生物活性

Fosbretabulin Disodium is a phosphorylated microtubule depolymerizing molecule which induces tumor necrosis by regression of unstable nascent tumor neo-vessels. Studies suggest that Fosbretabulin Disodium selectively inhibits endothelial tumor cells via disrupting VE-cadherin. In addition, Fosbretabulin Disodium can increase the permeability of endothelial cells while inhibiting their migration and suppress capillary tube formation via disruption of VE-cadherin. Furthermore, Fosbretabulin Disodium can change the morphology of endothelial cells by altering their tubulin cytoskeleton.

Cytotoxic IC50 value of CA-4 in human bladder cancer cells is below 4 nM.[2]

体外研究

体内研究

激酶实验

细胞实验

Reagents[3]

When HUVECs were incubated with FGF-2, 1% heparin (Sigma-Aldrich) was systematically added in the cell culture medium. CA4P was dissolved in PBS to 0.01 M and stored at 4°C. Further dilutions to working concentrations were made before use.

Cell culture

HUVECs and SMCs were isolated from umbilical cord veins with collagenase and were cultured in M199 medium containing 10% (vol/vol) FBS, 20 μg/ml endothelial cell growth factor, 50 μg/ml heparin, 100μg/ml penicillin, and 100 μg/ml streptomycin in a humidified incubator at 37°C with air/5% CO2. HUVEC and SMC monolayers from passages 2–4 were used in these studies.

Cell proliferation assay

For the proliferation assay, we used the minimal concentration of FBS (1%) diluted in X-VIVO medium (Cambrex Corp.) to allow sufficient viability of endothelial cells. Briefly, after detachment, the cells were seeded at a concentration of 2 × 104HUVECs in each well of 24-well plates, allowed to adhere overnight, and then incubated with or without cytokines (5 ng/ml FGF-2 or 5 ng/ml VEGF-A). CA4P was added at concentrations indicated in Results. After incubation for 12, 24, 36, and 48 hours, cells were detached by trypsin/EDTA and manually counted using trypan blue exclusion. For the coculture experiment, HUVECs and SMCs were stained using red fluorescent cell linker PKH26 and green fluorescent cell linker PKH2, respectively, according to the manufacturer’s instructions, and 2.5 × 104HUVECs and 2.5 × 104 SMCs were seeded together in each well of 24-well plates in X-VIVO supplemented with 5% FBS. Cells were then differentially counted under a fluorescent microscope. Each fluorescent cell was checked for the presence of trypan blue by switching to white light.

Apoptosis analysis

FGF-2–stimulated endothelial cells were treated with CA4P (0, 10, and 50 nM) for 12, 24, or 36 hours and analyzed for the presence of apoptotic cells using the ApoAlert Annexin V-FITC PI Apoptosis Kit (BD), following the manufacturer’s instructions. Apoptotic analysis was performed using a Beckman-Coulter Elite Flow Cytometer.

动物实验

Animals and Tumors[4]

A C3H mammary carcinoma grown subcutaneously in the right rear foot of 10- to 14-week-old female CDF1 mice was used in this study. Tumor volume was calculated from the formula: Vtumor =π/6[(d1-0.5) (d2-0.5)]3/2, where d1and d2values represent two orthogonal diameters [mm], and 0.5 mm is subtracted from each measured diameter to compensate for skin thickness. Experiments were performed when tumors had reached a size of approximately 250 to 350 mm3 (typically 3 weeks after inoculation). C3H tumors of this size contain only little necrosis, are uniformly firm without ulcerations, and permit normal use of the foot, but are big enough to accommodate the needle for IFP measurements. The mice were kept in standard housing conditions with free access to food and water; lighting was controlled in a 12-hour light/dark cycle. National and institutional guidelines for animal welfare and experimental conduct were followed.

Drug Preparation

CA4DP was dissolved in isotonic normal saline at a concentration of 12.5 mg/ml such that injection of 0.2 ml corresponded to a dose of 100 mg/kg in a 25-g mouse. A single intraperitoneal injection of CA4DP at 100 mg/kg has been shown to be nontoxic and to elicit changes in tumor perfusion without significantly affecting tumor growth rates. CA4DP solutions were stored in the dark at +4OC and used within 1 week of preparation.

Laser Doppler Flowmetry (LDF) Perfusion Measurement

LDF was carried out using a setup with a Periflux Laser Doppler flowmeter 4001and a custom-built probe with four integrated laser/receiver units (outer diameter, 6mm; fiber separation, 250 mm; timeconstant, 0.2 s; PF415-175; Perimed). Laser light at a wavelength of 780 nm was transmitted into the skin from the 42OC heated probe. The probe was held steady in the desired position by a micromanipulator. LDF signal was recorded continuously for 2 to 3 minutes while the calculated perfusion in arbitrary perfusion units (PU) was monitored graphically. During recording, care was taken to position the probe such that respiratory movements did not influence the readings. As soon as steady state had been reached (typically in 60–90 seconds), three individual values were noted from the display. The median of these three values was used for further data analysis. The LDF measurement apparatus was calibrated to 250 PU in a ‘‘motility standard’’ reference solution before the measurements, and calibration was regularly confirmed. Calibration was stable over time.

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] JA Woods JH, GR Pettit, BW Fox, AT McGown. The interaction with tubulin of a series of stilbenes based on combretastatin A-4. Br J Cancer 1995;71(4):705-711.
[2] Shen CH, Shee JJ, Wu JY, Lin YW, Wu JD, Liu YW. Combretastatin A-4 inhibits cell growth and metastasis in bladder cancer cells and retards tumour growth in a murine orthotopic bladder tumour model. Br J Pharmacol. 2010;160(8):2008-2027.
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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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