生物活性

Tivantinib is a novel and selective human Met inhibitor with IC50 of 0.1 μM. Tivantinib (ARQ 197), a highly selective orally administerednon-ATP-competitive c-Met allosteric inhibitor, has proven antiproliferativeeffects in hepatocellular carcinoma currently under phase 3 clinical trial.

Kinase activity of Tivantinib

Cytotoxicityof ARQ 197[3]

Antiproliferative activity of ARQ 197

OSCC: Oral squamous cell carcinoma, HCC: hepatocellularcarcinoma, NSCLC: non-small-cell lung cancer.

体外研究

体内研究

激酶实验

c-Met SDS-PAGE in vitro kinase assay[3]

Recombinant c-Met protein (100ng) waspreincubated with increasingcon centrations of ARQ 197 for 30 minutes at roomtemperature. Following preincubation, 100μmol/L of poly-Glu-Tyr substrate andvarious concentrations of ATP containing 5μCi of [γ-³²P] ATP wereadded to the reaction mixture. The reaction was incubated for 5 minutes at roomtemperature and then stopped by the addition of 5μL of SDS-polyacrylamide gel,reducing sample buffer. The samples were then loaded onto a 7.5% acrylamide geland SDS-PAGE was performed. The phosphorylated poly-Glu-Tyr substrates wereultimately visualized by autoradiography. c-Met activity was quantified bydensitometry using the Scion Image software.

细胞实验

Cell lines culture and reagents[8]

Huh7, HepG2, Hep3B, Chang cells (hepatocellularcarcinoma), TFK1 (human cholangiocarcinoma), DLD1 cells (human coloncarcinoma), PL5 and PANC1 cells (human pancreatic carcinoma) were used for invitro experiments. All cell lines were cultivated in standard cell culturemedia as recommended by the providers. Tivantinib (ARQ197) was dissolved in100% DMSO and stored at −20°C. Tigatuzumab (CS-1008) was provided as a 10mg/mlsolution and stored at 4°C.

Cellviability assay

Cells were seeded onto 96-well plates atdifferent cell densities to avoid overgrowth (0.6–3.5 × 10³/well)and were treated with increasing concentrations of tivantinib or vehicle. Toinvestigate the effect of tivantinib on cell viability at low concentrations,cells were kept in culture for6 days. At day 6 cells were washed with PBS,underwent osmotic-lysis in 100μl ddH2O, and then incubated in 5% CO₂ incubator for 45minutes. Fluorescence was measured after addition of 0.2% Sybrgreen.

Colonyformation assay

4–5 × 10³ cells were plated onto6-well plates. After 24-h incubation with tivantinib cells were allowed to growfor 3 weeks. Colony formation was evaluated after the cells were fixed in 9%paraformaldehyde and stained with crystal violet for 30 minutes. Total cellcolonies in each well were counted after being photographed.

Apoptosisand cell cycle assay

8.0 × 10⁴−1.5 × 10⁵ cells were seeded in12-well plates and treated after overnight incubation. Fluorescenceactivated cell sorting was performed to detect the sub-G1 cell fraction todetermine apoptosis and the respective different phases of cell cycle afterpropidium iodide (PI) staining. In addition, apoptosis was assessedmorphologically by Hoechst 33342 staining and fluorescence microscopy.

动物实验

Animal care[4]

Female BALB/c athymic nude mice, 5 to 6weeks old were used for in vivo studies. All animals were fed a standard dietad libitum and housed in a temperature-controlled animal facility with a 12/12h light/dark cycle.

Xenografttransplantation experiments

MHCC97L xenograft models were establishedby subcutaneous injection of tumor cells (5 × 10⁷/ml) in PBS with Matrigelat a 1:1 ratio. These cell suspensions were injected in a total volume of 0.2ml into the right flank of each mouse and allowed to grow for approximately twoweeks to reach a tumour size of roughly 80 to 200 mm³. The mice werethen randomised into three groups (n= 6/group): Vehicle control (orally),tivantinib (100mg/kg/d, orally) or tivantinib (200mg/kg/d, orally). The tumordimensions and body weights were measured every 3 days starting with the firstday of treatment. The tumor volume (mm³) was calculated using thefollowing formula: (l × w²)/2, where l and w refer to the larger andsmaller dimensions collected at each measurement. The mice were sacrificed, andsolid tumors were measured and excised after 15 days of treatment. The tumorgrowth inhibition (TGI) rate was calculated according to the following formula:(1 - T/C) × 100, where T indicates the mean weight of the tumor test groups andC indicates the mean tumor weight of the vehicletreated group. To evaluate theeffect of tivantinib on inhibition of MET expression in vivo, the xenograftsestablished by MHCC97L cells were not initiated treatment until tumors reached300 to 400 mm³ in size. The mice were then randomized into threegroups (n = 3/group): ddH2O (orally), tivantinib (200mg/kg/d, orally),orJNJ-38877605 (20mg/kg/d, orally) for three days. The mice were sacrificed 3 hafter the last treatment, and solid tumors were excised, split into two pieces,and either processed for paraffin embedding or homogenized in tumor lysisbuffer for Western blot analysis.

不同实验动物依据体表面积的等效剂量转换表（数据来源于FDA指南）

例如，已知某工具药用于小鼠的剂量为88 mg/kg , 则用于大鼠的剂量换算方法：将88 mg/kg 乘以小鼠的Km系数（3），再除以大鼠的Km系数（6），得到该药物用于大鼠的等效剂量44 mg/kg。

参考文献

[1] Trojan J, Zeuzem S. Tivantinib in hepatocellular carcinoma. Expert Opin Investig Drugs. 2013;22(1):141-147.
[2] Remsing Rix LL, Kuenzi BM, Luo Y, et al. GSK3 alpha and beta are new functionally relevant targets of tivantinib in lung cancer cells. ACS Chem Biol. 2014;9(2):353-358.
[3] Munshi N, Jeay S, Li Y, et al. ARQ 197, a novel and selective inhibitor of the human c-Met receptor tyrosine kinase with antitumor activity. Mol Cancer Ther. 2010;9(6):1544-1553.
[more]

体内溶解度

Clinical Trial Information ( data from http://clinicaltrials.gov )

注：以上所有数据均来自公开文献，并不保证对所有实验均有效，数据仅供参考。

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