Gillian M. Tozer

Guidelines for the welfare and use of animals in cancer research

Animal experiments remain essential to understand the fundamental mechanisms underpinning malignancy and to discover improved methods to prevent, diagnose and treat cancer. Excellent standards of animal care are fully consistent with the conduct of high quality cancer research. Here we provide updated guidelines on the welfare and use of animals in cancer research. All experiments should incorporate the 3Rs: replacement, reduction and refinement. Focusing on animal welfare, we present recommendations on all aspects of cancer research, including: study design, statistics and pilot studies; choice of tumour models (e.g., genetically engineered, orthotopic and metastatic); therapy (including drugs and radiation); imaging (covering techniques, anaesthesia and restraint); humane endpoints (including tumour burden and site); and publication of best practice.

Disrupting tumour blood vessels

Low-molecular-weight vascular-disrupting agents (VDAs) cause a pronounced shutdown in blood flow to solid tumours, resulting in extensive tumour-cell necrosis, while they leave the blood flow in normal tissues relatively intact. The largest group of VDAs is the tubulin-binding combretastatins, several of which are now being tested in clinical trials. DMXAA (5,6-dimethylxanthenone-4-acetic acid) — one of a structurally distinct group of drugs — is also being tested in clinical trials. A full understanding of the action of these and other VDAs will provide insights into mechanisms that control tumour blood flow and will be the basis for the development of new therapeutic drugs for targeting the established tumour vasculature for therapy.

Combretastatin A4 Phosphate Has Tumor Antivascular Activity in Rat and Man as Demonstrated by Dynamic Magnetic Resonance Imaging

PURPOSE Combretastatin A4 phosphate (CA4P) is a novel vascular targeting agent. Dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) studies were performed to examine changes in parameters related to blood flow and vascular permeability in tumor and normal tissue after CA4P treatment. MATERIALS AND METHODS Changes in kinetic DCE-MRI parameters (transfer constant [Ktrans] and area under contrast medium-time curve [AUC]) over 24 hours after treatment with CA4P were measured in 18 patients in a phase I trial and compared with those obtained in the rat P22 carcinosarcoma model, using the same imaging technique. Rats were treated with 30 mg/kg of CA4P; patients received escalating doses from 5 to 114 mg/m2. RESULTS A similar pattern and time course of change in tumor and normal tissue parameters was seen in rats and humans. Rat tumor Ktrans was reduced by 64% 6 hours after treatment with CA4P (30 mg/kg). No significant reductions in kidney or muscle parameters were seen. Significant reductions were seen in tumor Ktrans in six of 16 patients treated at >or= 52 mg/m2, with a significant group mean reduction of 37% and 29% at 4 and 24 hours, respectively, after treatment. The mean reduction in tumor initial area under the gadolinium-diethylenetriamine pentaacetic acid concentration-time curve (AUC) was 33% and 18%, respectively, at these times. No reduction was seen in muscle Ktrans or in kidney AUC in group analysis of the clinical data. CONCLUSION CA4P acutely reduces Ktrans in human as well as rat tumors at well-tolerated doses, with no significant changes in kidney or muscle, providing proof of principle that this drug has tumor antivascular activity in rats and humans.

The biology of the combretastatins as tumour vascular targeting agents

The tumour vasculature is an attractive target for therapy. Combretastatin A‐4 (CA‐4) and A‐1 (CA‐1) are tubulin binding agents, structurally related to colchicine, which induce vascular‐mediated tumour necrosis in animal models. CA‐1 and CA‐4 were isolated from the African bush willow, Combretum caffrum, and several synthetic analogues are also now available, such as the Aventis Pharma compound, AVE8062. More soluble, phosphated, forms of CA‐4 (CA‐4‐P) and CA‐1 (CA‐1‐P) are commonly used for in vitro and in vivo studies. These are cleaved to the natural forms by endogenous phosphatases and are taken up into cells. The lead compound, CA‐4‐P, is currently in clinical trial as a tumour vascular targeting agent. In animal models, CA‐4‐P causes a prolonged and extensive shut‐down of blood flow in established tumour blood vessels, with much less effect in normal tissues. This paper reviews the current understanding of the mechanism of action of the combretastatins and their therapeutic potential.

TIE2-expressing macrophages limit the therapeutic efficacy of the vascular-disrupting agent combretastatin A4 phosphate in mice

Vascular-disrupting agents (VDAs) such as combretastatin A4 phosphate (CA4P) selectively disrupt blood vessels in tumors and induce tumor necrosis. However, tumors rapidly repopulate after treatment with such compounds. Here, we show that CA4P-induced vessel narrowing, hypoxia, and hemorrhagic necrosis in murine mammary tumors were accompanied by elevated tumor levels of the chemokine CXCL12 and infiltration by proangiogenic TIE2-expressing macrophages (TEMs). Inhibiting TEM recruitment to CA4P-treated tumors either by interfering pharmacologically with the CXCL12/CXCR4 axis or by genetically depleting TEMs in tumor-bearing mice markedly increased the efficacy of CA4P treatment. These data suggest that TEMs limit VDA-induced tumor injury and represent a potential target for improving the clinical efficacy of VDA-based therapies.

Microtubule depolymerizing vascular disrupting agents: novel therapeutic agents for oncology and other pathologies

Vascular disrupting agents (VDAs) are a relatively new group of ‘vascular targeting’ agents that exhibit selective activity against established tumour vascular networks, causing severe interruption of tumour blood flow and necrosis to the tumour mass. Microtubule depolymerizing agents form by far the largest group of small molecular weight VDAs many of which, including lead compound disodium combretastatin A‐4 3‐O‐phosphate (CA‐4‐P), are under clinical development for cancer. Although distinct from the angiogenesis inhibitors, VDAs can also interfere with angiogenesis and therefore constitute a potential group of novel drugs for the treatment of pathological conditions characterized by excessive angiogenesis, in addition to cancer. The endothelial cytoskeleton is the primary cellular target of this family of drugs, and some progress in understanding the molecular and signalling mechanisms associated with their endothelial disrupting activity has been made in the last few years. Susceptibility of tumour vessels to VDA damage is ascribed to their immature pericyte‐defective nature, although the exact molecular mechanisms involved have not been clearly defined. Despite causing profound damage to tumours, VDAs fail to halt tumour growth unless used together with conventional treatments. This failure is attributed to resistance mechanisms, primarily associated with cells that remain viable within the tumour rim, and enhanced angiogenesis. The focus is now to understand mechanisms of susceptibility and resistance to identify novel molecular targets and develop strategies that are more effective.

From bench to bedside for gene-directed enzyme prodrug therapy of cancer.

Gene therapy of cancer offers the possibility of a targeted treatment that destroys tumors and metastases, but not normal tissues. In gene-directed enzyme prodrug therapy (GDEPT), or suicide gene therapy, the gene encoding an enzyme is delivered to tumor cells, followed by administration of a prodrug, which is converted locally to a cytotoxin by the enzyme. The producer cells as well as surrounding bystanders are subsequently killed. Promising results have meant that suicide gene therapy has reached multicenter phase III clinical trials. This review will discuss the development, efficiency, mode of action and pharmacokinetics of seven GDEPT systems in vitro and in vivo. We will review the latest data of those systems in clinical trials (herpes simplex virus thymidine kinase/gancyclovir, bacterial cytosine deaminase/5-fluorocytosine, bacterial nitroreductase/CB1954 and cytochrome P450/cyclophosphamide), as well as the development of more recent and experimental systems which are not yet in clinical trials (P450 reductase/tirapazamine, carboxypeptidase/CMDA, horseradish peroxidase/indole-3-acetic acid or paracetamol and others).

Effect of nitric-oxide synthesis on tumour blood volume and vascular activity: a phase I study

BACKGROUND Nitric oxide has been implicated in tumour angiogenesis and in the maintaining of vasodilator tone of tumour blood vessels. The tumour vascular effects of inhibition of nitric-oxide synthesis have not been investigated in patients with cancer. METHODS Seven women and 11 men (12 with non-small-cell lung cancer, five prostate cancer, and one cervical cancer) were recruited onto a phase I dose-escalation study and received a single dose of the nitric oxide synthase inhibitor, N-nitro-L-arginine (L-NNA). Dose escalation was done by a modified Fibonacci scale with three patients at each dose level, starting with 0.1 mg/kg. Changes in dynamic contrast-enhanced CT measures of tumour relative blood volume and transfer constant (K) were measured at 1 h and 24 h after L-NNA administration. FINDINGS In the 18 patients, toxic effects were self-limiting cardiovascular changes: three patients had Common Toxicity Criteria version 2.0 grade 1 hypertension; two had grade 1 sinus bradycardia; and one had grade 1 palpitation. L-NNA area under the curve (AUC) increased linearly with dose from 163 micromol min(-1) L(-1) at 0.1 mg/kg L-NNA to 2150 micromol min(-1) L(-1) at 0.9 mg/kg L-NNA. In eight patients that underwent dynamic CT scanning, tumour blood volume decreased 1 h after L-NNA treatment (mean 42.9% [range 12.0-62.1]; paired t test p=0.0070), which was sustained for up to 24 h (mean 33.9% [range 6.5-64.9]; p=0.035). This decrease in blood volume was associated with an increase in the number of non-perfused pixels from 7.3% (SD 5.5) at baseline to 25.1% (15.3; p=0.0089) at 1 h, and 18.2% (12.9; p=0.050) at 24 h. There was a significant correlation between L-NNA plasma AUC and the reduction in tumour blood volume at 24 h after L-NNA (r=0.83; p=0.010). INTERPRETATION We have shown in vivo in patients with cancer that nitric oxide has a role in maintaining tumour blood supply, and we provide early clinical evidence that inhibition of nitric-oxide synthesis has tumour antivascular activity.

Blood vessel maturation and response to vascular-disrupting therapy in single vascular endothelial growth factor-A isoform-producing tumors

Tubulin-binding vascular-disrupting agents (VDA) are currently in clinical trials for cancer therapy but the factors that influence tumor susceptibility to these agents are poorly understood. We evaluated the consequences of modifying tumor vascular morphology and function on vascular and therapeutic response to combretastatin-A4 3-O-phosphate (CA-4-P), which was chosen as a model VDA. Mouse fibrosarcoma cell lines that are capable of expressing all vascular endothelial growth factor (VEGF) isoforms (control) or only single isoforms of VEGF (VEGF120, VEGF164, or VEGF188) were developed under endogenous VEGF promoter control. Once tumors were established, VEGF isoform expression did not affect growth or blood flow rate. However, VEGF188 was uniquely associated with tumor vascular maturity, resistance to hemorrhage, and resistance to CA-4-P. Pericyte staining was much greater in VEGF188 and control tumors than in VEGF120 and VEGF164 tumors. Vascular volume was highest in VEGF120 and control tumors (CD31 staining) but total vascular length was highest in VEGF188 tumors, reflecting very narrow vessels forming complex vascular networks. I.v. administered 40 kDa FITC-dextran leaked slowly from the vasculature of VEGF188 tumors compared with VEGF120 tumors. Intravital microscopy measurements of vascular length and RBC velocity showed that CA-4-P produced significantly more vascular damage in VEGF120 and VEGF164 tumors than in VEGF188 and control tumors. Importantly, this translated into a similar differential in therapeutic response, as determined by tumor growth delay. Results imply differences in signaling pathways between VEGF isoforms and suggest that VEGF isoforms might be useful in vascular-disrupting cancer therapy to predict tumor susceptibility to VDAs.

Nitric oxide in biological fluids: analysis of nitrite and nitrate by high-performance ion chromatography

The analysis of nitric oxide-derived nitrite and nitrate ions in biological fluids represents a proven strategy for determining nitric oxide participation in a diverse range of physiological and pathophysiological processes in vivo. In this article we describe a versatile method for the simultaneous measurement of NO2- and NO3- anions in both plasma and isolated tumour models based on anion-exchange chromatography with spectrophotometric detection (214 nm). This method compares well with the capillary electrophoresis technique, exhibiting an equivalent sensitivity for NO2-/NO3- anions and short run-times, i.e. not greater than 4 min. Comparisons are also made with two alternative but less satisfactory methods which employ ion-exchange or reversed-phase ion-pair chromatography with conductimetric as well as spectrophotometric detection. Technical problems associated with each method, particularly those arising from nitrate contamination, have been addressed.