Microtubule; tubulin polymerization; β-tubulin (Kd = 0.4 μM)

FosbretabuLin disodium 抑制白血病 P-388、胰腺 BXPC-3、神经母细胞 SK-N-SH、甲状腺 SW1736、肺 NSC NCI-H460、前列腺 DU-145 和咽 FADU 的增殖，EC50 为 0.0029、0.23 和 0.00025分别为 0.00061、0.00035、0.00072 和 0.00045 μg/mL[8]。

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使用Combretastatin A4/考布他汀 A4 磷酸盐 (≥ 50 μM) 时，前向散射大大减少，膜联蛋白 V 结合细胞的比例显着增加。考布他汀 A4 磷酸盐不会显着增加溶血量。 Combretastatin A4 磷酸盐浓度为数百 μM 时可显着增强 Fluo3 荧光。当细胞外 Ca2+ 被去除时，Combretastatin A4 磷酸盐 (100 μM) 对膜联蛋白-V 结合的影响大大降低，但并未完全消除。考布他汀 A4 磷酸盐 (≥ 50 μM) 不会显着增加 ROS 和神经酰胺，但会显着降低 GSH 丰度和 ATP 水平 [2]。共封装阿霉素-考布他汀-A4磷酸盐（1:10）的聚合物胶囊对人鼻咽上皮癌（KB）细胞具有强协同细胞毒性[3]。这些重要分子的表达和 3-D 细胞中 VM 的数量不受考布他汀 A4 磷酸盐预处理的影响 [4]。

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将人红细胞暴露于Combretastatin A4/CA4P（≥50µM）48小时后，膜联蛋白-V结合细胞的百分比显著增加，前向散射显著降低Combretastatin A4/CA4P没有明显增加溶血。100µM CA4P显著增加了Fluo3荧光。通过去除细胞外Ca2+，CA4P（100µM）对膜联蛋白-V结合的影响显著减弱，但并未完全消除。CA4P（≥50µM）显著降低了GSH丰度和ATP水平，但没有显著增加ROS或神经酰胺。 结论：Combretastatin A4CA4P引发红细胞膜的细胞收缩和磷脂紊乱，这种作用至少部分是由于细胞外Ca2+的进入和能量消耗。[2]

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使用三维培养的体外模型来测试Combretastatin A4/CA4P对Walker 256细胞管形成的影响。进行Western blot分析以评估缺氧诱导因子（HIF）-1α和VM相关标志物的表达。在体外缺氧条件下48小时，W256细胞形成与VM标志物表达增加相关的VM网络。CA4P预处理不影响三维培养中VM的量以及这些关键分子的表达[4]。

在大鼠中，注射 fosbretabuLin disodium（100 mg/kg；腹膜内注射）可减少肿瘤血流量，并在注射后 1 小时和 6 小时提高平均动脉血压 (MABP)。在大鼠系统中评估了使用微管蛋白失稳剂考布他汀A-43-0-磷酸二钠（CA-4-P）靶向肿瘤血管的潜力。这种方法旨在关闭已建立的肿瘤血管系统，导致广泛的肿瘤细胞坏死。在皮下植入的P22癌肉瘤和一系列正常组织中评估了CA-4-P的早期血管效应。通过摄取放射性标记的碘安替比林来测量血流，并使用定量放射自显影来测量肿瘤切片中血流的空间异质性。CA-4-P（100mg/kg i.P.）在治疗后1和6小时导致平均动脉压显著升高，肿瘤血流量大幅减少，6小时后减少了约100倍。脾脏是受影响最严重的正常组织，6小时时血流量减少了7倍。血管阻力的计算显示，心脏和肾脏出现了一些血管变化，但血流量没有明显变化。定量放射自显影显示CA-4-P增加了肿瘤血流的空间异质性。该药物对周围肿瘤区域的影响小于中心区域。在一氧化氮合酶抑制剂N（ω）-硝基-L-精氨酸甲酯存在的情况下，给予CA-4-P（30mg/kg）可增强CA-4-P在肿瘤组织中的作用。与任何正常组织的不到7倍相比，该组合将肿瘤血管阻力增加了300倍。这表明，组织产生的一氧化氮可以防止CA-4-P对血管的破坏作用。在使用无细胞灌注液的孤立肿瘤灌注中，也可以获得肿瘤血管阻力的显著变化，尽管这些变化远小于体内观察到的变化。这表明CA-4-P的作用包括红细胞粘度、血管内凝血和中性粒细胞粘附以外的机制。CA-4-P和考布他汀A-4（CA-4）在肿瘤中的摄取比在骨骼肌组织中更有效，前者的CA-4-P去磷酸化为CA-4更快。这些结果有望将CA-4-P用作肿瘤血管靶向剂[9]。

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治疗30分钟后，给予120 mg/10 mL/kg Combretastatin A4磷酸二钠的大鼠具有更高的DBP和MBP。用康布他汀 A4 磷酸二钠 120 mg/10 mL/kg 治疗的大鼠显示出康布他汀 A4 及其磷酸盐的以下毒代动力学特征：康布他汀 A4 的 Cmax、T1/2 和 AUC0-inf 值为 156± 13 μM、5.87±1.69 h、89.4±10.1 h·μM[1]。 W256 肿瘤在考布他汀 A4 磷酸盐治疗后显示出明显的瘤内缺氧，这与 VM 发展的增加有关。 cercopetastatin A4 磷酸盐仅延迟肿瘤生长两天，但肿瘤生长很快恢复。第 8 天时，VM 密度与肿瘤重量和体积呈正相关。通过 HIF-1α/EphA2/PI3K/基质金属蛋白酶 (MMP) 信号通路，磷酸西西他汀 A4 刺激 W256 肿瘤中缺氧和 VM 形成，从而损害肿瘤更新[4]。

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在本研究中，我们设计了可生物降解的多聚体，用于联合递送抗血管生成药物Combretastatin A4磷酸盐（CA4P）和阿霉素（DOX），以破坏肿瘤新生血管系统并抑制癌症细胞增殖，目的是实现协同抗肿瘤效果。以甲氧基聚乙二醇-b-聚乳酸（mPEG-PLA）嵌段共聚物为药物载体，通过溶剂蒸发法制备了共包封DOX和CA4P的聚合物体（Ps-DOX-CA4P）。所得Ps-DOX-CA4P具有囊泡形状，大小均匀，约为50nm，DOX与CA4P的共包封率可控。更重要的是，Ps-DOX-CA4P（1:10）对人鼻咽表皮癌（KB）细胞显示出强烈的协同细胞毒性（组合指数CI=0.31）。此外，Ps-DOX-CA4P在裸鼠KB组织异种移植物中显著积累。与这些观察结果一致，Ps-DOX-CA4P（1:10）由于体内肿瘤血管系统的快速破坏和持续的肿瘤细胞增殖抑制而具有显著的抗肿瘤效力。总体研究结果表明，在多聚体中联合递送抗血管生成药物和化学治疗剂是癌症治疗的一种潜在的有前景的策略。[3]

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在体内，W256肿瘤在Combretastatin A4/CA4P治疗后表现出明显的瘤内缺氧，并伴有VM形成增加。CA4P在2天内仅表现出肿瘤生长的延迟，但随后肿瘤迅速再生。VM密度与第8天的肿瘤体积和肿瘤重量呈正相关。CA4P引起缺氧，通过HIF-1α/EphA2/PI3K/基质金属蛋白酶（MMP）信号通路诱导W256肿瘤中VM的形成，从而导致受损肿瘤的再生[4]。

体外微管蛋白聚合测定[5,6]
根据Wang等人描述的方法，将猪脑微管蛋白（纯度>97%）与普通微管蛋白缓冲液（80 mM PIPES、2.0 mM MgCl2、0.5 mM EGTA和1 mM GTP）混合，在4°C下达到3 mg/mL的最终浓度。在96孔板中混合微管蛋白溶液和测试化合物后，立即在37°C的SYNERGY 4微孔板读取器中孵育微管蛋白聚合测定，并在340nm下每30秒监测65分钟。以紫杉醇作为微管蛋白聚合的阳性对照，秋水仙碱和ABI-274作为微管蛋白解聚的阳性对照进行重复实验。

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用于亲和性测定的SPR[5,6]
在配备有葡聚糖SPR传感器芯片（Reichert Polycarboxylate Hydrogel chip P/N 13206067）的Reicher4SPR系统中使用SPR技术分析与微管蛋白的结合亲和力。然后，将50μg/mL微管蛋白固定在传感器芯片表面，以获得12μRIU。芯片上的四个流动池中的一个作为阴性对照。在传感器芯片表面上注射不同浓度的4v或秋水仙碱进行缔合分析，然后进行离解分析。实验数据在25°C下使用运行缓冲液PBST（8 mM Na2HPO4、136 mM NaCl、2 mM KH2PO4、2.6 mM KCl和0.05%（v/v）Tween 20，pH 7.4）获得。平衡离解常数（KD）是用TraceDrawer软件通过稳态拟合模式计算的。

细胞阻抗评估[1]
参照和修改早期研究中的方法，使用xCELLigence心脏分析仪对hiPS-CM的细胞阻抗进行了分析。简而言之，iCell hiPS CM是从Cellular Dynamics International购买的。根据制造商的方案，使用iCell hiPS CM专用的平板培养基和维持培养基，在96孔xCELLigence Cardio E-plate中以20000个细胞/孔和37°C在5%CO2中解冻和培养hiPS CM。在潜伏期，根据制造商的说明，使用xCELLigence心脏分析仪连续监测阻抗值。以12.9ms的间隔连续采样阻抗，并在每个测量点以20秒的扫描持续时间进行监测。孵育14天后，将测试化合物（100 nM、1μM和10μM CA4DP；100 nM，1μM，和10μMCombretastatin A4/CA4；CA4DP为0.1%H2O，CA4[载体]为0.1%DMSO）（n=3孔）加入培养物中。然后，使用专用软件计算阻抗细胞指数（CI）和搏动率16、18、20。CI和打浆率的数据由添加试验化合物前的值进行归一化。给药后36小时的CI用于检测细胞毒性作用。给药后15分钟、3小时和12小时的搏动率用于检测收缩性的变化。

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背景/目的：Combretastatin A4/康布他汀A4磷酸二钠（CA4P）用于治疗恶性肿瘤。该物质之前已被证明会引发自杀性细胞死亡或凋亡。与有核细胞的凋亡类似，红细胞也可能进入自杀性死亡或红细胞凋亡，其特征是细胞收缩和细胞膜紊乱，磷脂酰丝氨酸易位到红细胞表面。红细胞下垂的刺激因素包括细胞质Ca2+活性（[Ca2+]i）、神经酰胺、氧化应激和ATP耗竭的增加。本研究探讨了CA4P是否会诱导红细胞下垂，如果是，则深入了解相关机制。 方法：采用流式细胞术从膜联蛋白-V结合、前向散射的细胞体积、Fluo3荧光的[Ca2+]i、DCF荧光的活性氧（ROS）丰度、CMF荧光的谷胱甘肽（GSH）丰度和荧光抗体的神经酰胺丰度估算细胞表面的磷脂酰丝氨酸暴露量。此外，利用基于萤光素酶的测定法定量细胞质ATP水平，并根据上清液中的血红蛋白浓度估算溶血[2]。

Animal/Disease Models: Male BD9 rats (7-9 weeks) bearing the sc implanted P22 tumor[9]
Doses: 100 mg/kg
Route of Administration: A single ip injections
Experimental Results: Dramatically raised the MABP by about 30%, and decreased the heart rate at 1 h after administration. decreased the blood flow in the tumor.

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Evaluation of histopathological changes [1]
A total of 14 rats were divided into four groups as described in Table 1. At 6 weeks of age, CA4DP/Combretastatin A4 (four doses of 30 or 60 mg/10 mL/kg at intervals of 24 hours or two doses of 120 mg/10 mL/kg at an interval of 72 hours) or saline (two doses at an interval of 72 hours) was administered via the caudal vein by bolus infusion. On the day after the last administration, the rats were anesthetized with isoflurane, and necropsy was performed. Also, one rat administered four doses of CA4DP 60 mg/10 mL/kg died unexpectedly before necropsy because of CA4DP toxicity. The cause of death was thought to be the cardiotoxicity of CA4DP because severe myocardial necrosis had been observed in this rat. After exsanguination, the hearts of the rats were removed and immediately fixed in 10% neutral phosphate-buffered formalin. The fixed hearts were cross-sectioned in two planes through the ventricles as described in a previous report7. The fixed hearts was embedded in paraffin and sectioned at a thickness of 4-6 μm. The specimens were stained with hematoxylin and eosin (HE). Observation of these specimens was performed using a light microscope.

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Evaluation of ECG data [1]
Two rats were used (animal No. 1 and No. 2). At 5 weeks of age, a small telemetry device (weight = 3.9 g, volume = 1.9 cc) for transmitting ECG data was implanted in the dorsal subcutaneous region under anesthesia with pentobarbital sodium. Paired wire electrodes that came with the telemetry device were placed under the skin of the dorsal and ventral thorax to record the apex-base (A–B) lead ECG. One week after surgery, ECG signals were recorded from each rat in a cage that had been placed on a signal-receiving board. ECG data were continuously sampled at 1 ms intervals, and all data analyses of ECG-wave components were performed using an ECG processor analyzing system on a personal computer in series with an analog-digital converter; the ECG data were stored on an external hard disk. During the period of ECG recording, CA4DP/Combretastatin A4 50 mg/10 mL/kg was administered to both rats via the caudal vein by bolus infusion, 3 times at intervals of 24 hours. ECG was recorded until 12 hours after the third administration. The consecutive ECG waves for 4 seconds were averaged, and the ECG wave components (RR interval, QRS duration, PR interval, and QT interval) were analyzed.

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Evaluation of BP [1]
A total of 9 rats were used. At 6 weeks of age, rats were anesthetized with isoflurane, and placed in a supine position. The femoral artery was exposed, and a polyethylene catheter filled with heparinized saline was inserted. The catheter was connected to transducer amplification equipment via a pressure transducer, and the arterial pressure was recorded. BP was continuously sampled at 1 ms intervals, and all data analyses were performed using an ECG processor analyzing system on a personal computer in series with an analog-digital converter. During the period of BP recording, CA4DP/Combretastatin A4 120 mg/10 mL/kg or saline 10 mL/kg was administered as a single dose via the caudal vein by bolus infusion (n = 5 for CA4DP and n = 4 for saline). BP was recorded until 30 minutes after administration. Consecutive BP waves for 4 seconds were averaged, and the BP components (systolic BP [SBP], diastolic BP [DBP], and mean BP [MBP]) and heart rate (HR) were analyzed.

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Toxicokinetic analysis [1]
Rats were administered a single intravenous dose of CA4DP/Combretastatin A4 at 120 mg/10 mL/kg by bolus infusion (n = 3). Blood was taken via the jugular vein and collected in heparin-coated tubes at 10 minutes and 1, 3, 6, and 24 hours after administration. Plasma was separated by centrifugation immediately after sampling. After centrifugation, an aliquot of plasma was mixed with the equivalent volume of 1% formic acid and stored at −20°C. The thawed plasma samples were purified by solid-phase extraction, and the plasma concentrations of combretastatin A4 phosphate (free base of CA4DP; CA4P) and combretastatin A4 (the metabolite of CA4DP; CA4) were determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Toxicokinetic parameters [maximum concentration (Cmax), terminal half-life (T1/2), and area under the concentration-time curve from time zero to infinity (AUC0-inf)] were obtained by non-compartmental analysis using Phoenix WinNonlin 6.3.

Metabolism / Metabolites
Combretastatin A4 has known human metabolites that include (2S,3S,4S,5R)-3,4,5-trihydroxy-6-[2-methoxy-5-[(Z)-2-(3,4,5-trimethoxyphenyl)ethenyl]phenoxy]oxane-2-carboxylic acid.

[1]. Combretastatin A4 disodium phosphate-induced myocardial injury. J Toxicol Pathol. 2016 Jul;29(3):163-71.

[2]. Stimulation of Eryptosis by Combretastatin A4 Phosphate Disodium (CA4P). Cell Physiol Biochem. 2016;38(3):969-8.

[3]. Co-Encapsulation of Combretastatin-A4 Phosphate and Doxorubicin in Polymersomes for Synergistic Therapy of Nasopharyngeal Epidermal Carcinoma. J Biomed Nanotechnol. 2015 Jun;11(6):997-1006.

[4]. Combretastatin A4 phosphate treatment induces vasculogenic mimicry formation of W256 breast carcinoma tumor in vitro and in vivo. Tumour Biol. 2015 Nov;36(11):8499-510.

[5]. Structure-Activity Relationship Study of Novel 6-Aryl-2-benzoyl-pyridines as Tubulin Polymerization Inhibitors with Potent Antiproliferative Properties. J Med Chem. 2020 Jan 23;63(2):827-846.

[6]. Discovery of novel 2-aryl-4-benzoyl-imidazole (ABI-III) analogues targeting tubulin polymerization as antiproliferative agents. J Med Chem . 2012 Aug 23;55(16):7285-9.

[7]. 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 Aug;160(8):2008-27.

[8]. Antineoplastic agents. 445. Synthesis and evaluation of structural modifications of (Z)- and (E)-combretastatin A-41. J Med Chem. 2005 Jun 16;48(12):4087-99.

[9]. Combretastatin A-4 phosphate as a tumor vascular-targeting agent: early effects in tumors and normal tissues. Cancer Res. 1999 Apr 1;59(7):1626-34.

Fosbretabulin has been investigated for the treatment of Anaplastic Thyroid Cancer.
Fosbretabulin Disodium is the disodium salt of a water-soluble phosphate derivative of a natural stilbenoid phenol derived from the African bush willow (Combretum caffrum) with potential vascular disrupting and antineoplastic activities. Upon administration, the prodrug fosbretabulin is dephosphorylated to its active metabolite, the microtubule-depolymerizing agent combretastatin A4, which binds to tubulin dimers and prevents microtubule polymerization, resulting in mitotic arrest and apoptosis in endothelial cells. In addition, this agent disrupts the engagement of the endothelial cell-specific junctional molecule vascular endothelial-cadherin (VE-cadherin) and so the activity of the VE-cadherin/beta-catenin/Akt signaling pathway, which may result in the inhibition of endothelial cell migration and capillary tube formation. As a result of fosbretabulin's dual mechanism of action, the tumor vasculature collapses, resulting in reduced tumor blood flow and ischemic necrosis of tumor tissue.
Fosbretabulin is a water-soluble prodrug derived from the African bush willow (Combretum caffrum) with antineoplastic activity. Fosbretabulin is dephosphorylated to its active metabolite, combretastatin A4, which binds to tubulin and inhibits microtubule polymerization, resulting in mitotic arrest and apoptosis in endothelial cells. As apoptotic endothelial cells detach from their substrata, tumor blood vessels collapse; the acute disruption of tumor blood flow may result in tumor necrosis.

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Combretastatin A4 is a stilbenoid.
Combretastatin A4 has been reported in Combretum caffrum with data available.
Combretastatin A-4 is an inhibitor of microtubule polymerization derived from the South African willow bush which causes mitotic arrest and selectively targets and reduces or destroys existing blood vessels, causing decreased tumor blood supply.
See also: Fosbretabulin (annotation moved to).
Combretastatin A-4 is an inhibitor of microtubule polymerization derived from the South African willow bush which causes mitotic arrest and selectively targets and reduces or destroys existing blood vessels, causing decreased tumor blood supply.

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Histopathological and electrocardiographic features of myocardial lesions induced by combretastatin A4 disodium phosphate (CA4DP) were evaluated, and the relation between myocardial lesions and vascular changes and the direct toxic effect of CA4DP on cardiomyocytes were discussed. We induced myocardial lesions by administration of CA4DP to rats and evaluated myocardial damage by histopathologic examination and electrocardiography. We evaluated blood pressure (BP) of CA4DP-treated rats and effects of CA4DP on cellular impedance-based contractility of human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs). The results revealed multifocal myocardial necrosis with a predilection for the interventricular septum and subendocardial regions of the apex of the left ventricular wall, injury of capillaries, morphological change of the ST junction, and QT interval prolongation. The histopathological profile of myocardial lesions suggested that CA4DP induced a lack of myocardial blood flow. CA4DP increased the diastolic BP and showed direct effects on hiPS-CMs. These results suggest that CA4DP induces dysfunction of small arteries and capillaries and has direct toxicity in cardiomyocytes. Therefore, it is thought that CA4DP induced capillary and myocardial injury due to collapse of the microcirculation in the myocardium. Moreover, the direct toxic effect of CA4DP on cardiomyocytes induced myocardial lesions in a coordinated manner.[1]

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The purpose of this study was to investigate the effect of combretastatin A4 phosphate (CA4P) on vasculogenic mimicry (VM) channel formation in vitro and in vivo after a single-dose treatment and the underlying mechanism involved in supporting VM. In vitro model of three-dimensional cultures was used to test the effect of CA4P on the tube formation of Walker 256 cells. Western blot analysis was conducted to assess the expression of hypoxia-inducible factor (HIF)-1α and VM-associated markers. W256 tumor-bearing rat model was established to demonstrate the effect of CA4P on VM formation and tumor hypoxia by double staining and a hypoxic marker pimonidazole. Anti-tumor efficacy of CA4P treatment was evaluated by tumor growth curve. Under hypoxic conditions for 48 h in vitro, W256 cells formed VM network associated with increased expression of VM markers. Pretreatment with CA4P did not influence the amount of VM in 3-D culture as well as the expression of these key molecules. In vivo, W256 tumors showed marked intratumoral hypoxia after CA4P treatment, accompanied by increased VM formation. CA4P exhibited only a delay in tumor growth within 2 days but rapid tumor regrowth afterward. VM density was positively related to tumor volume and tumor weight at day 8. CA4P causes hypoxia which induces VM formation in W256 tumors through HIF-1α/EphA2/PI3K/matrix metalloproteinase (MMP) signaling pathway, resulting in the consequent regrowth of the damaged tumor.[4]

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We recently reported the crystal structure of tubulin in complex with a colchicine binding site inhibitor (CBSI), ABI-231, having 2-aryl-4-benzoyl-imidazole (ABI). Based on this and additional crystal structures, here we report the structure-activity relationship study of a novel series of pyridine analogues of ABI-231, with compound 4v being the most potent one (average IC50 ∼ 1.8 nM) against a panel of cancer cell lines. We determined the crystal structures of another potent CBSI ABI-274 and 4v in complex with tubulin and confirmed their direct binding at the colchicine site. 4v inhibited tubulin polymerization, strongly suppressed A375 melanoma tumor growth, induced tumor necrosis, disrupted tumor angiogenesis, and led to tumor cell apoptosis in vivo. Collectively, these studies suggest that 4v represents a promising new generation of tubulin inhibitors. [5]

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Novel ABI-III compounds were designed and synthesized based on our previously reported ABI-I and ABI-II analogues. ABI-III compounds are highly potent against a panel of melanoma and prostate cancer cell lines, with the best compound having an average IC(50) value of 3.8 nM. They are not substrate of Pgp and thus may effectively overcome Pgp-mediated multidrug resistance. ABI-III analogues maintain their mechanisms of action by inhibition of tubulin polymerization.[6]

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A series of cis- and trans-stilbenes related to combretastatin A-4 (1a), with a variety of substituents at the 3'-position of the aryl B-ring, were synthesized and evaluated for inhibitory activity employing six human cancer cell lines (NCI-H460 lung carcinoma, BXPC-3 pancreas, SK-N-SH neuroblastoma, SW1736 thyroid, DU-145 prostate, and FADU pharynx-squamous sarcoma) as well as the P-388 murine lymphocyte leukemia cell line. Several of the cis-stilbene derivatives were significantly inhibitory against all cell lines used, with potencies comparable to that of the parent 1a. All were potent inhibitors of tubulin polymerization. The corresponding trans-stilbenes had little or no activity as tubulin polymerization inhibitors and were relatively inactive against the seven cancer cell lines. In terms of inhibition of both cancer cell growth and tubulin polymerization, the dimethylamino and bromo cis-stilbenes were the most potent of the new derivatives, the latter having biological activity approaching that of 1a. As part of the present study, the X-ray crystal structure of the 3'-O-phosphate of combretastatin A-4 (1b) was successfully elucidated. Compound 1b has been termed the "combretastatin A-4 prodrug", and it is currently undergoing clinical trials for the treatment of human cancer patients.[8]

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The potential for tumor vascular-targeting by using the tubulin destabilizing agent disodium combretastatin A-4 3-0-phosphate (CA-4-P) was assessed in a rat system. This approach aims to shut down the established tumor vasculature, leading to the development of extensive tumor cell necrosis. The early vascular effects of CA-4-P were assessed in the s.c. implanted P22 carcinosarcoma and in a range of normal tissues. Blood flow was measured by the uptake of radiolabeled iodoantipyrine, and quantitative autoradiography was used to measure spatial heterogeneity of blood flow in tumor sections. CA-4-P (100 mg/kg i.p.) caused a significant increase in mean arterial blood pressure at 1 and 6 h after treatment and a very large decrease in tumor blood flow, which-by 6 h-was reduced approximately 100-fold. The spleen was the most affected normal tissue with a 7-fold reduction in blood flow at 6 h. Calculations of vascular resistance revealed some vascular changes in the heart and kidney for which there were no significant changes in blood flow. Quantitative autoradiography showed that CA-4-P increased the spatial heterogeneity in tumor blood flow. The drug affected peripheral tumor regions less than central regions. Administration of CA-4-P (30 mg/kg) in the presence of the nitric oxide synthase inhibitor, N(omega)-nitro-L-arginine methyl ester, potentiated the effect of CA-4-P in tumor tissue. The combination increased tumor vascular resistance 300-fold compared with less than 7-fold for any of the normal tissues. This shows that tissue production of nitric oxide protects against the damaging vascular effects of CA-4-P. Significant changes in tumor vascular resistance could also be obtained in isolated tumor perfusions using a cell-free perfusate, although the changes were much less than those observed in vivo. This shows that the action of CA-4-P includes mechanisms other than those involving red cell viscosity, intravascular coagulation, and neutrophil adhesion. The uptake of CA-4-P and combretastatin A-4 (CA-4) was more efficient in tumor than in skeletal muscle tissue and dephosphorylation of CA-4-P to CA-4 was faster in the former. These results are promising for the use of CA-4-P as a tumor vascular-targeting agent.[9]

C18H21O8P

396.32834

396.097

C, 54.55; H, 5.34; O, 32.29; P, 7.82

222030-63-9

168555-66-6 (disodium);222030-63-9 (free acid);404886-32-4 ( tromethamine);

5351387

Typically exists as solid at room temperature

1.342g/cm3

611.8ºC at 760mmHg

323.8ºC

7.99E-16mmHg at 25°C

1.609

3.362

113.49

2

8

8

27

508

0

O=P(O)(OC1=CC(/C=C\C2=CC(OC)=C(OC)C(OC)=C2)=CC=C1OC)O

WDOGQTQEKVLZIJ-WAYWQWQTSA-N

InChI=1S/C18H21O8P/c1-22-14-8-7-12(9-15(14)26-27(19,20)21)5-6-13-10-16(23-2)18(25-4)17(11-13)24-3/h5-11H,1-4H3,(H2,19,20,21)/b6-5-

[2-methoxy-5-[(Z)-2-(3,4,5-trimethoxyphenyl)ethenyl]phenyl] dihydrogen phosphate

Fosbretabulin; 222030-63-9; Combretastatin A-4 phosphate; Phosbretabulin; Fosbretabulin [INN]; combretastatin A4 phosphate; (Z)-2-Methoxy-5-(3,4,5-trimethoxystyryl)phenyl dihydrogen phosphate; fosbretabulina; Fosbretabulin disodium; 168555-66-6; Combretastatin A4 disodium phosphate; CA4DP; CA 4P; Combretastatin A4 Phosphate Disodium Salt; Fosbretabulin disodium [USAN]; CA-4DP;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

注意: 如下所列的是一些常用的体内动物实验溶解配方，主要用于溶解难溶或不溶于水的产品（水溶度<1 mg/mL）。 建议您先取少量样品进行尝试，如该配方可行，再根据实验需求增加样品量。

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注射用配方1: DMSO : Tween 80： Saline = 10 : 5 : 85 (如： 100 μL DMSO → 50 μL Tween 80 → 850 μL Saline)
*生理盐水/Saline的制备：将0.9g氯化钠/NaCl溶解在100 mL ddH ₂ O中，得到澄清溶液。
注射用配方 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL DMSO → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)
注射用配方 3: DMSO : Corn oil = 10 : 90 (如： 100 μL DMSO → 900 μL Corn oil)
示例: 以注射用配方 3 (DMSO : Corn oil = 10 : 90) 为例说明, 如果要配制 1 mL 2.5 mg/mL的工作液, 您可以取 100 μL 25 mg/mL 澄清的 DMSO 储备液，加到 900 μL Corn oil/玉米油中, 混合均匀。

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注射用配方 4: DMSO : 20% SBE-β-CD in Saline = 10 : 90 [如：100 μL DMSO → 900 μL (20% SBE-β-CD in Saline)]
*20% SBE-β-CD in Saline的制备（4°C，储存1周）：将2g SBE-β-CD (磺丁基-β-环糊精) 溶解于10mL生理盐水中，得到澄清溶液。
注射用配方 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (如： 500 μL 2-Hydroxypropyl-β-cyclodextrin (羟丙基环胡精)→ 500 μL Saline)
注射用配方 6: DMSO : PEG300 : Castor oil : Saline = 5 : 10 : 20 : 65 (如： 50 μL DMSO → 100 μL PEG300 → 200 μL Castor oil → 650 μL Saline)
注射用配方 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (如： 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
注射用配方 8: 溶解于Cremophor/Ethanol (50 : 50), 然后用生理盐水稀释。
注射用配方 9: EtOH : Corn oil = 10 : 90 (如： 100 μL EtOH → 900 μL Corn oil)
注射用配方 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL EtOH → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)

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口服配方 1: 悬浮于0.5% CMC Na (羧甲基纤维素钠)
口服配方 2: 悬浮于0.5% Carboxymethyl cellulose (羧甲基纤维素)
示例: 以口服配方 1 (悬浮于 0.5% CMC Na)为例说明, 如果要配制 100 mL 2.5 mg/mL 的工作液, 您可以先取0.5g CMC Na并将其溶解于100mL ddH2O中，得到0.5%CMC-Na澄清溶液；然后将250 mg待测化合物加到100 mL前述 0.5%CMC Na溶液中，得到悬浮液。

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口服配方 3: 溶解于 PEG400 (聚乙二醇400)
口服配方 4: 悬浮于0.2% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 5: 溶解于0.25% Tween 80 and 0.5% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 6: 做成粉末与食物混合

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注意: 以上为较为常见方法，仅供参考， InvivoChem并未独立验证这些配方的准确性。具体溶剂的选择首先应参照文献已报道溶解方法、配方或剂型，对于某些尚未有文献报道溶解方法的化合物，需通过前期实验来确定（建议先取少量样品进行尝试），包括产品的溶解情况、梯度设置、动物的耐受性等。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。