The hypoxia-selective cytotoxin NLCQ-1 (NSC 709257) controls metastatic disease when used as an adjuvant to radiotherapy.

BACKGROUND: Metastases cause most cancer-related deaths. We investigated the use of hypoxia-selective cytotoxins as adjuvants to radiotherapy in the control of metastatic tumour growth. METHODS: The NLCQ-1, RB6145 and tirapazamine were assessed against the spontaneously metastasising KHT model. Subcutaneous KHT tumours (250 mm(3)) were irradiated with 25 Gy (single fraction) to control primary growth. Equitoxic drug treatments (NLCQ-1 (10 mg kg(-1)) once daily; RB6145 (75 mg kg(-1)) and tirapazamine (13 mg kg(-1)) twice daily) were administered 3-6 days post-radiotherapy when hypoxic cells were evident in lung micrometastases. Mice were culled when 50% of controls exhibited detrimental signs of lung metastases. RESULTS: In total, 95% of control mice presented with lung disease. This was significantly reduced by NLCQ-1 (33%; P=0.0002) and RB6145 (60%; P=0.02). Semi-quantitative grading of lung disease revealed a significant improvement with all treatments, with NLCQ-1 proving most efficacious (median grades: control, 4; NLCQ, 0 (P<0.0001); RB6145, 1 (P<0.001), tirapazamine, 3 (P=0.007)). Positron emission tomography (PET) was evaluated as a non-invasive means of assessing metastatic development. Primary and metastatic KHT tumours showed robust uptake of [(18)F]fluorodeoxyglucose ([(18)F]FDG). Metastatic burden discernable by [(18)F]FDG PET correlated well with macroscopic and histological lung analysis. CONCLUSION: The hypoxia-selective cytotoxin NLCQ-1 controls metastatic disease and may be a successful adjuvant to radiotherapy in the clinical setting.

Inhibition of vascular endothelial growth factor signalling using cediranib (RECENTIN; AZD2171) enhances radiation response and causes substantial physiological changes in lung tumour xenografts.

A number of pre-clinical studies have suggested that blocking vascular endothelial growth factor (VEGF) signalling can be beneficial in combination with radiotherapy. This study investigated the effects of cediranib, a highly potent orally available inhibitor of VEGF receptor tyrosine kinase activity in combination with radiation in Calu-6 lung xenografts. In nude mice, Calu-6 tumours were established and treatments initiated at a volume of 250 mm(3). Tumour-localized radiotherapy was given as three or five daily fractions of 2 Gy. Cediranib (3 mg kg(-1)) was administered 2 h prior to each fraction and continued post radiotherapy (concomitant regimen) or was initiated immediately after the completion of radiotherapy (sequential regimen). The endpoint was the time taken for tumour volume to quadruple (RTV4). Combined treatments resulted in a significantly enhanced growth delay compared with either modality alone. The therapeutic benefit was the same irrespective of the scheduling regimen. Tumour regression was observed post radiotherapy, which was associated with high levels of apoptosis and necrosis, and pronounced antivascular effects in histological samples. The amplified antivascular effect of cediranib when given after radiation suggests that pre-irradiated endothelium is sensitized to cediranib. Concomitant 5-day treatment with both cediranib and radiation reduced vessel density, perfusion and increased in tumour hypoxia. This was not associated with an acquired radioresistance suggesting that the maintenance of cediranib treatment post radiotherapy prevents the contribution of hypoxic cells to tumour regrowth. Collectively, these data support the contention that VEGFR inhibition can enhance radiation response in pre-clinical models and provide a rationale to develop cediranib in combination with radiotherapy in the clinical setting.

Oxygen-sensitive enzyme-prodrug gene therapy for the eradication of radiation-resistant solid tumours.

Overwhelming clinical and experimental data demonstrate that tumour hypoxia is associated with aggressive disease and poor treatment outcome as hypoxic cells are refractive to radiotherapy and some forms of chemotherapy. However, hypoxia is rare in physiologically normal tissues representing a tumour-specific condition. To selectively target this therapeutically refractive cell population, we have combined bioreductive chemotherapy with hypoxia-directed gene therapy. We have transfected the human fibrosarcoma cell line, HT1080, with a hypoxia-regulated expression vector encoding the human flavoprotein cytochrome c P450 reductase (HRE-P450R). This conferred hypoxia-dependent sensitivity to the alkylating nitroimidazole prodrug RSU1069 in vitro, with a greater than 30-fold increase in oxic/hypoxic cytotoxicity ratio compared with controls. Xenografts of both the HRE-P450R and empty vector transfectants had comparable hypoxic fractions and were refractive to single dose radiotherapy of up to 15 Gy. However, combining a prodrug of RSU1069 with a reduced radiotherapy dose of 10 Gy represents a curative regimen (50% tumour-free survival; day 100) in the HRE-P450R xenografts. In complete contrast, 100% mortality was apparent by day 44 in the empty vector control xenografts treated in the same way. Thus, an oxygen-sensitive gene-directed enzyme prodrug therapy approach may have utility when incorporated into conventional radiotherapy and/or chemotherapy protocols for loco-regional disease in any tissue where hypoxia is a contra-indication to treatment success. doi:10.1038/sj.gt.3301702

ZD1839 ('Iressa'), a specific oral epidermal growth factor receptor-tyrosine kinase inhibitor, potentiates radiotherapy in a human colorectal cancer xenograft model.

The effect of ZD1839 ('Iressa'), a specific inhibitor of the tyrosine kinase activity of the epidermal growth factor receptor, on the radiation response of human tumour cells (LoVo colorectal carcinoma) was evaluated in vitro and in vivo. ZD1839 (0.5 microM, incubated days 1-5) significantly increased the anti-proliferative effect of fractionated radiation treatment (2 Gy day(-1), days 1-3) on LoVo cells grown in vitro (P=0.002). ZD1839 combined with either single or fractionated radiotherapy in mice bearing LoVo tumour xenografts, also produced a highly significant increase in tumour growth inhibition (P< or = 0.001) when compared to treatment with either modality alone. The radio-potentiating effect of ZD1839 was more apparent when radiation was administered in a fractionated protocol. This phenomenon may be attributed to an anti proliferative effect of ZD1839 on tumour cell re-population between radiotherapy fractions. These data suggest radiotherapy with adjuvant ZD1839 could enhance treatment response. Clinical investigation of ZD1839 in combination with radiotherapy is therefore warranted.

17beta-Oestradiol treatment modulates nitric oxide synthase activity in MDA231 tumour with implications on growth and radiation response.

The putative oestrogen receptor negative human breast cancer cell line MDA231, when grown as tumours in mice continually receiving 17beta-oestradiol, showed substantially increased growth rate when compared to control animals. Further, we observed that 17beta-oestradiol treatment could both increase the growth rate of established MDA231 tumours as well as decreasing the time taken for initiating tumour growth. We have also demonstrated that this increase in growth rate is accompanied by a four-fold increase in nitric oxide synthase activity, which was predominantly the inducible form. Inducible-nitric oxide synthase expression in these tumours was confirmed by immunohistochemical analysis and appeared localized primarily in areas between viable and necrotic regions of the tumour (an area that is presumably hypoxic). Prophylactic treatment with the nitric oxide synthase inhibitor nitro-L-arginine methyl ester resulted in significant reduction in this apparent 17beta-oestradiol-mediated growth promoting effect. Tumours derived from mice receiving 17beta-oestradiol-treatment were characterized by a significantly lower fraction of perfused blood vessels and an indication of an increased hypoxic fraction. Consistent with these observations, 17beta-oestradiol-treated tumours were less radio-responsive compared to control tumours when treated with a single radiation dose of 15 Gy. Our data suggests that long-term treatment with oestrogen could significantly alter the tumour oxygenation status during breast tumour progression, thus affecting response to radiotherapy.

Uptake of teicoplanin by isolated rat hepatocytes: comparison with in vivo hepatic distribution.

Tissue distribution of teicoplanin, a large glycopeptide antibiotic, is slow but at equilibrium its whole body distribution volume is relatively large (Vss = 1.18-2.78 liter/kg), despite a high binding to plasma albumin. In vivo distribution into liver is extensive. Previous in vitro homogenate studies suggest that teicoplanin binds to cell membranes but only enters some cells. This possibility was investigated with isolated hepatocytes incubated for 4 h with [14C]teicoplanin alone and in the presence of additional teicoplanin (1 and 100 microg/ml). Uptake was determined after separating the cells by rapid centrifugation through a dibutyl phthalate layer and assessing viability by the trypan blue exclusion test. Teicoplanin cell uptake curves, initially rapid followed by slower distribution (which agrees with in vivo findings), were adequately described by a closed two-compartment model. Whereas entry into hepatocytes was independent of drug concentration, binding to the cell exterior membrane was concentration-dependent. The equilibrium distribution ratio (Kpu(c) +/- S.D.; 42 +/- 10) was somewhat smaller than estimated in vivo (106 +/- 9), but similar to that reported previously in vitro using liver homogenates (54 +/- 11). Also, the estimated membrane permeability-surface area product was larger in vitro than in vivo (PSu +/- S.D.; 5.5 +/- 2.9 versus 0.74 +/- 0.10 ml/min per whole liver). The most likely explanation for this difference is that in vivo only a small fraction of the total cell surface area is exposed to the perisinusoidal space, where exchange occurs.

Use of a single-pass in situ perfused rat hindlimb to study tissue distribution kinetics. Method development and experiences with Evans blue.

A single-pass in situ rat hindlimb preparation has been developed as an alternative to the isolated perfused preparation to study tissue drug distribution kinetics with minimal disturbance of the animal physiology. Evans blue (EB), a vascular marker, was administered (in saline or plasma) as a bolus into the femoral artery, and the total outflow blood was collected at timed intervals from the corresponding femoral vein. Donor blood was infused through the jugular vein at a rate that matched the loss. No changes in the blood flow or development of edema were observed. The outflow profile was characterized using statistical moments. Regardless of the injected solution, the estimated vascular volume (10% of the hindlimb wet weight) was higher than that reported for the isolated rat hindlimb (4.1%) but closer to the in vivo blood volume (6.8-8.1% of body weight for 250-g rat) and sum of vascular volumes of its composite tissues (6.9% of tissue weights). The large normalized variance (CV(2)) of the outflow (2.2+/-0.1) confirms the known heterogeneity of the hindlimb. Entrapment in the limb (approximately 4%) and escape to the rest of the body could explain the incomplete recovery (79-89%) of the dye.

In vitro studies of teicoplanin binding to rat tissues and erythrocytes.

Teicoplanin is a large polar antibiotic with a large distribution volume (Vss = 1.2-2.8 l/kg) despite extensive binding to plasma albumin. To understand this observation, binding of 3H-teicoplanin to 10% rat tissue homogenates was determined in vitro by ultracentrifugation in the presence of teicoplanin (1-30 microg/ml). Binding and efflux from erythrocytes were also studied. The in vitro total-to-unbound tissue concentration ratio (Kpu) differed widely but for each tissue was concentration independent. Upon correction for the plasma unbound fraction, there was discrepancy between the in vitro and in vivo tissue-to-plasma concentration ratios (KP), the latter calculated from previously published tissue and plasma concentration-time data using the area method. Moreover, calculation of Vss from in vitro Kp (8.10 l/kg) overestimated the in vivo value. These results suggest that in vivo teicoplanin binds to cell membranes and enters some but not all cells, such as erythrocytes.

Tissue water content in rats measured by desiccation.

Tissue water content was determined by desiccation to constant weight at 40 degrees-50 degrees C in 14 tissues from two groups of rats weighing 200-250 and 270-430 g, respectively. The water content (mean +/- SE; ml/g) was highest in testes (0.861 +/- 0.002) and lowest in adipose (0.183 +/- 0.017) followed by bone (0.446 +/- 0.017) and skin (0.651 +/- 0.007). The average water content in the remaining tissues was 0.763 (+/- 0.003). Upon correction for the water content of residual tissue blood, significant difference between the uncorrected and corrected tissue water was observed for spleen, lungs, kidneys, heart, liver, and brain. Tissue water was independent of body weight, and was the same for right and left kidneys as well as testes and bone. Whereas the position of the muscle (back, abdomen, hindlimb) and adipose tissue (perirenal and subcutaneous) had no influence on water content, for skin, a slight difference was found between back and abdomen. In general, the current results are in agreement with composite literature values, but provide in one study data for all tissues used in the development of physiologically based pharmacokinetic models of rat.

Detection of abnormal peripheral blood mononuclear cell cytokine networks in human IgA nephropathy.

Dysregulated cytokine expression has been implicated in the pathogenesis of IgA nephropathy, but the mechanisms and selectivity of this response are poorly understood. In this study we have examined the expression of a range of immunoregulatory cytokine mRNAs by peripheral blood mononuclear cells (PBMNCs) from 45 patients with IgA nephropathy stratified empirically according to urinary red cell excretion: 10 in remission, and 35 with active disease (21 mild, 14 moderate), and 17 normal, and 15 disease, controls. We used a semi-quantitative polymerase chain reaction (PCR) technique. None of the patients had experienced recent episodes of macroscopic hematuria. Simultaneous analysis of monocyte class II antigen (DR) expression was also performed by two-color immunoflow cytometry. TGF-beta 1 mRNA was detected in 68% (24 of 35) of patients with active, and 70% (7 of 10) inactive IgA nephropathy, but in only 18% (3 of 17) normal (P < 0.005), and 27% (4 of 15) disease controls. IL-6 transcripts were identified in 37% (13 of 35) of patients with active IgA nephropathy, compared with 6% (1 of 17) normal controls (P = 0.015), with no significant increase in IgA remission, or disease control groups. TNF-alpha mRNA was detected in 29% (5 of 17) of normal and 53% (8 of 15) disease controls, but in only 7% (3 of 35) of patients with IgA nephropathy (P = 0.015). There was no significant change in TGF-beta 2, gamma-IFN, IL-2, IL-4, IL-1 alpha or IL-1 beta detection between groups.(ABSTRACT TRUNCATED AT 250 WORDS)

The detection of intracytoplasmic interleukin-1 alpha, interleukin-1 beta and tumour necrosis factor alpha expression in human monocytes using two colour immunofluorescence flow cytometry.

Two colour flow cytometry was used to analyse in situ cytokine expression by human monocytes. Whole blood was cultured in siliconised glass bottles, with or without E. coli lipopolysaccharide (LPS), for various times, and the mononuclear cells (MNCs) then exposed to a variety of permeabilisation procedures prior to flow cytometric analysis. Paraformaldehyde (PF)/saponin fixation preserved cellular morphology, and caused a reproducible degree of permeabilisation (estimated by propidium iodide inclusion: mean 94%, range 86-99% (n = 33)). After fixation with 4% PF and permeabilisation with 1% saponin at 0 degrees C in PBS containing 20% human serum, MNCs were incubated with phycoerythrin(PE)-conjugated mouse anti-CD14 (monocyte phenotype) and polyclonal rabbit anti-human interleukin-1 alpha (IL-1 alpha), IL-1 beta, tumour necrosis factor alpha (TNF-alpha), or control rabbit IgG. Binding of rabbit antibodies was detected using goat anti-rabbit IgG fluorescein isothiocyanate (FITC). FITC fluorescence was increased in CD14 PE positive cells with the three anti-cytokine antibodies following LPS stimulation, compared with controls. There was a reproducible dose related response in monocyte IL-1 beta and TNF-alpha expression following LPS stimulation, with early peaks in TNF-alpha (2 h), compared with IL-1 beta (4 h), and IL-1 alpha (12 h). Specificity of this cytokine detection system was confirmed by inhibition studies using the corresponding recombinant human cytokines, by an absence of staining in CD14 negative or unpermeabilised MNCs, and by the characteristic cytoplasmic localisation of the different cytokines visualised with UV immunochemistry. Hence, the methods described here provide a reproducible, semiquantitative and specific assay for the detection of cell associated monokines. The technique may be applicable to the analysis of a variety of different cytokines in other phenotypically defined cell populations.