生物活性

Olaparib is a potent inhibitor of both PARP-1 and PARP-2 （with IC50 = 5 nM and 1 nM, respectively）. Olaparib is strongly sensitive to KB2P cells due to suppression of base excision repair by PARP inhibition, which may result in the conversion of single-strand breaks to double-strand breaks during DNA replication, activating BRCA2-dependent recombination pathways.

体外研究

体内研究

15% Captisol

激酶实验

In Vitro Isolated Enzyme Assay[1]

This assay determined the ability of testcompounds to inhibit PARP-1 enzyme activity. PARP-2 activity inhibition was measuredby using a variation of the PARP-1 assay in which PARP-2 protein (recombinant)was bound down by a PARP-2 specific antibody in a 96-well white-walled plate.PARP-2 activity was measured following 3H-NAD+ DNA additions. After washing, scintillantwas added to measure ³H-incorporated ribosylations. For tankyrase-1,an AlphaScreen assay was developed in which HIS-tagged recombinant TANK-1protein was incubated with biotinylated NAD⁺ in a 384-wellProxiPlate assay. Alpha beads were added to bind the HIS and biotin tags tocreate a proximity signal, whereas the inhibition of TANK-1 activity was directlyproportional to the loss of this signal. All experiments were repeated at leastthree times.

细胞实验

Cell culture[2]

Human lung carcinoma cells Calu-3, Calu-6,and A549 were cultured in advanced Dulbecco’s modified Eagle medium/F12 mediumsupplemented with 5% fetal bovine serum, 2 mM glutamax, and 50μg/mLpenicillin/streptomycin in a humidified atmosphere with 7.5% CO₂.

Clonogenicsurvival assays

Exponentially growing cells were seededinto 6-well plates, and exposed to 21% O₂ (normoxia) or 1% O₂ (hypoxia) in a Whitley H35 Hypoxystation for 16 hours. An amount of 5μmol/Lolaparib or 0.05% DMSO was added to the medium 1 hour before irradiation (0-6Gy) with a cesium-137 source at1.69 Gy/min (GSR D1 irradiator) under normoxicor hypoxic conditions. Twenty-four hours after irradiation, cells were washed,and fresh medium was added. Plates were incubated for 6 to 13 days to allow coloniesto form. Colonies were then fixed and stained with0.5% crystal violet in 5%acetic acid and 75% methanol. Colonies (≥50 cells) were counted. Clonogenic survival curves and thesensitization enhancement ratio at 50% (SER50) were generated using OriginProversion 8.5.1 software.

动物实验

Animal Model[3]

Male Balb/c nu/nu mice weighing about 20 gat six-weeks old each were used in this study. They were maintained underspecific pathogen-free conditions throughout the experiments with constant roomtemperature, and a 12 h night and day cycle. The mice were continuouslysupplied with normal food chow and sterilized water ad libitum. They wereanesthetized and HSC-2, Ca9-22, and SAS cells were injected into their dorsalskin subcutaneously (5 x 10⁶ cells in 200μL growthmedium per mouse). Within two weeks, tumors usually enlarged to the diameter ofmore than 7 mm. Among the three cancer cell lines, only HSC-2 cells couldstably develop tumors. Therefore, in vivo experiments were performed usingHSC-2 cell-derived xenografted tumors.

AnimalExperimental Protocol

Once the tumor diameter had reached 7 mm,the mice were randomly assigned to the following groups: (a) control (200μLsaline); (b) cisplatin (2 mg/kg per body weight, dissolved in 200μL sterilizedwater); (c) AZD2281 (25 mg/kg per body weight, dissolved in 200μLsterilized water); or (d) combination (both cisplatin and AZD2281). Thechemicals were administered intraperitoneally every three days, five times.Although AZD2281 is administered orally in the clinic, intraperitonealinjection was recommended by the manufacturer because of easier manipulationand the ethical constraints associated with oral gavage administration to mice.Tumor size and body weight were measured at the time of administration. Thetumor volume was calculated using following equation

Three days after the last administration,all surviving mice were sacrificed.

HistopathologicalAnalysis

Mice were treated as described above andthen sacrificed. Tumors were removed and immediately fixed in 10% formalin/PBS for48 h, followed by immersion in 70% alcohol for 48 h and embedding in paraffin.Tissue sections (4μm) were mounted on silane-coated Slides, deparaffinized with xylene,and rehydrated with graded alcohol solutions. The specimens were pathologicallyanalyzed by hematoxylin and eosin staining.

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] Menear KA AC, Boulter R, Cockcroft XL, Copsey L, Cranston A, Dillon KJ, Drzewiecki J, Garman S, Gomez S, Javaid H, Kerrigan F, Knights C, Lau A, Loh VM Jr, Matthews IT, Moore S, O'Connor MJ, Smith GC, Martin NM. 4-[3-(4-cyclopropanecarbonylpiperazine-1-carbonyl)-4-fluorobenzyl]-2H-phthalazin-1-one: a novel bioavailable inhibitor of poly(ADP-ribose) polymerase-1. J Med Chem. 2008;51(20):6581-6591.
[2] Jiang Y, Verbiest T, Devery AM, et al. Hypoxia Potentiates the Radiation-Sensitizing Effect of Olaparib in Human Non-Small Cell Lung Cancer Xenografts by Contextual Synthetic Lethality. Int J Radiat Oncol Biol Phys. 2016;95(2):772-781.
[3] Yasukawa M, Fujihara H, Fujimori H, et al. Synergetic Effects of PARP Inhibitor AZD2281 and Cisplatin in Oral Squamous Cell Carcinoma in Vitro and in Vivo. Int J Mol Sci. 2016;17(3):272.

体内溶解度
7 mg/mL

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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