c-Met (IC50 = 4 nM)

EMD 1214063 treatment causes c-Met phosphorylation in Hs746T xenograft tumors to be more than 90% inhibited for at least 72 hours at doses of 10 mg/kg or higher. When treated with doses of 100 mg/kg, EMD 1214063 causes a more than 50% reduction in cyclin D1 expression, which lasts for 96 hours. Following treatment with EMD 1214063, a brief induction of p27 and cleaved caspase-3 are also seen. Treatment with EMD 1214063 (15 mg/kg, daily) results in total regression of gastric carcinoma xenografts Hs746T, wherein c-Met is overexpressed, amplified, and activated in a manner that is independent of ligands. [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.

Human gastric carcinoma xenografts Hs746T
15 mg/kg
daily

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Xenograft models of antitumor efficacy[1]
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, Distribution and Excretion
The absolute bioavailability of tepotinib following oral administration is approximately 72%. At the recommended dosage of 450mg once daily, the median Tmax is 8 hours and the mean steady-state Cmax and AUC0-24h were 1,291 ng/mL and 27,438 ng·h/mL, respectively. Co-administration with a high-fat, high-calorie meal increases the AUC and Cmax of tepotinib by approximately 1.6-fold and 2-fold, respectively.
Following oral administration, approximately 85% of the given dose is excreted in the feces with the remainder excreted in the urine. Unchanged parent drug accounts for roughly half of the dose excreted in the feces, with the remainder comprising the demethylated M478 metabolite, a glucuronide metabolite, the racemic M506 metabolite, and some minor oxidative metabolites. Unchanged parent drug also accounts for roughly half of the dose excreted in the urine, with the remainder comprising a glucuronide metabolite and a pair of N-oxide diastereomer metabolites.
The mean apparent volume of distribution is 1,038L.
The apparent clearance of tepotinib is 23.8 L/h.

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Metabolism / Metabolites
Tepotinib is metabolized primarily by CYP3A4 and CYP2C8, with some apparent contribution by unspecified UGT enzymes. The metabolite M506 is the major circulating metabolite, comprising approximately 40.4% of observed drug material in plasma, while the M668 glucuronide metabolite has been observed in plasma at much lower quantities (~4% of an orally administered dose). A total of 10 phase I and phase II metabolites have been detected following tepotinib administration, most of which are excreted in the feces.

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Biological Half-Life
Following oral administration, the half-life of tepotinib is approximately 32 hours.

Hepatotoxicity
In the prelicensure clinical trials of tepotinib 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 44% of tepotinib treated patients and were above 5 times the upper limit of normal (ULN) in 4%. In these trials that enrolled 255 patients, dose interruptions due to ALT or AST elevations occurred in 3%, but permanent discontinuations in less than 1%. The liver test abnormalities had a median onset of 30 days 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 tepotinib 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 tepotinib during breastfeeding. Because tepotinib is 98% bound to plasma proteins, the amount in milk is likely to be low. However, because of its potential toxicity in the breastfed infant and its half-life of 32 hours, the manufacturer recommends that breastfeeding be discontinued during tepotinib 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
Tepotinib is approximately 98% protein-bound in plasma, primarily to serum albumin and alpha-1-acid glycoprotein. Plasma protein binding is independent of drug concentration at clinically relevant exposures.

[1]. Clin Cancer Res . 2013 Jun 1;19(11):2941-51.

Tepotinib is a MET tyrosine kinase inhibitor intended to treat a variety of MET-overexpressing solid tumors. It was originally developed in partnership between EMD Serono and the University of Texas M.D. Anderson Cancer Center in 2009 and has since been investigated in the treatment of neuroblastoma, gastric cancers, non-small cell lung cancer, and hepatocellular carcinoma. MET is a desirable target in the treatment of certain solid tumors as it appears to play a critical role, both directly and indirectly, in the growth and proliferation of tumors in which it is overexpressed and/or mutated. Tepotinib was first approved in Japan in March 2020 for the treatment of non-small cell lung cancers (NSCLC) with MET alterations, and was subsequently granted accelerated approval by the US FDA in February 2021, under the brand name Tepmetko, for the treatment of adult patients with metastatic NSCLC and MET exon 14 skipping alterations. It is the first oral MET-targeted tyrosine kinase inhibitor to allow for once-daily dosing, an advantage that may aid in easing the pill burden often associated with chemotherapeutic regimens. In February 2022, tepotinib was approved for use in Europe.
Tepotinib is a Kinase Inhibitor. The mechanism of action of tepotinib is as a Mesenchymal Epithelial Transition Inhibitor, and P-Glycoprotein Inhibitor.
Tepotinib is an orally available, small molecule inhibitor of the mesenchymal-epithelial transition (MET) factor tyrosine kinase receptor that is used to treat selected cases of non-small cell lung cancer (NSCLC). Serum aminotransferase elevations are common during therapy with tepotinib, but it has not been linked to instances of clinically apparent liver injury with jaundice.
Tepotinib is an orally bioavailable inhibitor of MET tyrosine kinase with potential antineoplastic activity. Tepotinib selectively binds to MET tyrosine kinase and disrupts MET signal transduction pathways, which may induce apoptosis in tumor cells overexpressing this kinase. The receptor tyrosine kinase MET (also known as hepatocyte growth factor receptor or HGFR), is the product of the proto-oncogene c-Met and is overexpressed or mutated in many tumor cell types; this protein plays key roles in tumor cell proliferation, survival, invasion, and metastasis, and tumor angiogenesis.
See also: Tepotinib Hydrochloride (active moiety of).

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Drug Indication
Tepotinib is indicated for the treatment of adult patients with metastatic non-small cell lung cancer (NSCLC) who have mesenchymal-epithelial transition (_MET_) exon 14 skipping alterations.
Tepmetko as monotherapy is indicated for the treatment of adult patients with advanced non-small cell lung cancer (NSCLC) harbouring alterations leading to mesenchymal-epithelial transition factor gene exon 14 (METex14) skipping, who require systemic therapy following prior treatment with immunotherapy and/or platinum-based chemotherapy.
Treatment of non-small cell lung carcinoma (NSCLC)

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Mechanism of Action
Mesenchymal-epithelial transition factor (MET) is a receptor tyrosine kinase found overexpressed and/or mutated in a variety of tumor types, thus making it a desirable target in their treatment. MET plays a critical role in the proliferation, survival, invasion, and mobilization of tumor cells, and aberrant MET activation is thought to contribute to the development of more aggressive cancers with poorer prognoses. Tepotinib is a kinase inhibitor directed against MET, including variants with exon 14 skipping - it inhibits MET phosphorylation and subsequent downstream signaling pathways in order to inhibit tumor cell proliferation, anchorage-independent growth, and migration of MET-dependent tumor cells. Tepotinib has also been observed to down-regulate the expression of epithelial-mesenchymal transition (EMT) promoting genes (e.g. MMP7, COX-2, WNT1, MUC5B, and c-MYC) and upregulate the expression of EMT-suppressing genes (e.g. MUC5AC, MUC6, GSK3β, and E-cadherin) in c-MET-amplified gastric cancer cells, suggesting that the tumor-suppressing activity of tepotinib is driven, at least in part, by the negative regulation of c-MET-induced EMT. It has also been shown to inhibit melatonin 1B and nischarin at clinically relevant concentrations, though the relevance of this activity in regards to tepotinib's mechanism of action is unclear.

C29H28N6O2

492.57

492.227

C, 70.71; H, 5.73; N, 17.06; O, 6.50

1100598-32-0

1100598-32-0; 1103508-80-0 (HCl);1946826-82-9

25171648

White to light yellow solid powder

1.3±0.1 g/cm3

626.5±65.0 °C at 760 mmHg

332.7±34.3 °C

0.0±1.8 mmHg at 25°C

1.660

2.72

0

7

7

37

880

0

O(C1=C([H])N=C(C2=C([H])C([H])=C([H])C(=C2[H])C([H])([H])N2C(C([H])=C([H])C(C3=C([H])C([H])=C([H])C(C#N)=C3[H])=N2)=O)N=C1[H])C([H])([H])C1([H])C([H])([H])C([H])([H])N(C([H])([H])[H])C([H])([H])C1([H])[H]

AHYMHWXQRWRBKT-UHFFFAOYSA-N

InChI=1S/C29H28N6O2/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

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

EMD-1214063; MSC-2156119; EMD1214063; EMD 1214063; MSC2156119; MSC 2156119; Tepotinib

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)