Charles S. Parkins

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.

Repair in mouse lung for up to 20 fractions of X rays or neutrons

Local irradiation of the mouse thorax followed by the measurement of lung damage up to 17 months after irradiation has been carried out with up to 20 fractions of 3 MeV neutrons or of 240 kV X rays. Doses per fraction down to 0.28 and 1.5 Gy respectively were used. Repair capacity and RBE values were assessed by measuring breathing rate and lethality at monthly intervals up to 17 months. Only a small sparing of neutron damage was found. Sparing with X rays continued to increase as the size of each fraction was decreased, and was the main influence on the RBE values. The single-dose RBE was approximately 1.8, increasing to approximately 5 at the lowest dose per fraction measured. Dose-response curves derived for each fraction were well fitted by the formula alpha d + beta d2 where the repair parameter alpha/beta has values of 2-4 Gy after X irradiation. A slight fall of alpha/beta with time after X irradiation was observed, from about 4 Gy for pneumonitis to about 2 Gy for late fibrosis. This was significant for lethality but not for the increase of breathing rate. With neutrons the value of alpha was much higher than with X rays and a trend of increasing value of alpha at later times after irradiation was seen. Use of the linear quadratic dose-response formula predicts a continuing increase in the sparing of X-ray damage in lung as doses per fraction are decreased below those used here, and a limiting low-dose RBE of about 7.

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.

Nitric oxide production by tumour tissue: impact on the response to photodynamic therapy

The role of nitric oxide (NO) in the response to Photofrin-based photodynamic therapy (PDT) was investigated using mouse tumour models characterized by either relatively high or low endogenous NO production (RIF and SCCVII vs EMT6 and FsaR, respectively). The NO synthase inhibitors Nω-nitro- L -arginine (L-NNA) or Nω-nitro- L -arginine methyl ester (L-NAME), administered to mice immediately after PDT light treatment of subcutaneously growing tumours, markedly enhanced the cure rate of RIF and SCCVII models, but produced no obvious benefit with the EMT6 and FsaR models. Laser Doppler flowmetry measurement revealed that both L-NNA and L-NAME strongly inhibit blood flow in RIF and SCCVII tumours, but not in EMT6 and FsaR tumours. When injected intravenously immediately after PDT light treatment, L-NAME dramatically augmented the decrease in blood flow in SCCVII tumours induced by PDT. The pattern of blood flow alterations in tumours following PDT indicates that, even with curative doses, regular circulation may be restored in some vessels after episodes of partial or complete obstruction. Such conditions are conducive to the induction of ischaemia-reperfusion injury, which is instigated by the formation of superoxide radical. The administration of superoxide dismutase immediately after PDT resulted in a decrease in tumour cure rates, thus confirming the involvement of superoxide in the anti-tumour effect. The results of this study demonstrate that NO participates in the events associated with PDT-mediated tumour destruction, particularly in the vascular response that is of critical importance for the curative outcome of this therapy. The level of endogenous production of NO in tumours appears to be one of the determinants of sensitivity to PDT.

Induction of systemic neutrophil response in mice by photodynamic therapy of solid tumors.

Abstract Photodynamic therapy (PDT) of solid tumors elicits a strong, acute inflammatory response characterized by a rapid and massive infiltration of activated neutrophils into the tumor. The present study investigated the impact of PDT on the systemic and local (treatment site) kinetics of neutrophil trafficking and activity in mouse SCCVII and EMT6 tumor models. Differential leukocyte counts in the peripheral blood of treated mice revealed a pronounced neutrophilia developing rapidly after Photofrin® porfimer sodium (Photofrin)- or tetra(m-tetrahydroxyphenyl)chlorin (mTHPC)-based PDT. Significant neutrophilia was also observed upon PDT treatment of normal dorsal skin but not on the footpad of tumor-free mice. The changes in circulating neutrophil numbers were accompanied by an efflux of these cells from the bone marrow. An increased proportion of cells with high L-selectin (CD62L antigen) expression was found among bone-marrow–residing neutrophils 6–24 h after PDT, and in neutrophils in the peripheral circulation and treated tumors 24 h after therapy. Complement inhibition completely prevented the development of PDT-induced neutrophilia. The results of the present study demonstrate that treatment of solid tumors by PDT induces a strong and protracted increase in systemic neutrophil numbers mediated by complement activation. This reaction reflects rapid and massive mobilization and activation of neutrophils for the destruction of PDT-treated tumor tissue.

A murine model of lip epidermal/mucosal reactions to X-irradiation

A new radiobiological test system has been developed for lip epidermal/mucosal reactions in mice. This is intended for use in investigations of the effect of non-standard fractionation and of modifying drugs on oral radiation reactions in human cancer patients. An arbitrary scale of scores was devised, with separate scores for oedema of the lips and for erythema or exudation. After single doses of 13-20 Gy, the mouse lip epidermal reactions began at 5 days, reached a peak about 10-13 days, and had fallen to low values, but not to zero, by 21 days. Several different periods for averaging the reaction scores were tested for relative steepness and variability, the most useful being 10-12 days inclusive or the 12th day score alone. The use of longer periods of averaging led to apparent saturation of the scores. It was found that large doses of X-rays repeated at 21-23 day intervals did not lead to escalating waves of reactions unless each dose was greater than 17 Gy. With these larger doses, escalation of reactions occurred even if the intervals were extended.

Neutrophils as inflammatory and immune effectors in photodynamic therapy-treated mouse SCCVII tumours.

Neutrophils have become recognised as important contributors to the effectiveness of tumour eradication by photodynamic therapy (PDT). In this study, we have used the mouse SCCVII squamous cell carcinoma model to investigate the activity of neutrophils in tumours treated by PDT. Tumour levels of neutrophilic myeloperoxidase (MPO) demonstrated not only a massive and sustained sequestration of these cells in PDT-treated tumours but also revealed their activated state evidenced by the presence of released MPO. Among the adhesion molecules expressed on tumour vascular endothelium, ICAM-1 appears to be of primary importance in the invasion of neutrophils into PDT-treated tumours, because its functional blocking with monoclonal antibodies reduced the tumour cure rate. A marked upregulation of its ligands CD11b/CD18 and CD11c/CD18 found on neutrophils associated with PDT-treated tumours supports this assumption. To evaluate the role of inflammatory cytokines regulating neutrophil activity, neutralising antibodies were given to mice before PDT treatment. The results suggest that IL-1beta activity is critical for the therapeutic outcome, since its neutralisation diminished the cure rates of PDT-treated tumours. No significant effect was observed with anti-IL-6 and anti-TNF-alpha treatment. Further flow cytometry-based examination of neutrophils round in PDT-treated tumours revealed that these cells express MHC class II molecules, which suggests their engagement as antigen-presenting cells and involvement in the development of antitumour immune response.

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.

WR-2721 protection of pneumonitis and fibrosis in mouse lung after single doses of x rays

The radioprotective effect of WR-2721 has been studied in mouse lung after single doses of radiation. Using the breathing rate assay and lethality, radioprotection was assessed at monthly intervals between 3 and 18 months after irradiation during both pneumonitis and chronic fibrosis. The degree of radioprotection was greater for fibrosis than for pneumonitis using both assays. In replicate experiments, dose modifying factors (DMFs) ranging from 1.2 to 1.4 were obtained for pneumonitis and 1.5 and 1.6 for fibrosis. The differences in DMFs for the two phases of lung damage were significant. A difference in the time course of expression of damage was seen in both the breathing rate and lethality assays between mice irradiated with and without WR-2721: the damage ended sooner in the drug-treated mice. This difference is best explained by protection of all damage after 5 months by WR-2721. No evidence of drug toxicity was found. We conclude that WR-2721 protects against chronic lung fibrosis caused by radiation at least as well as against the earlier appearing pneumonitis after single doses of radiation. Thus, if WR-2721 is dose modifying and if late tissue complications are dose limiting in clinical radiotherapy, then a therapeutic benefit would be obtained by the use of this drug in clinical radiotherapy, provided that the radioprotection of tumors did not exceed a factor of 1.5-1.6.

Effect of misonidazole on radiation injury to mouse spinal cord

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