D. J. Chaplin

Magnetic resonance imaging and spectroscopy of combretastatin A4 prodrug-induced disruption of tumour perfusion and energetic status

The effects of combretastatin A4 prodrug on perfusion and the levels of 31P metabolites in an implanted murine tumour were investigated for 3 h after drug treatment using nuclear magnetic resonance imaging (MRI) and spectroscopy (MRS). The area of regions of low signal intensity in spin-echo images of tumours increased slightly after treatment with the drug. These regions of low signal intensity corresponded to necrosis seen in histological sections, whereas the expanding regions surrounding them corresponded to haemorrhage. Tumour perfusion was assessed before and 160 min after drug treatment using dynamic MRI measurements of gadolinium diethylenetriaminepentaacetate (GdDTPA) uptake and washout. Perfusion decreased significantly in central regions of the tumour after treatment. This was attributed to disruption of the vasculature and was consistent with the haemorrhage seen in histological sections. The mean apparent diffusion coefficient of water within the tumour did not change, indicating that there was no expansion of necrotic regions during the 3 h after drug treatment. Localized 31P-MRS showed that there was decline in cellular energy status in the tumour after treatment with the drug. The concentrations of nucleoside triphosphates within the tumour fell, the inorganic phosphate concentration increased and there was a significant decrease in tumour pH for 80 min after drug treatment. The rapid, selective and extensive damage caused to these tumours by combretastatin A4 prodrug has highlighted the potential of the agent as a novel cancer chemotherapeutic agent. We have shown that the response of tumours to treatment with the drug may be monitored non-invasively using MRI and MRS experiments that are appropriate for use in a clinical setting.

Electroporation of human microvascular endothelial cells: evidence for an anti-vascular mechanism of electrochemotherapy

Recent studies have indicated that the antitumour effectiveness of electrochemotherapy, a combination of chemotherapeutic drugs with application of high voltage electric pulses applied to the tumour nodule (electroporation), result in a significant reduction in tumour blood flow and may therefore be mediated by an anti-vascular mechanism. The aim of this study was to evaluate the cytotoxicity of electroporation with bleomycin or cisplatin on cultured human microvascular endothelial cells (HMEC-1). The sensitivity of HMEC-1 cells to a 5 min treatment by electroporation with bleomycin or cisplatin (8 electric pulses, pulse duration 100 μs, frequency 1 Hz, electric field intensity 1400 V cm−1) was compared to the sensitivity of cells treated continuously for 3 days with drugs alone. HMEC-1 cells were moderately sensitive to continuous exposure to cisplatin, but showed greater sensitivity to bleomycin. Combination of a 5 min drug exposure with electric pulses increased cytotoxicity of cisplatin by ∼10-fold for cisplatin and ∼5000-fold for bleomycin. The electroporation of HMEC-1 cells with bleomycin for a 5 min exposure was ∼250-fold better than a continuous exposure to the drug alone. The results of this study indicate that the anti-tumour action of electrochemotherapy is likely to be due, in part, to the highly sensitive response of vascular endothelial cells. Further studies are necessary to identify the determinants of endothelial response and its relationship to the anti-vascular action of electrochemotherapy in vivo.

Determinants of anti-vascular action by combretastatin A-4 phosphate: role of nitric oxide

The anti-vascular action of the tubulin binding agent combretastatin A-4 phosphate (CA-4-P) has been quantified in two types of murine tumour, the breast adenocarcinoma CaNT and the round cell sarcoma SaS. The functional vascular volume, assessed using a fluorescent carbocyanine dye, was significantly reduced at 18 h after CA-4-P treatment in both tumour types, although the degree of reduction was very different in the two tumours. The SaS tumour, which has a higher nitric oxide synthase (NOS) activity than the CaNT tumour, showed ~10-fold greater resistance to vascular damage by CA-4-P. This is consistent with our previous findings, which showed that NO exerts a protective action against this drug. Simultaneous administration of CA-4-P with a NOS inhibitor, Nω-nitro-L-arginine (L-NNA), resulted in enhanced vascular damage and cytotoxicity in both tumour types. Administration of diethylamine NO, an NO donor, conferred protection against the vascular damaging effects. Following treatment with CA-4-P, neutrophil infiltration into the tumours, measured by myeloperoxidase (MPO) activity, was significantly increased. Levels of MPO activity also correlated with the levels of vascular injury and cytotoxicity measured in both tumour types. Neutrophilic MPO generates free radicals and may therefore contribute to the vascular damage associated with CA-4-P treatment. MPO activity was significantly increased in the presence of L-NNA, suggesting that the protective effect of NO against CA-4-P-induced vascular injury may be, at least partially, mediated by limiting neutrophil infiltration. The data are consistent with the hypothesis that neutrophil action contributes to vascular injury by CA-4-P and that NO generation acts to protect the tumour vasculature against CA4-P-induced injury. The protective effect of NO is probably associated with an anti-neutrophil action.

Phase I Clinical and Pharmacokinetic Evaluation of the Vascular-Disrupting Agent OXi4503 in Patients with Advanced Solid Tumors

Purpose: Preclinical studies show that OXi4503 (combretastatin A1 diphosphate, CA1P) is more potent than other clinically evaluated vascular-disrupting agents. Experimental Design: Escalating doses of OXi4503 were given intravenously over 10 minutes on days 1, 8, and 15 every 28 days to patients with advanced solid tumors. Results: Doses were escalated in single-patient cohorts from 0.06 to 1.92 mg/m2, then expanded cohorts to 15.4 mg/m2 in 43 patients. Common adverse drug reactions were hypertension, tumor pain, anemia, lymphopenia, and easily controllable nausea/vomiting and fatigue. Five patients experienced different drug-related dose-limiting toxicities, atrial fibrillation, increased troponin, blurred vision, diplopia, and tumor lysis. Prophylactic amlodipine failed to prevent adverse events. Pharmacokinetics showed dose-dependent linear increases in peak plasma concentrations and area under the curve value of OXi4503. One partial response was seen in a heavily pretreated patient with ovarian cancer. Dynamic contrast-enhanced MRI confirmed a dose effect and showed significant antivascular effects in 10 of 13 patients treated at doses of 11 mg/m2 or higher. Conclusions: The maximum tolerated dose was 8.5 mg/m2 but escalation to 14 mg/m2 was possible with only temporary reversible cerebrovascular toxicity by excluding hypertensive patients. As a tumor response was seen at 14 mg/m2 and maximum tumor perfusion reductions were seen at doses of 11 mg/m2 or higher, the recommended phase II dose is from 11 to 14 mg/m2. Clin Cancer Res; 18(5); 1415–25. ©2012 AACR.

The relationship between extracellular lactate and tumour pH in a murine tumour model of ischaemia-reperfusion.

We have studied the relationship between extracellular lactate (LACTe) and extracellular pH (pHe) in murine tumours after vascular occlusion (clamping) followed by reperfusion. In tumours occluded at ambient room temperature, LACTe, measured by microdialysis, increased linearly with time and correlated strongly with the acidification of the extracellular compartment (r=0.97, P<0.03, n=4). Significant decrease in LACTe was evident following removal of occlusion at room temperature and is consistent with vascular reperfusion. Occlusion at 35 degrees C, i.e. to maintain tumour temperature during occlusion, resulted in an initial increase in LACTe, which mirrored a rapid reduction in pHe. However further reductions in pHe occurred without increase in LACTe. During vascular occlusion, tumour adenine nucleotide pool decreased and AMP accumulated. AMP subsequently decreased in the 35 degrees C group and this may contribute to the observed differences in accumulation of LACTe, and capacity to recover from vascular occlusion, between the two treatment groups. These data show that extracellular lactate concentration is a good predictor for tumour pH when adequate energy sources are available within the tumour. However, under conditions of more severe stress, resulting in abolition of primary energy stores and cell death, the pHe continues to decline in the absence of a corresponding accumulation of extracellular lactate. This emphasizes the fact that other processes, apart from lactate production, can contribute to reduction in extracellular pH.

Reduced capacity of tumour blood vessels to produce endothelium-derived relaxing factor : significance for blood flow modification

The effect of nitric oxide-dependent vasodilators on vascular resistance of tumours and normal tissue was determined with the aim of modifying tumour blood flow for therapeutic benefit. Isolated preparations of the rat P22 tumour and normal rat hindlimb were perfused ex vivo. The effects on tissue vascular resistance of administration of sodium nitroprusside (SNP) and the diazeniumdiolate (or NONO-ate) NOC-7, vasodilators which act via direct release of nitric oxide (NO), were compared with the effects of acetylcholine (ACh), a vasodilator which acts primarily via receptor stimulation of endothelial cells to release NO in the form of endothelium-derived relaxing factor (EDRF). SNP and NOC-7 effectively dilated tumour blood vessels after preconstriction with phenylephrine (PE) or potassium chloride (KCl) as indicated by a decrease in vascular resistance. SNP also effectively dilated normal rat hindlimb vessels after PE/KCl constriction. Vasodilatation in the tumour preparations was accompanied by a significant rise in nitrite levels measured in the tumour effluent. ACh induced a significant vasodilation in the normal hindlimb but an anomalous vasoconstriction in the tumour. This result suggests that tumours, unlike normal tissues are incapable of releasing NO (EDRF) in response to ACh. Capacity for EDRF production may represent a difference between tumour and normal tissue blood vessels, which could be exploited for selective pharmacological manipulation of tumour blood flow.

Abstract A4: Combretastatin A-4 Phosphate (CA4P) is effective for the treatment of functional pancreatic neuroendocrine tumors (PNETs) in a transgenic mouse model.

Background: PNETs are rare highly vascular tumors that are increasing in incidence. Management of advanced tumors, both functional and nonfunctional, can be challenging, as systemic treatment options are limited. CA4P is a systemically delivered vascular disrupting agent that has shown clinical activity for the treatment of other advanced endocrine cancers. We evaluated the efficacy of systemic administration of CA4P for the treatment of functional insulinomas in a transgenic mouse model of PNETs. Methods: Twelve month-old mice with homozygous deletion of the Men1 gene in the pancreas and confirmation of a functional insulinoma by presence of elevated serum insulin were divided into two groups (4 mice per group). The treatment group received CA4P, 100 mg/kg, IP on Monday, Wednesday and Friday for four weeks. The control group received PBS by the same route, frequency and for the same duration. Serum insulin was measured by enzyme-linked immunosorbent assay (ELISA) pre and at intervals during treatment. After four weeks, mice were euthanized and whole pancreata were dissected, formalin-fixed with paraffin embedding, and sectioned (5 μm) on a rotating microtome. Hematoxylin and eosin staining was performed and the tumor cross-sectional area in the sections with maximal tumor diameter was determined in three sections for each mouse in a blinded fashion. Tumor size was assessed by measuring the length and width of the tumor in two dimensions and then calculating the maximum tumor area for each mouse and for each group. Statistical analysis was performed with ANOVA and Student9s t-test. Results: Treatment was well tolerated in all mice without treatment related death or weight loss. At baseline, mean serum insulin was elevated, but equivalent in both groups (CA4P group 3.695 ± 0.669 µg/L, versus PBS group 3.485 ± 0.666 µg/L, p=0.672). Treatment with CA4P resulted in a significant and sustained decrease in circulating insulin, with maximum effect seen by day 17 (CA4P group 0.213 ± 0.075 µg/L, versus PBS group 4.578 ± 0.161 µg/L, p Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A4. Citation Format: Ziqiang Yuan, Nikolas Zaphiros, Asha Adem, Norvilia Etienne, Jai Balkissoon, Dai Chaplin, Steven K. Libutti. Combretastatin A-4 Phosphate (CA4P) is effective for the treatment of functional pancreatic neuroendocrine tumors (PNETs) in a transgenic mouse model. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A4.