生物活性

Ceritinib is potent inhibitor against ALK with IC50 of 0.2 nM, shows 40- and 35-fold selectivity against IGF-1R and InsR, respectively. Ceritinib shows great anti-proliferative activity in Ba/F3-NPM-ALK and Karpas290 cells with IC50 of 26.0 nM and 22.8 nM. Ceritinib (LDK378) is a novel, oral ATP-competitive, highly potent, and selectivesecond-generation ALK inhibitor with demonstrated efficacy in patients withNSCLC who have acquired resistance to crizotinib.

Kinaseactivities of ceritinib[1]

Thecytotoxicity of ceritinib[2]

Growthinhibition of ceritinib[1]

体外研究

体内研究

激酶实验

Enzymatic Kinase Profiling Description[1]

All kinases were expressed as eitherhistidine- or GST-tagged fusion proteins using the baculovirus expressiontechnology except for the untagged ERK2 which was produced in E. coli.AURORA-A, JAK2, MK2, SYK, PKA and ERK2 were purchased. All other kinases weresupplied in-house. The kinase activity was measured in the Lab Chip mobility-shiftassay (PerkinElmer). The assay was performed at 30 °C for 60 min. The effect ofcompound on the enzymatic activity was obtained from the linear progress curvesin the absence and presence of compound and routinely determined from onereading (end point measurement).

细胞实验

Cell culture[2]

The human oral epidermoid carcinoma cellline KB and its vincristine-selected ABCB1 overexpressing derivative KBv200cells; the human breast carcinoma cell line MCF-7, its doxorubicin selectedABCB1 overexpressing derivative MCF-7/adr cells; the human colon carcinoma cellline S1 and its mitoxantrone-selected ABCG2-overexpressing derivative S1-M1-80cells and the human embryonic kidney cell line HEK293 and its stable pcDNA3.1,ABCB1 and ABCG2 stable gene transfect cell lines HEK293/pcDNA3.1, HEK293/ABCB1,HEK293/ABCG2-R2 were used. The transfected cells were cultured in mediumcontaining 2 mg/mL G418. All cell lines were cultured in DMEM or RPMI 1640supplemented with 10% FBS at 37°C in a humidified atmosphere of 5% CO₂.All cells were grown in drug-free culture medium for more than 2 weeks beforeassay.

Cell cytotoxicity assay

The MTT assay was used to assesscytotoxicity. Briefly, cells growing in logarithmic phase were seeded at adensity of 2000 ~ 6000 cells per well in 96-well plates. When the cells becameadherent 24 h later, a range of different concentrations of ceritinib wereadded to the wells. After 68 h, MTT (5 mg/ml, 20μL) was added into each well,and 4 h later, the medium was discarded and 150μL DMSO was added into the wellsto dissolve the formazan product from the metabolism of MTT. Finally, opticaldensity was measured at 540nm, with background subtraction at 670 nm by a Model550 Microplate Reader. The half maximal (50%) inhibitory concentration (IC50)value of a substance was calculated by the Bliss method. The fold reversal ofMDR was calculated. All experiments were repeated at least three times.

动物实验

In vivo antitumor assays[3]

RNU nude rats bearing the H2228 tumors wererandomized into four groups (n = 6 rats per group) with an average tumor sizeof 150mm³. Compounds 7, 9 and ceritinibwas formulated in 0.5%MC/0.5% Tween 80 and administered by oral gavage at a dosing volume of 10 mL/gof an animal body weight. Animals in each group received vehicle, compounds 7,9 or ceritinib every day for 14 consecutive days. During treatment, body weightwas monitored regularly. Tumor volume was calculated by the formula (V=ab²/2,where a and b stand for the longest and shortest diameter, respectively). Aftertreated for 14 days with drugs, the animals were sacrificed and solid tumorswere removed and weighted. The inhibition rate was calculated as [(averaged tumorweight of the control group e averaged tumor weight of drug-treatedgroup)/averaged tumor weight of the control group] x100%.

SD male rats (weight 200-220 g) wererandomized into three groups (n = 4 rats per group). Compounds 7, 9 andceritinib was formulated in 0.5% MC/0.5% Tween 80 and administered by oral gavageat a dosing volume of 10 mL/g of an animal body weight. Animals in each groupreceived compounds 7, 9 or ceritinib. Plasma samples were collected 0.5, 1, 3,5, 8 and 24 h after dosing. Aliquots of all biological matrixes weredeproteinized with ethyl acetate. The suspension was vortexed, mixed, andcentrifuged at 4000 rpm for 5 min. The organic phase was injected into the HPLCsystem.

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] Marsilje TH, Pei W, Chen B, et al. Synthesis, structure-activity relationships, and in vivo efficacy of the novel potent and selective anaplastic lymphoma kinase (ALK) inhibitor 5-chloro-N2-(2-isopropoxy-5-methyl-4-(piperidin-4-yl)phenyl)-N4-(2-(isopropylsulf onyl)phenyl)pyrimidine-2,4-diamine (LDK378) currently in phase 1 and phase 2 clinical trials. J Med Chem. 2013;56(14):5675-5690.
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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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