tirapazamine and Breast-Neoplasms

Several groups have shown that quantitation of tumor angiogenesis by counting blood vessels in primary breast cancer gives an independent assessment of prognosis. Poor prognosis is associated with high blood vessel counts. We have shown that the rate of cell division in endothelial cells is much higher in breast tumours than in normal breast. Breast cancer cell lines and primary human breast tumours express a wide range of vascular growth factors, including VEGF, placenta growth factor, pleiotrophin, TGF beta 1, acidic and basic FGF, and platelet-derived endothelial cell growth factor. Inhibiting angiogenesis by blocking vascular growth factors would be difficult with highly specific agents, but drugs with a broader spectrum of antagonism may be effective. We have developed several suramin analogues which are less toxic than suramin in vivo but more potent in inhibiting angiogenesis, and these have been developed for Phase I. A combination of anti-angiogenesis agents with drugs activated by hypoxia may also be useful, because anti-angiogenesis alone may not kill cells, whereas activation of hypoxic drugs could synergize. New endpoints may be necessary because inhibition of new blood vessel formation may not cause tumour regression. Thus, the endpoint of stable disease and biochemical assessment of inhibition of angiogenesis may be much more important in therapeutic studies and for drug development in the future. The prognostic importance of angiogenesis suggests that this should be a major new therapeutic target.

Topics: Angiogenesis Inducing Agents; Breast Neoplasms; Female; Genetic Therapy; Humans; Neovascularization, Pathologic; Platelet-Derived Growth Factor; Prognosis; Suramin; Thymidine Phosphorylase; Tirapazamine; Transfection; Triazines

Topics: A549 Cells; Animals; Antineoplastic Agents, Phytogenic; Breast Neoplasms; Cell Hypoxia; Colchicine; Drug Screening Assays, Antitumor; Female; Humans; Mice; Mice, Inbred BALB C; NADPH-Ferrihemoprotein Reductase; Prodrugs; Stilbenes; Tubulin

Hypoxia-activated prodrugs (HAPs) have the potential to selectively kill hypoxic cells and convert tumor hypoxia from a problem to a selective treatment advantage. However, HAPs are unsuccessful in most clinical trials owing to inadequate hypoxia within the treated tumors, as implied by a further substudy of a phase II clinical trial. Here, a novel strategy for the combination of HAPs plus vascular disrupting agent (VDA) nanomedicine for efficacious solid tumor therapy is developed. An effective VDA nanomedicine of poly(l-glutamic acid)-graft-methoxy poly(ethylene glycol)/combretastatin A4 (CA4-NPs) is prepared and can selectively enhance tumor hypoxia and boost a typical HAP tirapazamine (TPZ) therapy against metastatic 4T1 breast tumors. After treatment with the combination of TPZ plus CA4-NPs, complete tumor reduction is observed in 4T1 xenograft mice (initial tumor volume is 180 mm

Topics: Animals; Breast Neoplasms; Cell Line, Tumor; Drug Synergism; Mice; Mice, Inbred BALB C; Nanomedicine; Neoplasm Metastasis; Prodrugs; Stilbenes; Tirapazamine; Tumor Hypoxia; Xenograft Model Antitumor Assays

Tumor hypoxia, which is indispensable to tumor propagation and therapy resistance, has been one of the most important factors influencing clinical outcomes. To modulate the hypoxia microenvironment, we herein developed reactive oxygen species (ROS)-sensitive arylboronic ester-based biomimetic nanocarriers co-encapsulated with a photosensitizer chlorin e6 (Ce6) and a hypoxia-activated prodrug tirapazamine (TPZp) for tumor-specific release and synergistic photodynamic chemotherapy. In order to bypass macrophage uptake and improve tumor penetration, the nanocarriers were further modified with the red blood cell membrane and iRGD peptide (denoted as NPs@i-RBMCe6+TPZp). After administration, NPs@i-RBMCe6+TPZp exhibited prolonged blood circulation, selective tumor accumulation and excellent penetration into the tumor interior. Upon light irradiation, ROS were generated by Ce6 for photodynamic therapy (PDT), which subsequently caused dissociation of the ROS-responsive nanocarriers. An enhanced therapeutic effect was further achieved through the activation of TPZp in the aggravated local hypoxia microenvironment. The synergistic cancer therapy based on NPs@i-RBMCe6+TPZp significantly suppressed tumor growth with negligible side effects. The biomimetic nanocarriers have great potential to overcome hypoxia-limited PDT, and significantly improve the anticancer efficacy by synergistic tumor-targeted PDT and hypoxia-activated chemotherapy.

Topics: Animals; Antineoplastic Agents; Biomimetic Materials; Breast Neoplasms; Cell Hypoxia; Cell Line, Tumor; Cell Proliferation; Dose-Response Relationship, Drug; Drug Carriers; Drug Screening Assays, Antitumor; Female; Mice; Molecular Structure; Nanoparticles; Photochemotherapy; Photosensitizing Agents; Prodrugs; Reactive Oxygen Species; Structure-Activity Relationship; Tirapazamine

The recent clinical successes of antiangiogenic drug-based therapies have also served to highlight the problem of acquired resistance because, similar to other types of cancer therapy, tumors that initially respond eventually stop doing so. Consequently, strategies designed to delay resistance or treat resistant subpopulations when they arise have assumed considerable importance. This requires a better understanding of the various possible mechanisms for resistance. In this regard, reduced oxygenation is thought to be a key mediator of the antitumor effects of antiangiogenic therapies; accordingly, increased hypoxia tolerance of the tumor cells presents a potential mechanism of resistance. However, hypoxia can also be exploited therapeutically through the use of hypoxic cell cytotoxins, such as tirapazamine. With this in mind, we measured the oxygenation of PC-3 human prostate cancer xenografts subjected to chronic low-dose metronomic (LDM) antiangiogenic chemotherapy using cyclophosphamide given through the drinking water. We found that LDM cyclophosphamide impairs the oxygenation of PC-3 xenografts even during relapse, coinciding with reduced microvessel density. Combination of LDM cyclophosphamide with tirapazamine results in significantly improved tumor control in the PC-3, HT-29 colon adenocarcinoma, and MDA-MB-231 breast cancer human xenograft models without having a negative effect on the favorable toxicity profile of LDM cyclophosphamide. These results provide further evidence that reduced vascular dependence/increased hypoxia tolerance may be a basis for eventual resistance of tumors exposed to long-term LDM chemotherapy.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Cell Growth Processes; Cell Hypoxia; Cell Line, Tumor; Colonic Neoplasms; Cyclophosphamide; Female; Humans; Male; Mice; Mice, Inbred BALB C; Neoplasms; Neovascularization, Pathologic; Oxygen; Prostatic Neoplasms; Tirapazamine; Triazines; Xenograft Model Antitumor Assays

The diaziridiny/benzoquinone RH1 is shortly to enter a phase I clinical trial. The drug was originally designed as a substrate for the enzyme DT-diaphorase (DTD) such that metabolic activation of the drug would lead to toxicity. To evaluate this, we have measured the toxicity of RH1 in a pair of non-small cell lung cancer (NSCLC) cell lines of widely differing levels of DTD and in MDA231 breast cancer cells which have been engineered to overexpress DTD. In addition, we have explored the importance of the putative one-electron reductase, P450 reductase, by assessing the toxicity of RH1 in MDA231 cells engineered to overexpress the enzyme. All drug exposures were carried out under hypoxic and aerobic conditions. Those cells with the highest levels of DTD, i.e. D7 versus MDA231 wt and H460 versus H596, are substantially more sensitive to RH1 than the cell lines expressing low DTD activity. Those cells with the lowest levels of DTD activity, i.e. MDA231 wt, R4 and H596, show much greater sensitivity to RH1 under hypoxic conditions compared to aerobic conditions. Finally, overexpression of P450 reductase, i.e. comparing MDA231 wt with R4, has little, if any, impact on the toxicity of RH1 under hypoxic or aerobic conditions. In summary, RH1 can be effective in killing cells containing high levels of DTD and may be useful in treating tumors expressing this enzyme.

Topics: Aerobiosis; Antineoplastic Agents; Aziridines; Benzoquinones; Breast Neoplasms; Carcinoma, Non-Small-Cell Lung; Cell Hypoxia; Cell Line, Tumor; Cell Survival; Colony-Forming Units Assay; Humans; Inhibitory Concentration 50; Lung Neoplasms; NAD(P)H Dehydrogenase (Quinone); NADPH-Ferrihemoprotein Reductase; Spectrophotometry; Tirapazamine; Transfection; Triazines

The bioreductive drug tirapazamine (TPZ, SR 4233, WIN 59075) is a lead compound in a series of potent cytotoxins that selectively kill hypoxic rodent and human solid tumour cells in vitro and in vivo. Phases II and III trials have demonstrated its efficacy in combination with both fractionated radiotherapy and some chemotherapy. We have evaluated the generality of an enzyme-directed approach to TPZ toxicity by examining the importance of the one-electron reducing enzyme NADPH:cytochrome P450 reductase (P450R) in the metabolism and toxicity of this lead prodrug in a panel of seven human non-small-cell lung cancer cell lines. We relate our findings on TPZ sensitivity in these lung lines with our previously published results on TPZ sensitivity in six human breast cancer cell lines (Patterson et al (1995) Br J Cancer 72: 1144-1150) and with the sensitivity of all these cell types to eight unrelated cancer chemotherapeutic agents with diverse modes of action. Our results demonstrate that P450R plays a significant role in the activation of TPZ in this panel of lung lines, which is consistent with previous observations in a panel of breast cancer cell lines (Patterson et al (1995) Br J Cancer 72: 1144-1150; Patterson et al (1997) Br J Cancer 76: 1338-1347). However, in the lung lines it is likely that it is the inherent ability of these cells to respond to multiple forms of DNA damage, including that arising from P450R-dependent TPZ metabolism, that underlies the ultimate expression of toxicity.

Topics: Antineoplastic Agents; Breast Neoplasms; Carcinoma, Non-Small-Cell Lung; Drug Screening Assays, Antitumor; Female; Humans; Lung Neoplasms; Tirapazamine; Triazines; Tumor Cells, Cultured

P450 reductase (NADPH: cytochrome c (P450) reductase, EC 1.6.2.4) plays an important role in the reductive activation of the bioreductive drug tirapazamine (SR4233). Thus, in a panel of human breast cancer cell lines, expression of P450 reductase correlated with both the hypoxic toxicity and the metabolism of tirapazamine [Patterson et al (1995) Br J Cancer 72: 1144-1150]. To examine this dependence in more detail, the MDA231 cell line, which has the lowest activity of P450 reductase in our breast cell line panel, was transfected with the human P450 reductase cDNA. Isolated clones expressed a 78-kDa protein, which was detected with anti-P450 reductase antibody, and were shown to have up to a 53-fold increase in activity of the enzyme. Using six stable transfected clones covering the 53-fold range of activity of P450 reductase, it was shown that the enzyme activity correlated directly with both hypoxic and aerobic toxicity of tirapazamine, and metabolism of the drug under hypoxic conditions. No metabolism was detected under aerobic conditions. For RSU1069, toxicity was also correlated with P450 reductase activity, but only under hypoxic conditions. Measurable activity of P450 reductase was found in a selection of 14 primary human breast tumours. Activity covered an 18-fold range, which was generally higher than that seen in cell lines but within the range of activity measured in the transfected clones. These results suggest that if breast tumours have significant areas of low oxygen tension, then they are likely to be highly sensitive to the cytotoxic action of tirapazamine and RSU 1069.

Topics: Antineoplastic Agents; Breast Neoplasms; Female; Humans; Misonidazole; NADPH-Ferrihemoprotein Reductase; Radiation-Sensitizing Agents; Tirapazamine; Transfection; Triazines; Tumor Cells, Cultured

P450 reductase (NADPH:cytochrome P450 reductase, EC 1.6.2.4) is known to be important in the reductive activation of the benzotriazene-di-N-oxide tirapazamine (SR 4233). Using a panel of six human breast adenocarcinoma cell lines we have examined the relationship between P450 reductase activity and sensitivity to tirapazamine. The toxicity of tirapazamine was found to correlate strongly with P450 reductase activity following an acute (3 h) exposure under hypoxic conditions, the drug being most toxic in the cell lines with the highest P450 reductase activity. A similar correlation was also observed following a chronic (96 h) exposure to the drug in air but not following acute (3 h) exposure in air. We have also determined the ability of lysates prepared from the cell lines to metabolise tirapazamine to its two-electron reduced product, SR 4317, under hypoxic conditions using NADPH as an electron donor. The rate of SR 4317 formation was found to correlate both with P450 reductase activity and with sensitivity to tirapazamine, the highest rates of SR 4317 formation being associated with the highest levels of P450 reductase activity and the greatest sensitivity to the drug. These findings indicate a major role for P450 reductase in determining the hypoxic toxicity of tirapazamine in breast tumour cell lines.

Topics: Antineoplastic Agents; Biotransformation; Breast Neoplasms; Cell Hypoxia; Chromatography, High Pressure Liquid; Drug Resistance, Neoplasm; Humans; NADPH-Ferrihemoprotein Reductase; Oxidation-Reduction; Oxidoreductases; Oxygen; Prodrugs; Tirapazamine; Triazines; Tumor Cells, Cultured

SR-4233 (Tirapazamine) is a hypoxic cell selective cytotoxic agent currently in Phase I clinical trial. Although SR-4233 is selectively cytotoxic toward hypoxic cells some cytotoxicity toward normally oxygenated cells also occurs. SR-4233 (500 microM, 1 h) killed about 70% of normally oxygenated and 99% of hypoxic human MCF-7 breast carcinoma cells. Using a polarographic chamber and a Clark O2 electrode the O2 consumption of MCF-7 cells was measured in the presence or absence of SR-4233 (500 microM) or other inhibitors or uncouplers of oxidative phosphorylation. MCF-7 cells exhibited increased O2 consumption in the presence of SR-4233 alone and after treatment with oligomycin but not after treatment with retenone. The pattern of O2 consumption observed after treatment with SR-4233 was very similar to that seen when the cells were treated with the classical uncoupler FCCP. After 1 h of exposure to SR-4233 (500 microM) the cells were not responsive to treatment with oligomycin or FCCP for at least 3 h, but by 24 h post exposure to SR-4233 the cells had regained responsiveness to both FCCP and oligomycin. These results indicate that in normally oxygenated cells SR-4233 acts as an uncoupler of oxidative phosphorylation so that the cells continue to consume O2 but no ATP is produced. This condition can lead to ATP depletion especially in respiration intensive tissues and may provide an explanation for the muscle cramping observed in some patients treated with SR-4233.

Topics: Adenocarcinoma; Breast Neoplasms; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Electron Transport Complex IV; Female; Humans; In Vitro Techniques; Oxygen Consumption; Tirapazamine; Triazines; Tumor Cells, Cultured; Uncoupling Agents

This study examined the efficacy of combining cyclophosphamide and the hypoxic cytotoxin, tirapazamine, in the treatment of human breast cancer xenografts grown in nude mice. A single dose of tirapazamine was followed 2 h later by a single dose of cyclophosphamide. As determined from tumor regrowth delay, the effectiveness of combined therapy was greater than the additive effects of each treatment given alone. Possible mechanisms of this synergistic interaction include enhancement of DNA damage, inhibition of repair of DNA damage, or induction of apoptosis. Apart from some loss in body weight, the only other toxicity of interest in mice treated with tirapazamine was necrosis of the skin on the distal tail, which appeared to be vascular in origin.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Body Weight; Breast Neoplasms; Cyclophosphamide; Drug Screening Assays, Antitumor; Drug Synergism; Female; Humans; Mice; Mice, Nude; Tail; Tirapazamine; Transplantation, Heterologous; Triazines