Combretastatin A4

J Med Chem . 2014 Apr 24;57(8):3369-3381.

Design, synthesis, and biological evaluation of novel pyridine-bridged analogues of combretastatin-A4 as anticancer agents[Pubmed: 24669888]

A series of novel pyridine-bridged analogues of combretastatin-A4 (CA-4) were designed and synthesized. As expected, the 4-atom linker configuration retained little cytotoxicities in the compounds 2e, 3e, 3g, and 4i. Activities of the analogues with 3-atom linker varied widely depending on the phenyl ring substitutions, and the 3-atom linker containing nitrogen represents the more favorable linker structure. Among them, three analogues (4h, 4s, and 4t) potently inhibited cell survival and growth, arrested cell cycle, and blocked angiogenesis and vasculature formation in vivo in ways comparable to CA-4. The superposition of 4h and 4s in the colchicine-binding pocket of tubulin shows the binding posture of CA-4, 4h, and 4s are similar, as confirmed by the competitive binding assay where the ability of the ligands to replace tubulin-bound colchicine was measured. The binding data are consistent with the observed biological activities in antiproliferation and suppression of angiogenesis but are not predictive of their antitubulin polymerization activities.

J Biomed Nanotechnol . 2015 Jun;11(6):997-1006.

Co-Encapsulation of Combretastatin-A4 Phosphate and Doxorubicin in Polymersomes for Synergistic Therapy of Nasopharyngeal Epidermal Carcinoma[Pubmed: 26353589]

In this study, we designed biodegradable polymersomes for co-delivery of an antiangiogenic drug combretastatin-A4 phosphate (CA4P) and doxorubicin (DOX) to collapse tumor neovasculature and inhibit cancer cell proliferation with the aim to achieve synergistic antitumor effects. The polymersomes co-encapsulating DOX and CA4P (Ps-DOX-CA4P) were prepared by solvent evaporation method using methoxy poly(ethylene glycol)-b-polylactide (mPEG-PLA) block copolymers as drug carriers. The resulting Ps-DOX-CA4P has vesicles shape with uniform sizes of about 50 nm and controlled co-encapsulation ratios of DOX to CA4P. More importantly, Ps-DOX-CA4P (1:10) showed strong synergistic cytotoxicity (combination index CI = 0.31) against human nasopharyngeal epidermal carcinoma (KB) cells. Furthermore, Ps-DOX-CA4P accumulated remarkably in KB tissues xenografts in nude mice. Consistent with these observations, Ps-DOX-CA4P (1:10) achieved significant antitumor potency because of fast tumor vasculature disruption and sustained tumor cells proliferation inhibition in vivo. The overall findings indicate that co-delivery of an antiangiogenic drug and a chemotherapeutic agent in polymersomes is a potentially promising strategy for cancer therapy.

Cell Physiol Biochem . 2016;38(3):969-981.

Stimulation of Eryptosis by Combretastatin A4 Phosphate Disodium (CA4P)[Pubmed: 26938611]

Background/aims: Combretastatin A4 phosphate disodium (CA4P) is utilized for the treatment of malignancy. The substance has previously been shown to trigger suicidal cell death or apoptosis. Similar to apoptosis of nucleated cells, erythrocytes may enter suicidal death or eryptosis, characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Stimulators of eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i), ceramide, oxidative stress and ATP depletion. The present study explored, whether CA4P induces eryptosis and, if so, to gain insight into mechanisms involved. Methods: Flow cytometry has been employed to estimate phosphatidylserine exposure at the cell surface from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, reactive oxygen species (ROS) abundance from DCF fluorescence, glutathione (GSH) abundance from CMF fluorescence and ceramide abundance from fluorescent antibodies. In addition cytosolic ATP levels were quantified utilizing a luciferin-luciferase-based assay and hemolysis was estimated from hemoglobin concentration in the supernatant. Results: A 48 hours exposure of human erythrocytes to CA4P (≥ 50 μM) significantly increased the percentage of annexin-V-binding cells and significantly decreased forward scatter. CA4P did not appreciably increase hemolysis. Hundred μM CA4P significantly increased Fluo3-fluorescence. The effect of CA4P (100 μM) on annexin-V-binding was significantly blunted, but not abolished, by removal of extracellular Ca2+. CA4P (≥ 50 μM) significantly decreased GSH abundance and ATP levels but did not significantly increase ROS or ceramide. Conclusions: CA4P triggers cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect at least in part due to entry of extracellular Ca2+ and energy depletion.

Tumour Biol . 2015 Nov;36(11):8499-8510.

Combretastatin A4 phosphate treatment induces vasculogenic mimicry formation of W256 breast carcinoma tumor in vitro and in vivo[Pubmed: 26026583]

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.

Magn Reson Med . 2016 Feb;75(2):866-872.

Monitoring Combretastatin A4-induced tumor hypoxia and hemodynamic changes using endogenous MR contrast and DCE-MRI[Pubmed: 25765253]

Purpose: To benchmark MOBILE (Mapping of Oxygen By Imaging Lipid relaxation Enhancement), a recent noninvasive MR method of mapping changes in tumor hypoxia, electron paramagnetic resonance (EPR) oximetry, and dynamic contrast-enhanced MRI (DCE-MRI) as biomarkers of changes in tumor hemodynamics induced by the antivascular agent Combretastatin A4 (CA4). Methods: NT2 and MDA-MB-231 mammary tumors were implanted subcutaneously in FVB/N and nude NMRI mice. Mice received 100 mg/kg of CA4 intraperitoneally 3 hr before imaging. The MOBILE sequence (assessing R1 of lipids) and the DCE sequence (assessing K(trans) hemodynamic parameter), were assessed on different cohorts. pO2 changes were confirmed on matching tumors using EPR oximetry consecutive to the MOBILE sequence. Changes in tumor vasculature were assessed using immunohistology consecutive to DCE-MRI studies. Results: Administration of CA4 induced a significant decrease in lipids R1 (P = 0.0273) on pooled tumor models and a reduction in tumor pO2 measured by EPR oximetry. DCE-MRI also exhibited a significant drop of K(trans) (P < 0.01) that was confirmed by immunohistology. Conclusion: MOBILE was identified as a marker to follow a decrease in oxygenation induced by CA4. However, DCE-MRI showed a higher dynamic range to follow changes in tumor hemodynamics induced by CA4.

NMR Biomed. 2014 Nov;27(11):1403-1412.

Dynamic contrast-enhanced MRI in mouse tumors at 11.7 T: comparison of three contrast agents with different molecular weights to assess the early effects of combretastatin A4[Pubmed: 25323069]

Dynamic contrast-enhanced (DCE)-MRI is useful to assess the early effects of drugs acting on tumor vasculature, namely anti-angiogenic and vascular disrupting agents. Ultra-high-field MRI allows higher-resolution scanning for DCE-MRI while maintaining an adequate signal-to-noise ratio. However, increases in susceptibility effects, combined with decreases in longitudinal relaxivity of gadolinium-based contrast agents (GdCAs), make DCE-MRI more challenging at high field. The aim of this work was to explore the feasibility of using DCE-MRI at 11.7 T to assess the tumor hemodynamics of mice. Three GdCAs possessing different molecular weights (gadoterate: 560 Da, 0.29 mmol Gd/kg; p846: 3.5 kDa, 0.10 mmol Gd/kg; and p792: 6.47 kDa, 0.15 mmol Gd/kg) were compared to see the influence of the molecular weight in the highlight of the biologic effects induced by Combretastatin A4 (CA4). Mice bearing transplantable liver tumor (TLT) hepatocarcinoma were divided into two groups (n = 5-6 per group and per GdCA): a treated group receiving 100 mg/kg CA4, and a control group receiving vehicle. The mice were imaged at 11.7 T with a T1 -weighted FLASH sequence 2 h after the treatment. Individual arterial input functions (AIFs) were computed using phase imaging. These AIFs were used in the Extended Tofts Model to determine K(trans) and vp values. A separate immunohistochemistry study was performed to assess the vascular perfusion and the vascular density. Phase imaging was used successfully to measure the AIF for the three GdCAs. In control groups, an inverse relationship between the molecular weight of the GdCA and K(trans) and vp values was observed. K(trans) was significantly decreased in the treated group compared with the control group for each GdCA. DCE-MRI at 11.7 T is feasible to assess tumor hemodynamics in mice. With K(trans) , the three GdCAs were able to track the early vascular effects induced by CA4 treatment.

J Toxicol Pathol . 2016 Jul;29(3):163-171.

Combretastatin A4 disodium phosphate-induced myocardial injury[Pubmed: 27559241]

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.