c-MET (IC50 = 0.13 nM)
Mesenchymal-epithelial transition factor (MET) tyrosine kinase (recombinant human MET Ki = 0.13 nM; recombinant mouse MET Ki = 0.17 nM);
Exhibited high selectivity over 270 other kinases, with Ki > 1000 nM for most kinases (e.g., EGFR, HER2, VEGFR2, FGFR1) [1]

INCB28060 has more than 10,000-fold selectivity over a broad panel of human kinases, picomolar enzymatic potency, and high specificity for c-MET. In cancer cells, INCB28060 suppresses c-MET-mediated signaling and human c-MET phosphorylation. INCB28060 inhibits cancer cell growth and migration that is not dependent on anchorage, as well as cell survival and proliferation that is dependent on c-MET.[1]

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Capmatinib HCl hydrate potently inhibited recombinant human MET kinase activity with a Ki of 0.13 nM and mouse MET with a Ki of 0.17 nM in ATP-competitive assays [1]
- In MET-amplified cancer cell lines: GTL-16 (gastric), H1993 (lung), MKN-45 (gastric), and EBC-1 (lung), Capmatinib HCl hydrate inhibited cell proliferation with IC50 values of 0.1 nM, 0.3 nM, 0.5 nM, and 0.7 nM, respectively [1]
- In MET 14-exon skipping mutant cell line H596 (lung), Capmatinib HCl hydrate showed antiproliferative activity with an IC50 of 0.4 nM [1]
- Western blot analysis revealed Capmatinib HCl hydrate (0.1-10 nM) dose-dependently inhibited MET phosphorylation (p-MET) and downstream signaling molecules p-ERK1/2 and p-AKT in GTL-16 and H1993 cells, with maximal inhibition at 1 nM [1]
- Capmatinib HCl hydrate (1 nM) reduced colony formation of GTL-16 cells by 85% compared to vehicle control, and induced apoptosis in H1993 cells (Annexin V-positive cells increased from 5% to 32% at 10 nM) [1]
- No significant antiproliferative activity was observed in MET-nonamplified cell lines (A549, MCF-7) with IC50 > 1000 nM [1]

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] Capmatinib (INCB28060) shows strong antitumor activity in c-MET–dependent mouse tumor models[1]
To assess the in vivo activities of Capmatinib (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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In nude mice bearing GTL-16 (MET-amplified gastric cancer) xenografts, oral administration of Capmatinib HCl hydrate (10 mg/kg, 30 mg/kg, 100 mg/kg, once daily for 21 days) dose-dependently inhibited tumor growth, with 100 mg/kg achieving 92% tumor growth inhibition (TGI) and 30% tumor regression [1]
- In H1993 (MET-amplified lung cancer) xenograft model, Capmatinib HCl hydrate (30 mg/kg, 100 mg/kg, po, qd × 21) resulted in TGI of 78% and 95%, respectively; 100 mg/kg group showed significant reduction in p-MET and p-ERK levels in tumor tissues [1]
- In MET 14-exon skipping mutant H596 xenografts, Capmatinib HCl hydrate (100 mg/kg, po, qd × 14) achieved 88% TGI compared to vehicle [1]
- No significant body weight loss or overt toxicity was observed in treated mice at doses up to 100 mg/kg [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 Capmatinib (INCB28060)dissolved in DMSO. According to DMSO titration, a solvent concentration of 4% is the highest that can be tolerated. The Capmatinib (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

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MET kinase activity inhibition assay: Recombinant human/mouse MET kinase domain was incubated with varying concentrations of Capmatinib HCl hydrate, ATP (at Km concentration), and a peptide substrate in reaction buffer at 30°C for 60 minutes. The reaction was terminated by adding a stop solution, and phosphorylated substrate was quantified using a time-resolved fluorescence resonance energy transfer (TR-FRET) assay. Ki values were calculated using nonlinear regression analysis [1]
- Kinase selectivity profiling: Capmatinib HCl hydrate (1 μM) was screened against a panel of 270 recombinant kinases. Inhibition rates were determined using radiometric or fluorescence-based assays, and Ki values were calculated for kinases with inhibition rates >20% [1]

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 Capmatinib (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 Capmatinib (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 Capmatinib (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 Capmatinib (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 Capmatinib (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 Capmatinib (INCB28060) as described above. Protein extracts were then prepared and subjected to Western blot analysis using a rabbit anti-cleaved PARP (Asp214) antibody.

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Cell proliferation assay: MET-amplified (GTL-16, H1993, MKN-45, EBC-1) and MET-nonamplified (A549, MCF-7) cell lines were seeded in 96-well plates and cultured for 24 hours. Cells were treated with serial dilutions of Capmatinib HCl hydrate (0.01 nM-10 μM) for 72 hours. Cell viability was assessed using a colorimetric assay based on mitochondrial dehydrogenase activity, and IC50 values were calculated [1]
- Western blot analysis: GTL-16 and H1993 cells were treated with Capmatinib HCl hydrate (0.1 nM-10 nM) for 24 hours, then lysed. Protein extracts were separated by SDS-PAGE, transferred to membranes, and probed with antibodies against p-MET, total MET, p-ERK1/2, total ERK1/2, p-AKT, and total AKT. Band intensities were quantified by densitometry [1]
- Colony formation assay: GTL-16 cells were seeded in 6-well plates at low density and treated with Capmatinib HCl hydrate (0.1 nM-10 nM) or vehicle. After 14 days of incubation, colonies were stained with crystal violet and counted. Colony formation efficiency was calculated relative to vehicle control [1]
- Apoptosis assay: H1993 cells were treated with Capmatinib HCl hydrate (1 nM-100 nM) for 48 hours, then stained with Annexin V-FITC and propidium iodide (PI). Apoptotic cells were analyzed by flow cytometry [1]

Eight-week-old female Balb/c nu/nu mice (Charles River) are inoculated subcutaneously with 4 × 10 6 tumor cells (S114 model) or with 5 × 10 6 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.

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MET-amplified xenograft models: Female nude mice (6-7 weeks old) were subcutaneously inoculated with GTL-16 (5×10⁶ cells), H1993 (1×10⁷ cells), or H596 (2×10⁶ cells) into the right flank. When tumors reached 100-150 mm³, mice were randomly divided into vehicle and Capmatinib HCl hydrate treatment groups (n=8/group). Capmatinib HCl hydrate was dissolved in 0.5% carboxymethylcellulose sodium (CMC-Na) and administered orally once daily at doses of 10 mg/kg, 30 mg/kg, or 100 mg/kg for 14-21 days. Tumor volume (measured twice weekly) and body weight (measured weekly) were recorded. At study end, tumors were excised, weighed, and processed for Western blot analysis of p-MET, p-ERK, and p-AKT [1]

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
Approximately 78% of the radioactive material is excreted in feces after oral administration of radiolabeled carmatinib, 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.
Metabolism/Metabolites
Carmatinib is primarily metabolized via CYP3A4 and aldehyde oxidase. Specific biotransformation pathways and metabolites remain to be elucidated.

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

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In rats, the absolute bioavailability of oral carmatinib hydrochloride hydrate (10 mg/kg) was 78%, Tmax was 1.0 hours, and Cmax was 2560 ng/mL [1]
-The terminal half-life (t1/2) of rats (intravenous injection, 2 mg/kg) was 4.2 hours, and that of dogs (intravenous injection, 1 mg/kg) was 6.8 hours [1]
-The volume of distribution (Vdss) of rats was 2.3 L/kg, and that of dogs was 3.1 L/kg, indicating its extensive tissue distribution [1]
-The plasma protein binding rates in humans, rats and dogs were 95%, 93% and 91%, respectively (concentration range: 0.1-10 mg/kg). μM) [1]
- In vitro metabolic studies using human liver microsomes showed that carmatinib hydrochloride hydrate is mainly metabolized by oxidation and has no significant inhibitory effect on CYP450 isoenzymes (CYP1A2, 2C9, 2C19, 2D6, 3A4) at concentrations up to 10 μM [1]

Effects During Pregnancy and Lactation
◉ Overview of use during lactation There is currently no information on the clinical use of carmatinib during lactation. Because carmatinib binds to plasma proteins at a rate of up to 96%, its content 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 No published information was found as of the revision date. ◉ Effects on lactation and breast milk No published information was found as of the revision date. Acute toxicity: Oral administration of up to 300 mg/kg of carmatinib hydrochloride hydrate to rats and up to 200 mg/kg of carmatinib hydrochloride hydrate to dogs did not cause death or obvious toxic symptoms (e.g., ataxia, somnolence, diarrhea) within 14 days [1].
- Subchronic toxicity (28 days, rats): Oral administration of 30 mg/kg, 100 mg/kg and 300 mg/kg daily did not show significant changes in body weight, food consumption, hematological parameters or organ weight. (liver, kidney, heart, lungs) [1]
- No significant nephrotoxicity, hepatotoxicity or cardiovascular toxicity was observed in the subchronic toxicity studies [1]

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

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

Carmatinib hydrochloride is the hydrochloride form of carmatinib, a highly bioavailable oral inhibitor of the proto-oncogene c-Met (also known as hepatocyte growth factor receptor (HGFR)) with potential antitumor activity. Carmatinib selectively binds to c-Met, thereby inhibiting c-Met phosphorylation and disrupting the c-Met signaling pathway. This may induce death in tumor cells that overexpress c-Met protein or express constitutively activated c-Met protein. c-Met is a receptor tyrosine kinase that is overexpressed or mutated in various tumor cell types and plays a crucial role in tumor cell proliferation, survival, invasion, metastasis, and tumor angiogenesis.
See also: Carmatinib (with active ingredient).

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Drug Indications
Tabrecta monotherapy is indicated for the treatment of adult patients with advanced non-small cell lung cancer (NSCLC) who carry a skipping mutation in exon 14 (METex14) of the mesenchymal epithelial transforming factor gene and who require systemic therapy after receiving immunotherapy and/or platinum-based chemotherapy.

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Camatinib hydrochloride hydrate (INCB-28060; INC280) is a potent, selective, and orally effective MET tyrosine kinase inhibitor [1] - its mechanism of action involves binding to the ATP-binding pocket of MET, thereby inhibiting MET phosphorylation. And downstream signaling pathways (PI3K/AKT and RAS/ERK), thereby inhibiting tumor cell proliferation, migration and invasion, and inducing apoptosis [1] - Preclinical data support its potential to treat cancers carrying MET amplification or activation mutations (e.g., exon 14 skipping mutations) [1] - Carmatinib hydrochloride hydrate has no cross-reactivity with other closely related tyrosine kinases, which gives it a good therapeutic window [1]

C23H21CL2FN6O2

503.3594

502.108

C, 54.88; H, 4.21; Cl, 14.09; F, 3.77; N, 16.70; O, 6.36

1865733-40-9

Capmatinib;1029712-80-8;Capmatinib dihydrochloride; 1197376-85-4 (2HCl); Capmatinib hydrochloride;1029714-89-3; 1450883-33-6 (fumarate); 1865733-40-9 (HCl hydrate);1197376-90-1 (besylate) ; 1029714-89-3 (xHCl)

122201352

White to light yellow solid powder

4

7

4

34

637

0

Cl[H].Cl[H].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.O([H])[H]

COWBUPJEEDYWKD-UHFFFAOYSA-N

InChI=1S/C23H17FN6O.2ClH.H2O/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*1H;1H2

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

Capmatinib hydrochloride; INC 280; INCB028060; INCB-028060; INCB 028060; INCB28060; INCB-28060; Capmatinib hydrochloride; 1865733-40-9; Capmatinib 2HCl.H2O; NVP-INC280-AAA; Tabrecta; C2A374O70X; UNII-C2A374O70X; Capmatinib hydrochloride [USAN]; INCB 28060; Capmatinib; NVP-INC 280AAA; INC280; INC-280

2934.99.9001

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 (e.g. under nitrogen), avoid exposure to moisture and light.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~20.83 mg/mL (~41.38 mM)
H2O : ~3.33 mg/mL (~6.62 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (4.13 mM) (saturation unknown) in 10% DMSO + 40% PEG300 +5% Tween-80 + 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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 (Please use freshly prepared in vivo formulations for optimal results.)