c-Met (IC50 = 4 nM)

Tepotinib hydrochloride inhibits IRAK4, TrkA, Axl, IRAK1, Mer and TrkA with IC50 of 615, 1017, 1566, 2037, 2272 and 5716 nM, respectively[1]. Tepotinib hydrochloride inhibits HGF-induced c-Met phosphorylation in A549 cells with an average IC50 of 6 nM[1]. Tepotinib (0.01 nM-30 μM) hydrochloride can inhibit tumor cell proliferation and migration in vitro[1].

Tepotinib hydrochloride induces tumor regression in xenograft models and inhibits c-Met phosphorylation in vivo [1].

EMD 1214063 and EMD 1204831 selectively suppressed the c-Met receptor tyrosine kinase activity. Their inhibitory activity was potent [inhibitory 50% concentration (IC50), 3 nmol/L and 9 nmol/L, respectively] and highly selective, when compared with their effect on a panel of 242 human kinases. Both EMD 1214063 and EMD 1204831 inhibited c-Met phosphorylation and downstream signaling in a dose-dependent fashion, but differed in the duration of their inhibitory activity[1].

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c-Met in vitro kinase assay[1]
Kinase inhibition by EMD 1214063 or EMD 1204831 (1 and 10 μmol/L) was assessed in vitro using a panel of 242 different kinases. Biochemical activity was measured in a flash-plate assay. His6-tagged recombinant human c-Met kinase domain (Aa 974–end; 20 ng) and biotinylated poly-Ala-Glu-Lys-Tyr (6:2:5:1; 500 ng) were incubated with or without the test compound for 90 minutes at room temperature in 100 μL buffer containing 0.3 μCi 33P-ATP, 2.5 μg polyethylene glycol 20.000, and 1% dimethyl sulfoxide (DMSO), as previously described. Radioactivity was measured with a TopCount microplate scintillation and luminescence counter. Inhibitory 50% concentration values (IC50) were calculated by nonlinear regression analysis using the RS/1 software program. [1]

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Phospho-c-Met-capture ELISA[1]
Total c-Met phosphorylation was assessed by c-Met–capture ELISA in Nunc-Immuno MicroWell 96-well solid plates. A549 human lung cancer cells were seeded 2 days before treatment, serum-starved for 20 hours, and treated on day 3 with different concentrations of EMD 1214063 or EMD 1204831 or 0.2% DMSO for 45 minutes at 37°C, 5% CO2. Upon stimulation with 100 ng/mL HGF for 5 minutes, cells were lysed with 70 μL per well ice-cold lysis buffer [20 nmol/L HEPES, pH 7,4; 10% (V/V) Glycerol; 150 nmol/L NaCl; 1% (V/V) Triton-X-100; 2 nmol/L EDTA] supplemented with protease and phosphatase inhibitors. In the wash-out experiments, A549 were treated with EMD 1214063 or EMD 1204831 for 45 minutes, washed, and incubated in serum-free medium for 14 hours, before stimulation with HGF (100 ng/mL). In the ELISA, the capture antibody was specific for the c-Met extracellular domain, whereas an antiphosphotyrosine biotin-labeled antibody was used for detection. Tyrosine phosphorylation was revealed using a streptavidin peroxidase conjugate and chemiluminescence read-out.

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Biochemical analysis[1]
Phosphorylation of c-Met, Gab-1, Akt, and Erk1/2 was analyzed by Western blot analysis in EBC-1 cells. In brief, cells were seeded at a density of 3 × 106 cells per well, serum-starved for 20 hours, and lysed on day 3 after incubation with EMD 1214063. Proteins were separated by SDS-PAGE and blotted onto nitrocellulose membranes. Membranes were blocked with Tris-buffered saline and incubated in primary antibody solution (anti-pMet, anti-pAkt, anti-pERK1/2, anti-Gab1) at 4°C overnight. Proteins were detected by chemiluminescence, with VersaDoc MP 5000 imaging system equipped with Quantity One 1-D analysis software.

Tepotinib (EMD-1214063) is a c-Met inhibitor that is both potent and selective. With an IC50 of 4 nM, it is >200 times more selective for c-Met than IRAK4, TrkA, Axl, IRAK1, and Mer.

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Wound healing test and proliferation assays[1]
Wound healing tests were carried out as previously described. In brief, a scratch was produced with a sterile pipette tip on a monolayer of NCI-H441 lung cancer cells. The effect of EMD 1210463 and EMD 1204831 on closure of the cell gap was monitored over 24 hours in the presence or absence of 100 ng/mL HGF. All proliferation and colony formation assays were conducted in 4 replicates and included 4 DMSO vehicle controls. IC50 values were determined by 4PL fitting in GraphPad Prism v5.

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Pharmacodynamic markers on ex vivo tumor samples[1]
c-Met autophosphorylation was investigated by Western blot analysis on frozen ex vivo tumor samples. The tumor tissue was mechanically homogenized, lysed using Precellys 24 homogenizer, or Precellys ceramic lysing tubes (PEQLab Ltd) according to the manufacturer's instructions. Further preparation of lysates and protein separation by SDS-PAGE were conducted as already described for EBC-1 cells. Histone H3 phosphorylation and biomarkers of cell-cycle arrest and apoptosis (cyclin D1, p27, and cleaved, activated capase-3) were analyzed by immunohistochemistry (IHC) on formalin-fixed, paraffin-embedded sections. IHC was conducted using Discovery staining instruments, with the OmniMap Kit, according to the manufacturer's instructions. Sections were counterstained with hematoxylin.

Animal/Disease Models:CD-1 or BALB/C nude mice bearing human cancer cell lines KP-4, or EBC-1[1]
Doses: 6 and 15 mg/kg for mice bearing NSCLC EBC-1; 25, 50 and 200 mg/kg for mice bearing pancreatic carcinoma cell line KP-4.
Route of Administration: Injected daily; for 14-18 days
Experimental Results: Daily administration of 5 or 15 mg/kg to EBC-1 tumor-bearing mice resulted in effective inhibition or complete tumor regression, respectively. Induced dose-dependent tumor growth inhibition in mice bearing human pancreatic carcinoma KP-4 tumors.

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The antitumor efficacy of EMD 1214063 or EMD 1204831 was investigated in mouse xenograft models. CD-1 or BALB/C nude mice were injected subcutaneously with human cancer cell lines KP-4, U87MG: 10 × 106 cells in 100 μL, Hs746T, EBC-1: 5 × 106 cells in 100 μL. As soon as the tumor reached the linear growth phase (70–150 mm3), tumor-bearing mice (10 mice/group) were injected daily with the indicated doses of EMD 1214063 or EMD 1204831, or vehicle. Body weight and tumor size [length (L) and width (W)] were measured twice weekly. The tumor volume was calculated using the formula L × W2/2. Statistical significance was determined by one-way ANOVA. P ≤ 0.05 were considered significant.

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Pharmacokinetic and pharmacodynamic studies[1]
Plasma and tumor drug concentrations were measured using high-performance liquid chromatography (HPLC) and mass spectrometry (MS). In brief, protein precipitation was carried out in methanol for plasma samples, and in ethanol/water 80:20 (v/v) using a Precellys 24 homogenizer for homogenized tumor samples. The HPLC/tandem mass spectrometry (MS-MS) system consisted of an Agilent 1100 Series HPLC system with a CTC HTC PAL Autosampler coupled to an Applied Biosystems API4000 mass spectrometer. HPLC separation was achieved on a reversed-phase column (Chromolith SpeedROD RP-18e, 50–3 mm) using gradient elution (eluent A: formic acid 0.1%; eluent B: acetonitrile). Selectivity was achieved using multiple reaction monitoring (MRM) for the MS/MS detection of the compounds. For the in vivo pharmacodynamic studies, all animal studies were conducted according to standard procedures approved by local animal welfare authorities. Mice were injected subcutaneously with 5 × 106 Hs746T cells (100 μL). Once the tumor volume had reached 600 to 1,000 mm3, mice were randomized into different experimental groups, receiving a single oral dose of 3, 10, 30, and 100 mg/kg of EMD 1214063, EMD 1204831, or vehicle. Tumor and plasma samples were collected at 3, 6, 12, 24, 48, 72, and 96 hours after treatment. Each experimental group comprised 4 mice per dose and time point. Samples of the tumor tissue were snap-frozen for pharmacokinetic and biomarker analyses, or formalin-fixed for immunohistochemical analysis.

Absorption
The absolute bioavailability of oral tepoltinib is approximately 72%. The recommended dose is 450 mg once daily, with a median time to peak concentration (Tmax) of 8 hours. The mean steady-state peak plasma concentration (Cmax) and the area under the curve (AUC0-24h) are 1291 ng/mL and 27438 ng·h/mL, respectively. Concomitant administration with high-fat, high-calorie foods can increase the AUC and Cmax of tepoltinib by approximately 1.6-fold and 2-fold, respectively.
Elimination Route
After oral administration, approximately 85% of the administered dose is excreted in the feces, with the remainder excreted in the urine. Approximately half of the fecal excretion is unmodified parent drug; the remainder includes a demethylated M478 metabolite, a glucuronide metabolite, a racemic M506 metabolite, and small amounts of oxidized metabolites. The unmodified original drug also accounts for approximately half of the urinary excretion, with the remainder comprising one glucuronide metabolite and a pair of N-oxide diastereomer metabolites.
Volume of Distribution
The mean apparent volume of distribution is 1038 L.
Clearance
The apparent clearance of tepotinib is 23.8 L/h.

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Metabolites
Tepotinib is primarily metabolized by CYP3A4 and CYP2C8, with some unidentified UGT enzymes also contributing. Metabolite M506 is the major circulating metabolite, accounting for approximately 40.4% of the total drug observed in plasma, while the glucuronide metabolite M668 is present in much lower amounts in plasma (approximately 4% of the oral dose). Ten phase I and II metabolites were detected after administration of tepotinib, most of which were excreted in feces.

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Biological Half-Life
The half-life of tepotinib after oral administration is approximately 32 hours.

Hepatotoxicity
Liver dysfunction was common in pre-marketing clinical trials of tepotinib in patients with solid tumors harboring MET mutations, but it was usually self-limiting and mild. 44% of patients treated with tepotinib experienced varying degrees of ALT elevation, with 4% experiencing ALT elevations exceeding 5 times the upper limit of normal (ULN). In these trials involving 255 patients, 3% discontinued treatment due to elevated ALT or AST, but less than 1% permanently discontinued. The median time to onset of liver dysfunction was 30 days 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 tepotinib product label recommends routine liver function tests every 2 weeks before treatment initiation, for the first 3 months of treatment, and monthly as clinically necessary thereafter.
Probability score: E (Rare, unproven but suspected 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 tepoltinib during lactation. Because tepoltinib binds to plasma proteins at a rate of up to 98%, its concentration in breast milk may be very low. However, considering its potential toxicity to breastfed infants and its 32-hour half-life, the manufacturer recommends discontinuing breastfeeding during tepoltinib 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.

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Protein binding rate
Tepoltinib has a protein binding rate of approximately 98% in plasma, primarily binding to serum albumin and α1-acid glycoprotein. At clinically relevant exposure levels, plasma protein binding rate is independent of drug concentration.

[1]. EMD 1214063 and EMD 1204831 constitute a new class of potent and highly selective c-Met inhibitors. Clin Cancer Res, 2013, 19(11), 2941-2951.

[2]. The Effect of Selective c-MET Inhibitor on Hepatocellular Carcinoma in the MET-Active, β-Catenin-Mutated Mouse Model. Gene Expr. 2018 May 18;18(2):135-147.

[3]. IS4-1 - A Phase I Dose-Escalation Study of emd 1214063, an Oral Selective CMET Inhibitor, in Patients with Advanced Solid Tumors. Annals of Oncology. Volume 23, Supplement 11, October 2012, Page xi21.

See also: tepotinib hydrochloride (note moved to).

C29H28N6O2.XHCL

492.57 (free base)

528.204

1103508-80-0

1100598-32-0;1946826-82-9

25171647

Typically exists as solid at room temperature

1

7

7

38

880

0

C(C1C=CC=C(C2=NC=C(OCC3CCN(C)CC3)C=N2)C=1)N1C(C=CC(C2C=CC=C(C#N)C=2)=N1)=O.Cl

YHHHGHDGBUUWIS-UHFFFAOYSA-N

InChI=1S/C29H28N6O2.ClH/c1-34-12-10-21(11-13-34)20-37-26-17-31-29(32-18-26)25-7-3-5-23(15-25)19-35-28(36)9-8-27(33-35)24-6-2-4-22(14-24)16-30;/h2-9,14-15,17-18,21H,10-13,19-20H2,1H3;1H

3-[1-[[3-[5-[(1-methylpiperidin-4-yl)methoxy]pyrimidin-2-yl]phenyl]methyl]-6-oxopyridazin-3-yl]benzonitrile;hydrochloride

EMD-1214063 hydrochloride; 8B73AZL5XP; 1103508-80-0; Tepotinib hydrochloride anhydrous; UNII-8B73AZL5XP; Benzonitrile, 3-(1,6-dihydro-1-((3-(5-((1-methyl-4-piperidinyl)methoxy)-2-pyrimidinyl)phenyl)methyl)-6-oxo-3-pyridazinyl)-, hydrochloride (1:1); SCHEMBL1295616; YHHHGHDGBUUWIS-UHFFFAOYSA-N; 3-(1-{3-[5-(1-methyl-piperidin-4-ylmethoxy)-pyrimidin-2-yl]-benzyl}-6-oxo-1,6-dihydro-pyridazin-3-yl)-benzonitrile hydrochloride;

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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.)