6mg/ml

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