In vivo drug screening method of radiosensitizers using tumor-bearing chick embryo.

In radiotherapy, tumor hypoxia is the main factor responsible for treatment resistance, and the development of radiosensitizers that can overcome this is imperative. However, many drugs that are effective in vitro and in vivo fail in clinical trials, and thus it is necessary to develop an animal model that can be used for the correct evaluation of pharmacokinetics and activity. Developing chicken eggs are commonly used in various research fields such as anticancer drug sensitivity tests and cardiotoxicity tests. We examined whether the radiosensitizing activity of etanidazole, as a hypoxic cell radiosensitizer, could be evaluated using tumor-bearing chick embryo. Following the transplantation of mouse mammary carcinoma EMT6 cells on day 11, a solid tumor was formed on day 15 and an evaluation of the time-course of the tumor revealed that the tumor weight was the highest on day 18. The maximum dose of etanidazole that did not affect tumor growth and fetal survival was 1.0mg and the maximum X-ray dose was 8Gy. Etanidazole was intravenously administered 10min prior to single dose X-ray irradiation. A significant tumor growth inhibitory effect was confirmed with 1.0mg of etanidazole in combination with 8Gy X-ray. In the case of mouse colon cancer colon26 cells, the combination of 3.0mg of etanidazole and 2Gy X-ray showed 2.79 times higher radiosensitizing activity than that observed for the control group. These results demonstrate that it is possible to evaluate the activity of radiosensitizers using tumor-bearing chick embryo.

Evaluation of the in vivo radiosensitizing activity of etanidazole using tumor-bearing chick embryo.

Chick embryos have been used as alternative experimental animals in various research fields, including virology, immunology, toxicology, oncology, and embryology. Until now, there have been no in vivo models using chick embryo to evaluate radiosensitizing activity. Here, the in vivo radiosensitizing activity of etanidazole, a well-known hypoxic cell radiosensitizer, was evaluated using tumor-bearing chick embryo. On the basis of tumor growth, drug administration and X-ray irradiation were performed on day 15 chick embryo, with the endpoint being day 18 chick embryo. In day 15 chick embryo, an X-ray irradiation dose of equal or less than 10 Gy did not cause significant tumor growth suppression. Intravenous administration of equal or less than 1.0 mg of etanidazole did not cause tumor growth suppression. Neither doses of equal or less than 8 Gy of irradiation nor 1.0 mg of etanidazole caused fatality of the chick embryo. On the basis of these results, we evaluated the radiosensitizing effect of a combination treatment with 8 Gy of irradiation and 1.0 mg of etanidazole. As noted above, 1.0 mg of etanidazole alone and 8 Gy of irradiation alone did not show tumor growth suppression. In contrast, a combination treatment with 8 Gy of irradiation and 1.0 mg of etanidazole showed 35% of significant tumor growth suppression. Thus, we succeeded in evaluating the in vivo radiosensitizing activity of etanidazole using tumor-bearing chick embryo. These results suggest that the use of tumor-bearing chick embryo may be part of a promising system for evaluating radiosensitizing activity.

Changes in tumor hypoxia induced by mild temperature hyperthermia as assessed by dual-tracer immunohistochemistry.

PURPOSE: To study the changes in hypoxia resulting from mild temperature hyperthermia (MTH) in a subcutaneous xenograft model using dual-tracer immunohistochemical techniques. MATERIALS AND METHODS: HT29 tumors were locally heated at 41 degrees C. Changes in tumor hypoxia were investigated by pimonidazole and EF5. Pimonidazole was given 1h preheating, EF5 at various times during or after treatment, 1h later the animals were sacrificed. Blood vessels were identified by CD31 staining, and perfusion by Hoechst 33342 injected 1 min pre-sacrifice. RESULTS: The overall hypoxic fraction was significantly decreased by MTH during and immediately after heating. However, MTH induced both increases and decreases in tumor hypoxia in different parts of the tumor. Specifically, MTH decreased hypoxia in the regions with relatively well-perfused blood vessels, but increased hypoxia in regions that were poorly perfused. At 24-h post heating, newly formed hypoxic regions surrounded previously-hypoxic foci, which in turn surrounded pimonidazole-stained debris. Quantitative analysis did not evince changes in tumor oxygenation due to MTH at 24h post-treatment. CONCLUSION: In this xenograft model, the effect of MTH on tumor oxygenation was variable, both spatially and kinetically. Overall tumor oxygenation was improved during and after heating, but the effect was short-lived.

Importance of antibody concentration in the assessment of cellular hypoxia by flow cytometry: EF5 and pimonidazole.

The binding kinetics of the hypoxia marker EF5 can be quantified by uptake of (14)C-labeled drug or calibrated flow cytometry using antibodies specific for drug adducts. Maximum EF5 binding is cell-line dependent and varies directly with drug exposure (area under the curve; concentration integrated over time) but inversely with pO(2) from 0 to >100 mmHg. For pimonidazole, binding is reported to be independent of the cell line and drug AUC, being zero above 10 mmHg, with an easily discriminated increase at lower pO(2). The basis for these kinetic differences is unknown, but the main experimental variable distinguishing the two marker techniques is antibody concentration ([Ab] - pimonidazole << EF5). In this study, EF5 and pimonidazole binding kinetics were compared as a function of pO(2) and antibody concentration in cells of two rat (9L and R3230) and two human (HT1080 and SiHa) cancer cell lines. For both markers, binding varied directly with AUC at all pO(2). The dynamic range of observed binding (maximum change from 0 to 76 mmHg oxygen) decreased with antibody concentration. The pO(2) dependence of binding for pimonidazole, but not EF5, varied dramatically with antibody concentration. Thus the data presented herein do not support the reported binding kinetics of pimonidazole. In particular, it is shown that the common use of antibody concentrations much lower than antigen concentrations can lead to unreliable estimations of adduct level and hence pO(2).

Radiosensitization of paclitaxel, etanidazole and paclitaxel+etanidazole nanoparticles on hypoxic human tumor cells in vitro.

Paclitaxel and etanidazole are hypoxic radiosensitizers that exhibit cytotoxic action at different mechanisms. The poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles containing paclitaxel, etanidazole and paclitaxel+etanidazole were prepared by o/w and w/o/w emulsification-solvent evaporation method. The morphology of the nanoparticles was investigated by scanning electron microscope (SEM). The drug encapsulation efficiency (EE) and release profile in vitro were measured by high-performance liquid chromatography (HPLC). The cellular uptake of nanoparticles for the human breast carcinoma cells (MCF-7) and the human carcinoma cervicis cells (HeLa) was evaluated by transmission electronic microscopy and fluorescence microscopy. Cell viability was determined by the ability of single cell to form colonies in vitro. The prepared nanoparticles were spherical shape with size between 80 and 150 nm. The EE was higher for paclitaxel and lower for etanidazole. The drug release was controlled over time. The cellular uptake of nanoparticles was observed. Co-culture of the two tumor cell lines with drug-loaded nanoparticles demonstrated that released drug effectively sensitized hypoxic tumor cells to radiation. The radiosensitization of paclitaxel+etanidazole nanoparticles was more significant than that of single drug-loaded nanoparticles.

Immunomodulatory effects of etanercept in an experimental model of spinal cord injury.

Etanercept is a tumor necrosis factor antagonist with anti-inflammatory effects. The aim of our study was to evaluate for the first time the therapeutic efficacy of in vivo inhibition of tumor necrosis factor-alpha (TNF-alpha) in experimental model of spinal cord trauma, which was induced by the application of vascular clips (force of 24 g) to the dura via a four-level T5-T8 laminectomy. Spinal cord injury in mice resulted in severe trauma characterized by edema, neutrophil infiltration, and cytokine production that it is followed by recruitment of other inflammatory cells, such as production of a range of inflammation mediators, tissue damage, apoptosis, and disease. Treatment of the mice with etanercept significantly reduced the degree of 1) spinal cord inflammation and tissue injury (histological score); 2) neutrophil infiltration (myeloperoxidase evaluation); 3) inducible nitric-oxide synthase, nitrotyrosine, cyclooxygenase-2, and cytokines expression (TNF-alpha and interleukin-1beta); and 4) apoptosis (terminal deoxynucleotidyl transferase dUTP nick-end labeling staining and Bax and Bcl-2 expression). In a separate set of experiment, we have also clearly demonstrated that TNF-alpha inhibitor significantly ameliorated the recovery of limb function (evaluated by motor recovery score). Taken together, our results clearly demonstrate that treatment with etanercept reduces the development of inflammation and tissue injury events associated with spinal cord trauma.

Etanidazole in pH-sensitive liposomes: design, characterization and in vitro/in vivo anti-Trypanosoma cruzi activity.

In this work, the hydrophilic, low molecular weight and trypanocidal drug etanidazole (ETZ) was loaded in pH-sensitive liposomes (L-ETZ). Liposomes were made of dioleoyl-phosphatidylethanolamine: cholesteryl hemisuccinate (DOPE:CHEMS, 6:4, mol:mol), of 380 nm size at 14% ETZ/total lipid (w/w) ratio. To follow their uptake and intracellular fate by fluorescence microscopy, pH-sensitive liposomes were loaded with the fluorophore/quencher pair HPTS/DPX. A fast and massive delivery of the liposomal aqueous content into the cytosol of murine J774 macrophages was observed. L-ETZ vesicles were phagocytosed by both uninfected and Trypanosoma cruzi-infected macrophages. A 72% of anti-amastigote activity (AA) was demonstrated on L-ETZ-treated J774 cells, whereas the same dose of free ETZ rendered 0% AA. Endovenous administration of L-ETZ at 14 microg/mouse dose provoked significant decrease in parasitemia levels of T. cruzi-infected mice. Conversely, inoculation of a 180-fold higher dose of free ETZ failed in reducing the number of bloodstream trypomastigotes. Hence, these results point to develop systems, such as L-ETZ, designed for selective delivery of drugs to the cytoplasm of phagocytic cells, thus enhancing the efficacy of molecules considered poorly active.

In vitro activity of Etanidazole against the protozoan parasite Trypanosoma cruzi.

We investigated the in vitro action of an hydrosoluble 2-nitroimidazole, Etanidazole (EZL), against Trypanosoma cruzi, the etiologic agent of Chagas disease. EZL displayed lethal activity against isolated trypomastigotes as well as amastigotes of T. cruzi (RA strain) growing in Vero cells or J774 macrophages, without affecting host cell viability. Although not completely equivalent to Benznidazole (BZL), the reference drug for Chagas chemotherapy, EZL takes advantage in exerting its anti-T. cruzi activity for longer periods without serious toxic side effects, as those recorded in BZL-treated patients. Our present results encourage further experiments to study in depth the trypanocidal properties of this drug already licensed for use in human cancers.

Measurement of absolute oxygen levels in cells and tissues using oxygen sensors and 2-nitroimidazole EF5.

We have established basic methods, using quantitative measures of EF5 binding, to estimate the actual pO2 of cells and tissues. In situations where the tissue can be dissociated into single cells, or for cell cultures, we can measure the distribution of cellular binding rates using flow cytometry and these can be compared with cells treated under pO2S controlled by the spinner vial or thin-film methods in vitro. The flow cytometer is calibrated by staining V79 cells treated with EF5 under "standard" conditions. For intact tissues treated with EF5 in vivo, we need to correct for possible variations in drug exposure (AUC). Frozen sections are stained for EF5 binding and are analyzed by a sensitive (cooled) CCD camera with linear output vs fluorescence [figure: see text] input. The camera has very consistent sensitivity, but the entire optical system, including the camera, can be calibrated by an absolute fluorescence standard (dye in hemocytometer). This system can also be used to measure the fluorescence of the flow cytometer standards, providing a direct link between the two assays. We can measure the maximum binding rate using the tissue cube method, but need to assume an "average" oxygen dependence of binding for intact tissues. The best-fit approximation for existing data is an inverse relationship between binding and pO2, with binding decreasing 50-fold between 0.1 and 10% oxygen. Using these methods, we routinely estimate the minimum pO2 (maximum binding) in experimental rodent and human tumors. In normal tissue models, an excellent correlation is found between near-maximal binding (severe hypoxia) and apoptosis (heart infarct and ductus arteriosus). Some normal tissues (e.g., skeletal muscle) are refractory to both cellular disaggregation and cube calibration methods. To extend the tissue imaging measurements to a complete two- or three-dimensional analysis of the distribution of tissue pO2s requires a substantial additional investment of imaging methods, which are currently being implemented.

Effects of hyperglycemia on oxygenation, radiosensitivity and bioenergetic status of subcutaneous RIF-1 tumors.

Since tissue oxygen tension is a balance between delivery and consumption of oxygen, considerable effort has been directed at increasing the former and/or decreasing the latter. Techniques to decrease the rate of cellular oxygen consumption (increasing the distance oxygen can diffuse into tissues) include increasing glycolysis by administering supra-physiologic levels of glucose. We have examined the effect of hyperglycemia produced by intravenous glucose infusion on the tissue oxygenation and radiation response of subcutaneously implanted murine radiation induced fibrosarcomas (RIF-1). A 0.3 M glucose solution was delivered via tail vein injection according to a protocol that maintained glucose at a plasma concentration of 17+/-1 mM. The effect of this treatment on radiation response (clonogenic and growth delay studies), tumor oxygenation (needle electrode pO2 and 2-[2-nitro-1H-imidazol-1-yl]-N-(2,2,3,3,3-pentafluoropropyl) acetamide (EF5) binding), and tumor bioenergetics and pH (31P NMR spectroscopy) was examined. Systemic measurements included hematocrit and blood glucose and lactate concentrations. The results of these studies suggest that these subcutaneously implanted RIF-1 tumors are both radiobiologically and metabolically hypoxic and that intravenous glucose infusion is not an effective method of modifying this metabolic state.

Hypoxia and etanidazole alter radiation-induced apoptosis in HL60 cells but not in MOLT-4 cells.

PURPOSE: To examine how hypoxia influences ionizing irradiation-induced apoptosis in cultured mammalian cells and how a hypoxic cell radiosensitizer sensitizes apoptosis under hypoxic conditions. MATERIALS AND METHODS: Two cell lines derived from human lymphocytes, HL60 and MOLT-4, were exposed to 15 Gy X-rays under aerobic and hypoxic conditions. Etanidazole was used as a hypoxic cell radiosensitizer. The apoptotic morphological changes of nuclei and the induction of ladder-like DNA fragmentation were assessed by fluorescence microscopy and agarose gel electrophoresis, respectively. RESULTS: In HL60 cells, apototic cell death and the activation of caspases 8, 9 and 3 were less induced under the hypoxic conditions than under the aerobic ones. Treatment of hypoxic cells with etanidazole enhanced X-ray-induced apoptosis and caspase activation. However, in MOLT-4 cells, neither hypoxia nor etanidazole influenced X-ray-induced apoptosis and caspase activation. In both cell lines, the frequency of X-ray-induced DNA double-strand breaks (DSB) under hypoxia was significantly smaller than that in aerobic conditions. Treatment of hypoxic cells with etanidazole enhanced them. CONCLUSION: These results suggested that X-ray-induced apoptosis in HL60 cells was initiated by DNA DSB and the treatment of hypoxic cells with etanidazole sensitized them through the enhancement of DSB induction, whereas X-ray-induced apoptosis in MOLT-4 cells occurred through damage other than to DNA.

Intravascular HBO(2) saturations, perfusion and hypoxia in spontaneous and transplanted tumor models.

Clinical trials utilizing strategies to manipulate tumor oxygenation, blood flow and angiogenesis are under way, although limited quantitative information exists regarding basic tumor pathophysiology. The current study utilized murine KHT fibrosarcomas, spontaneous mammary carcinomas and first-generation spontaneous transplants to examine heterogeneity in vascular structure and function, to relate these changes to the distribution of tumor hypoxia and to determine whether fundamental relationships among the different pathophysiological parameters exist. Three methods were included: (i) immunohistochemical staining of anatomical and perfused blood vessels, (ii) cryospectrophotometric measurement of intravascular oxyhemoglobin saturations and (iii) fluorescent detection of the EF5 hypoxic marker. While a distinct pattern of decreasing oxygenation with increasing distance from the tumor surface was observed for KHT tumors, striking intertumor variability was found in both spontaneous and first-generation transplants, with a reduced dependence on tumor volume. EF5 hypoxic marker uptake was also much more heterogeneous among individual spontaneous and first-generation tumors compared to KHT. Although mammary carcinomas demonstrated fewer anatomical blood vessels than fibrosarcomas, the proportion of perfused vessels was substantially reduced in KHT tumors, especially at larger tumor volumes. Vascular morphology, tissue histological appearance and pathophysiological parameters differed substantially between KHT tumors and both spontaneous and first-generation tumors. Such differences in vascular structure and function are also likely to correlate with altered response to therapies targeted to the vascular system. Finally, spontaneous differentiation status, tumor morphology, vascular configuration and function were well preserved in first-generation transplanted tumors, suggesting a close relationship between vascular development and function in early-generation transplants and spontaneous tumor models.

Tirapazamine-induced cytotoxicity and DNA damage in transplanted tumors: relationship to tumor hypoxia.

Tirapazamine (TPZ) is a hypoxia-selective bioreductive drug currently in Phases II and III clinical trials with both radiotherapy and chemotherapy. The response of tumors to TPZ is expected to depend both on the levels of reductive enzymes that activate the drug to a DNA-damaging and toxic species and on tumor oxygenation. Both of these parameters are likely to vary between individual tumors. In this study, we examined whether the enhancement of radiation damage to tumors by TPZ can be predicted from TPZ-induced DNA damage measured using the comet assay. DNA damage provides a functional end point that is directly related to cell killing and should be dependent on both reductive enzyme activity and hypoxia. We demonstrate that TPZ potentiates tumor cell kill by fractionated radiation in three murine tumors (SCCVII, RIF-1, and EMT6) and two human tumor xenografts (A549 and HT29), with no potentiation observed in a third xenograft (HT1080). Overall, there was no correlation of radiation potentiation and TPZ-induced DNA damage in the tumors, except that the nonresponsive tumor xenograft had significantly lower levels of DNA damage than the other five tumor types. However, there was a large tumor-to-tumor variability in DNA damage within each tumor type. This variability appeared not to result from differences in activity of the reductive enzymes but largely from differences in oxygenation between individual tumors, measured using fluorescent detection of the hypoxia marker EF5. The results, therefore, suggest that the sensitivity of individual tumors to TPZ, although not necessarily the response to TPZ plus radiation, might be assessed from measurements of DNA damage using the comet assay.

Substructure in the cell survival response at low radiation dose: effect of different subpopulations.

In order to obtain more accurate measurements of cell survival after low doses of radiation, we have used the cell sorter assay, in which a cell sorter is used to accurately count out the number of cells plated for colony formation. This method, combined with data averaging, permits measurements of survival with superior precision, which have revealed that there is substructure in the radiation response of asynchronously dividing Chinese hamster cells. The substructure, observed at doses of a few Gy, has features of a 2-component response, consistent with the presence of subpopulations of cells of different cell-cycle-related radiosensitivity. The absence of any substructure in the radiation response of homogeneous (tightly synchronized) cell populations lends strong support to this subpopulation explanation of the substructure. This assay has also been used on a variety of human tumour cell lines, most of which exhibited substructure similar to that of Chinese hamster cells. This paper outlines the application of the cell sorter assay to three different problems: (i) radiosensitizer mechanisms-etanidazole and RB 6145 are shown to enhance primarily the beta term and alpha term, respectively, of tumour cell kill, indicating that sensitizer efficacy may be tumour-specific and predictable from tumour response parameters; (ii) accurate measurement of Relative Biological Effectiveness (RBE) in a modulated clinical proton beam shows that the RBE is both dose- and depth-dependent; and (iii) measurements at lower doses clearly demonstrate a second order of substructure, termed the hypersensitive response, at doses < 1 Gy.

Mitochondrial respiratory chain features after gamma-irradiation.

Radiation provokes damage to DNA but also to membrane and protein structure. Radiolysis is a tool used very often in the study of free radical biological effects and of scavenger molecules effectiveness. Nitroimidazoles have been demonstrated to enhance the radiation effects on biological structures. The studies we have performed on isolated mitochondria irradiated, with and without nitroimidazoles, at a radiation dose equal to LD90, indicate that this treatment is not able to affect the structural and functional features investigated (ubiquinone-10, fatty acids, respiratory cytochrome levels or membrane fluidity and respiratory enzymatic activities), suggesting that an involvement of such externally produced radicals on membrane damage is unlikely. Moreover it was ascertained that the mitochondrial redox activities do not take part into the intracellular nitroimidazole reduction.

Hypoxic cell sensitization: low-dose intrinsic radiosensitivity is predictive for etanidazole efficacy in a panel of human tumour cell lines.

We used a cell sorter assay to evaluate the efficacy of the hypoxic cell sensitizer etanidazole over 3-4 logs of cell inactivation, with particular attention to the clinically relevant low-dose survival region. Analysis of the radiation responses in a panel of six human tumour cell lines under conditions of hypoxia and hypoxia with etanidazole revealed both a cell-line and radiation dose-dependence in the sensitizing ability of this drug. Fits of the linear-quadratic (LQ) model to the low-dose region of cell survival indicate that sensitization of hypoxic cells by etanidazole results primarily from a modification of the beta parameter. This results in selective sensitization of cell lines in which this parameter contributes significantly to cell kill (i.e. a low alpha/beta ratio) and implies that the efficacy of this drug may be tumour specific. Selective modification of beta also leads to a radiation dose-dependence of the sensitizing enhancement ratio (SER). Analysis of the alpha and beta parameters derived from fist to data at low doses of radiation, suggests that the dose-dependence of this sensitizer, and possibly others including oxygen, is cell-line dependent; cell lines exhibiting a low alpha/beta ratio (i.e. with a large shoulder) exhibit little or no SER dose dependence, while those with a high alpha/beta ratio (i.e. small shoulder) exhibit a reduced SER at low doses as compared to high doses. Furthermore, this analysis suggests that modelling of the low-dose radiation survival data under conditions of hypoxia, can be predictive for both the absolute sensitizing ability of etanidazole, and its dose dependence. Our results also indicate that measurement of the in vitro low-dose radiation survival response in a panel of human cell lines is a more effective assay for evaluating agents like etanidazole than simply high-dose measurements in rodent cell lines which have, in general, demonstrated more congruent survival responses.

Effect of BSO and etanidazole on neurofilament degradation in neonatal rat spinal cord cultures.

Peripheral neuropathy is the major dose-limiting toxicity of the hypoxic cell sensitiser, etanidazole. Previous work from this laboratory using culture neuronal cell lines suggested that nitroimidazole-induced degradation of neurofilament proteins might be the critical biological event mediating this neurotoxicity. The purpose of the present study was to develop the neurofilament degradation assay in an organotypic spinal cord culture system with the goal of developing strategies for optimising sensitiser efficacy as well as ameliorating nitroimidazole-induced neurotoxicity. Spinal cord cultures were treated with etanidazole and neurofilament protein degradation was analysed by immunoblot analysis. Spinal cord cultures exposed to etanidazole exhibited a dose-dependent loss of parent neurofilament proteins, with concomitant appearance of low molecular weight degradation products. The potential neurotoxic effect of L, S-buthionine sulphoximine (BSO), a compound that enhances the radiosensitising effectiveness of 2-nitroimidazoles, was also screened in this assay system. BSO alone, at concentrations up to 100 microM, did not promote neurofilament degradation. BSO (20 microM) enhanced the effect of etanidazole on neurofilament degradation by a dose-modifying factor of 1.6 +/- 0.5. Since 20 microM BSO is expected to enhance etanidazole radiosensitisation of hypoxic cells by a larger factor, this suggests that a therapeutic gain could be achieved using BSO in combination with etanidazole in radiation therapy.