human c-Met (Ki = 4.8 nM)

PF-04217903 mesylate (1-30 mg/kg; p.o.; daily for 16 days) inhibits tumor growth in a dose-dependent manner. This effect is correlated with the tumors' decreased c-Met phosphorylation[1]. PF-04217903 mesylate (5-50 mg/kg, p.o.; once daily for 3 days) induces apoptosis (cleaved caspase-3) in U87MG xenograft tumors at all dose levels while dose-dependently inhibiting phosphorylation of c-Met, Gab-1, Erk1/2, and AKT. In both the GTL-16 and U87MG models, PF-04217903 mesylate significantly and dose-dependently lowers human IL-8 levels, and in the GTL-16 model, it lowers human VEGFA levels. In U87MG xenograft tumors, PF-04217903 mesylate significantly increases phospho-PDGFRβ levels[1].

In 96-well plates, A549 cells expressing endogenous human WT c-Met are plated in growth medium and allowed to grow for the entire night. The growth medium is switched out for serum-free medium (containing 0.04% BSA) on the second day of the experiment. Each well receives serial dilutions of PF-04217903, and the cells are incubated for an hour at 37 °C. The cells are then treated with 40 ng/mL of HGF for 20 minutes. After giving the cells one wash in HBSS supplemented with 1 mM Na3VO4, lysis buffer is used to extract the protein from the cells. An ELISA technique that uses capture antibodies specific to c-Met and a detection antibody specific to phosphorylated tyrosine residues is used to measure the phosphorylation of c-Met. Protein lysates are added to antibody-coated plates, which are then incubated at 4 °C for an entire night before being seven times cleaned with 1% Tween 20 in PBS. Each plate is treated with 1:500 diluted horseradish peroxidase-conjugated anti-phosphotyrosine (HRP-PY20) for 30 minutes. After another round of washing the plates, the HRP-dependent colorimetric reaction is started with the addition of TMB peroxidase substrate, and it is halted with the addition of 0.09 N H2SO4. Using a spectrophotometer, the absorbance at 450 nm is used to determine the ELISA end points. By fitting a concentration-response curve with a four-parameter analytical method based on Microsoft Excel, the IC50 value is determined.

For four days, cells are exposed to varying concentrations of PF-04217903. Using a Coulter counter machine to count the contents of each well, cell proliferation is evaluated.

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Briefly, GTL-16 cells were plated at 20 000 cells per well in a 96-well plate and treated with either 0.5, 1, or 5 μM PF-04217903. The compound was replenished every 3–5 days as needed. Cells were grown in the presence of a drug for ∼4 months. The concentration of PF-04217903 was progressively increased once a month in 0.5 μM increments to a final concentration of 2.5 μM. Cells that survived in 2.5 μM PF-04217903 were expanded and subcloned. These resistant cells were referred to as R3 clones due to their rounded phenotype. Parental GTL16 and R3 cells were plated in 150 cm dishes in RPMI supplemented with 10% FBS and maintained at 37 °C in a humidified atmosphere at 5% CO2. At 70% confluency, GTL16 cells were starved overnight in RPMI/0.1% FBS. The following day each plate was treated with either DMSO control or 2 μM PF-04217903 for 6 or 24 h at 37 °C. Cells were lysed with modified RIPA buffer (150 mM NACl, 50 mM Tris-HCl, pH 7.4; 1% NP-40, 0.25% sodium deoxycholate, 1 mM EDTA) mixed with inhibitors and incubated on ice for 30 min. Lysis was completed by ultrasonication in 5–8 s pulses. Cell lysates were centrifuged at 15 000× g for 20 min (4 °C) to remove cellular debris. Protein yield of the supernatant was determined by BCA assay before storing the samples at −80 °C until phosphoprotein enrichment[4].

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Cell lines, including B16F1, Tib6, EL4, and LLC, and endothelial cells, HUVECs and C166, were seeded at 104 cells in each well of 24-well tissue culture–treated plates. Cells were grown in the standard media as described earlier. Cells were treated with different concentrations (2, 0.2, and 0.02 μmol/L) of sunitinib, PF-04217903, and combination of both compounds for 4 days. Efficacy of the compounds was measured by counting cells in a Coulter counter machine. Similar approach was applied to evaluate the role of HGF or VEGF on cell proliferation, using 3 different concentrations (10, 100, and 200 ng/mL) of each ligand[2].

Female nu/nu mice GTL-16 xenograft model
1, 3, 10, 30 mg/kg
Oral; daily for 16 days

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Nude mice were maintained under guidelines provided by the Pfizer IACUC. All the tumor cell lines (B16F1, EL4, LLC, and Tib6) in the current study were obtained from American Tissue Culture Collection and were cultured in RPMI 1640 supplemented with glutamine (2 mmol/L) and fetal bovine serum (FBS; 10%). All the cell lines in the current study were authenticated by the supplier. For implantation, tumor cells (1 × 106 cells per mouse) were resuspended in 100 μL of media and 100 μL of matrigel growth factor reduced and were subcutaneously implanted in one of the flanking areas. Tumor-bearing mice were treated once daily with sunitinib malate at 80 mg/kg or PF-04217903 (45 mg/kg) or the combination of both compounds, using oral route of administration. Tumors volumes were assessed using caliper measurement as described. HUVECs and C166 cells were purchased from Lonza Inc. and ATCC, respectively. For in vitro assays, HUVECs were grown in EBM2 media supplemented with a cocktail of growth factors provided by the supplier, and C166 were grown in DMEM supplemented with FBS (10%).

[1]. Biochemistry. 2009 Jun 16;48(23):5339-49.

[2]. Cancer Res. 2010 Dec 15;70(24):10090-100.

[3]. Eur J Cancer. 2011 May;47(8):1231-43..

[4]. J Proteome Res. 2011 Nov 4;10(11):5084-94.

2-[4-[3-(6-quinolinemethyl)-5-triazolo[4,5-b]pyrazinyl]-1-pyrazolyl]ethanol belongs to the quinoline class of compounds. PF-04217903 has been used in clinical trials for tumor treatment research. The MET tyrosine kinase inhibitor PF-04217903 is a small molecule tyrosine kinase inhibitor with high oral bioavailability and potential anti-tumor activity. PF-04217903 selectively binds to and inhibits c-Met, thereby disrupting the c-Met signaling pathway, which may lead to inhibition of tumor cell growth, migration, and invasion, and induce death in c-Met-expressing tumor cells. The c-Met receptor tyrosine kinase, also known as the hepatocyte growth factor (HGF) receptor, is overexpressed or mutated in various tumor cell types and plays an important role in tumor cell proliferation, survival, invasion, metastasis, and angiogenesis. c-Met receptor tyrosine kinase (RTK) is a key regulator of cancer, partly through oncogenic mutations. We characterized eight clinically significant mutants using biochemical, biophysical, and cellular methods. The c-Met catalytic domain exhibits high activity in the unphosphorylated state (k(cat) = 1.0 s(-1)), and the catalytic efficiency (k(cat)/K(m)) increases by 160-fold after activation, reaching 425,000 s(-1) M(-1). The basal enzyme activity (k(cat)) of c-Met mutants is 2-10 times higher than that of wild-type, but their maximal activity is similar to that of wild-type c-Met, while the maximal activity of the Y1235D mutant is reduced. Even small increases in basal activity can significantly promote the gain of enzyme activity, which is achieved by accelerating the rate of autophosphorylation. Biophysical analysis of the c-Met mutants showed minimal differences in melting temperature, indicating that these mutations did not alter the protein's stability. This paper presents an RTK activation model to describe how RTK responses are matched to the biological environment through enzymatic properties. Two clinical candidates for c-Met (PF-02341066 and PF-04217903) derived from aminopyridine and triazolopyrazine compounds, respectively, were investigated. Biochemically, each series yielded molecules with high selectivity for multiple kinases, with PF-04217903 (more than 1000-fold selectivity for 208 kinases) showing higher selectivity than PF-02341066. Although these prototype inhibitors have similar potency against wild-type c-Met (K_i = 6–7 nM), significant differences in potency were observed in clinically relevant mutants evaluated under biochemical and cellular conditions. In particular, PF-02341066 exhibited 180-fold higher activity against the Y1230C mutant c-Met than PF-04217903. These highly optimized inhibitors suggest that inhibitor design may require a balance between overall kinase selectivity and the ability to inhibit multiple mutant kinases (penetration) for kinases susceptible to mutations at active sites. [1]

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The molecular and cellular mechanisms of resistance/hyporesponsiveness to anti-angiogenic compounds are being extensively investigated. Both tumor cells and the stroma (non-tumor components) appear to be associated with inherent/acquired resistance to anti-angiogenic therapies. This study investigated the in vivo efficacy of sunitinib in experimental models to identify tumors resistant to/sensitive to the therapy. Analysis of tumor protein lysates showed that the concentration of hepatocyte growth factor (HGF) was higher in resistant tumors than in sensitive tumors. Furthermore, flow cytometry analysis revealed that c-Met expression was significantly higher in endothelial cells than in tumor cells, suggesting that HGF may target vascular endothelial cells in resistant tumors. In resistant tumors, sunitinib in combination with a selective c-Met inhibitor significantly inhibited tumor growth, superior to sunitinib or a c-Met inhibitor alone. Histological and in vitro analyses indicated that the combination therapy primarily targeted the vascular system of resistant tumors. Conversely, systemic injection of HGF in sensitive tumor models can confer sunitinib resistance by maintaining tumor angiogenesis. In summary, our study suggests that the HGF/c-Met pathway plays a role in the development of resistance to anti-angiogenic therapy and suggests a potential strategy to overcome resistance to vascular endothelial growth factor receptor tyrosine kinase inhibitors in the clinical setting. [2] Cetuximab (Erbitux®) targets the epidermal growth factor receptor (EGFR) and is approved for the treatment of colorectal and head and neck cancers. Despite the widespread expression of EGFR, only a subset of cancer patients respond to cetuximab therapy. In this study, we evaluated the in vivo response to cetuximab in 79 human xenografts from five tumor tissue types. We analyzed the basic characteristics of the tumors, including EGFR expression and activation, mutation status of KRAS, BRAF and NRAS, expression of EGFR ligands, and activation of HER3 (ErbB3) and hepatocyte growth factor receptor MET. Based on these results, we proposed a cetuximab response score that incorporates positive and negative factors that affect treatment response. Positive factors are the high expression and activation of EGFR and its ligand epidermal regulatory protein or bimodalin, while negative factors are markers of downstream pathway activation unrelated to EGFR. In cetuximab-resistant non-small cell lung cancer adenocarcinomas LXFA 526 and LXFA 1647, we found MET overexpression and strong MET activation due to gene amplification. In the corresponding cell lines, knockdown of the MET gene by siRNA showed that anchorage-independent growth and migration depended on MET. MET knockdown made LXFA 526L and LXFA 1647L cells more sensitive to EGF. MET inhibitors combined with cetuximab have an additive effect. Therefore, in some lung cancer patients, the combination of cetuximab and MET inhibitors may have important clinical significance. [3] In recent years, significant progress has been made in the development of anticancer drugs, including drugs that target protein tyrosine kinases (such as c-Met receptors). c-Met receptors are closely related to the occurrence and development of various cancers. However, despite these advances, drug resistance remains the leading cause of cancer treatment failure, and understanding the mechanisms of resistance remains a major challenge in treating patients with relapsed disease. PF-04217903 is a small-molecule c-Met kinase inhibitor that effectively inhibits c-Met-driven processes in various tumor cells, including growth (proliferation and survival), migration, invasion, and morphology. Resistance to PF-04217903 was observed in the GTL-16 gastric cancer cell line, which possesses constitutively activated c-Met receptors. This study employed mass spectrometry (MS)-based quantitative phosphorylated proteomics analysis to determine changes in signaling pathways in parental cells after c-Met inhibition and to investigate changes in protein levels and related classical pathways in parental cells and PF-04217903-resistant (R3) clones after c-Met inhibition. The quantitative mass spectrometry workflow included enrichment of phosphorylated proteins from cell lysates under six treatment conditions: in-solution enzymatic digestion, chemical labeling of peptides using a set of six isotope tandem mass spectrometry tags (TMT), hydrophilic interaction chromatography (HILIC) separation, phosphorylated peptide enrichment, and nano-liquid chromatography-tandem mass spectrometry (nano LC-MS/MS) analysis on an LTQ-Orbitrap mass spectrometer. Analysis of these quantitative datasets using Ingenuity Pathways Analysis (IPA) software revealed pathway changes under different treatment conditions, consistent with previously observed transcriptomic and phenotypic changes. Proteomics analysis also showed increased B-Raf expression in the R3 clone. Expression profiling confirmed upregulation of the B-Raf gene copy number and indicated the presence of B-Raf mutants. Using a bottom-up mass spectrometry approach, SND-1 was identified as a B-Raf fusion partner. The discovery of this novel B-Raf fusion protein provides a new target for the treatment of patients resistant to c-Met inhibitors and has potential clinical significance. [4]

C20H20N8O4S

468.492

468.132

C, 51.28; H, 4.30; N, 23.92; O, 13.66; S, 6.84

956906-93-7

PF-04217903;956905-27-4;PF-04217903 phenolsulfonate;1159490-85-3

24852079

White to off-white solid powder

2.258

2

10

5

33

617

0

O=S(C)(O)=O.OCCN1C=C(C2C=NC3N=NN(C=3N=2)CC2C=C3C(N=CC=C3)=CC=2)C=N1

HBEMHKVWZJTVOC-UHFFFAOYSA-N

InChI=1S/C19H16N8O.CH4O3S/c28-7-6-26-12-15(9-22-26)17-10-21-18-19(23-17)27(25-24-18)11-13-3-4-16-14(8-13)2-1-5-20-16;1-5(2,3)4/h1-5,8-10,12,28H,6-7,11H2;1H3,(H,2,3,4)

methanesulfonic acid;2-[4-[3-(quinolin-6-ylmethyl)triazolo[4,5-b]pyrazin-5-yl]pyrazol-1-yl]ethanol

PF04217903 mesylate; PF 04217903; PF-04217903; PF-04217903 methanesulfonate; PF-04217903 mesylate; PF 04217903 Mesylate; PF-04217903.MsOH; PF-04217903 (methanesulfonate); CHEMBL2170804; 956906-93-7 (mesylate); PF4217903 mesylate; PF-4217903; PF 4217903 mesylate

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, avoid exposure to moisture.

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: ≥ 3 mg/mL (6.40 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 30.0 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: ≥ 3 mg/mL (6.40 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (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 30.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 3: 1% DMSO+30% polyethylene glycol+1% Tween 80: 30 mg/mL

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