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The effect of serum starvation on the expression and phosphorylation of PKC-alpha and p53 in Chinese hamster V79 cells was investigated. Serum starvation led to growth arrest, rounding up of cells and the appearance of new PKC-alpha and p53 bands on Western blots. Prolonged incubation (> or = 48 hr) in serum-deprived medium led to cell detachment and death. Moving cells to fresh medium containing 10% serum before, but not after, cell detachment reversed the changes observed in PKC-alpha and p53, and also prevented later cell detachment. Radiolabelling studies showed that the higher-molecular-weight PKC-alpha and p53 bands result from increased phosphorylation, while a lower-molecular-weight PKC-alpha band reflects newly synthesized protein. Immunocomplex kinase assays have shown that the increased phosphorylation of PKC-alpha is associated with its increased activity. To study the relationship between PKC-alpha, p53 and cell death, cells were treated either with TPA, to down-regulate PKC or with staurosporine, to inhibit PKC activity. Staurosporine, a potent PKC inhibitor and inducer of programmed cell death, caused the appearance of new PKC-alpha and p53 bands similar to those induced by serum starvation. If serum starvation was preceded by prolonged (48 hr) TPA treatment to down-regulate PKC-alpha, cell detachment and death did not take place within the same time frame. Intracellular fractionation of cells demonstrated that increased expression of PKC-alpha and the appearance of the associated higher and lower molecular-weight bands occurred in the nucleus. These data highlight the association of PKC-alpha and p53 with cellular events leading to cell death.

Photodynamic therapy (PDT) is a developing approach to the treatment of solid tumours which requires the combined action of light and a photosensitizing drug in the presence of adequate levels of molecular oxygen. We have developed a novel series of photosensitizers based on zinc phthalocyanine which are water-soluble and contain neutral (TDEPC), positive (PPC) and negative (TCPC) side-chains. The PDT effects of these sensitizers have been studied in a mouse model bearing the RIF-1 murine fibrosarcoma line studying tumour regrowth delay, phosphate metabolism by magnetic resonance spectroscopy (MRS) and blood flow, using D2O uptake and MRS. The two main aims of the study were to determine if MRS measurements made at the time of PDT treatment could potentially be predictive of ultimate PDT efficacy and to assess the effects of sensitizer charge on PDT in this model. It was clearly demonstrated that there is a relationship between MRS measurements during and immediately following PDT and the ultimate effect on the tumour. For all three drugs, tumour regrowth delay was greater with a 1-h time interval between drug and light administration than with a 24-h interval. In both cases, the order of tumour regrowth delay was PPC > TDEPC = TCPC (though the data at 24 h were not statistically significant). Correspondingly, there were greater effects on phosphate metabolism (measured at the time of PDT or soon after) for the 1-h than for the 24-h time interval. Again effects were greatest with the cationic PPC, with the sequence being PPC > TDEPC > TCPC. A parallel sequence was observed for the blood flow effects, demonstrating that reduction in blood flow is an important factor in PDT with these sensitizers. © 1999 Cancer Research Campaign

Nitro-L-arginine inhibits the production of nitric oxide and can thereby cause vasoconstriction in vivo. One consequence of this is that nitro-L-arginine can increase hypoxia in a range of transplantable and spontaneous murine solid tumours. Bioreductive drugs such as RB6145 are more active under hypoxic conditions, and the combination of RB6145 with nitro-L-arginine in vivo shows greater anti-tumour activity than either agent individually. In mice given nitro-L-arginine at 10 mg kg(-1) i.p. up to 1 h before or after 300 mg kg(-1) i.p. RB6145, survival of KHT tumour cells was reduced by 3-4 logs when assessed by clonogenic assay 24 h after treatment. RB6145 or nitro-L-arginine alone caused no more than 20% cell kill. Similar effects were found in SCCVII tumours. The tumour response to the drug combination was tumour size dependent, with increased tumour cell sensitivity occurring when the tumour volume at the time of treatment was increased. Further, the response of KHT tumours to the combination of RB6145 and nitro-L-arginine was also dependent on the time interval between treatment and on when tumours were excised for determination of survival in vitro. The relative surviving fraction was about 0.3 for intervals less than 4 h but was reduced to 0.01 at 12 h and 0.001 at 24 h. These results were supported by histological examination of tumours, when necrosis at 2 h after treatment was less than 5% but increased to greater than 90% at 24 h. RB6145-induced normal tissue damage, as measured by CFU-A survival, was not altered by combining with nitro-L-arginine. Hence, this drug combination may provide therapeutic benefit. It is likely that the substantial anti-tumour effects are due to enhancement of bioreductive toxicity through increased tumour hypoxia, although additional mechanism(s) may also contribute to the overall response. Images Figure 4

The cellular stress response, whereby very low doses of cytotoxic agents induce resistance to much higher doses, is an evolutionary defence mechanism and is stimulated following challenges by numerous chemical, biological and physical agents including particularly radiation, drugs, heat and hypoxia. There is much homology in the effects of these agents which are manifest through the up-regulation of various genetic pathways. Low-dose radiation stress influences processes involved in cell-cycle control, signal transduction pathways, radiation sensitivity, changes in cell adhesion and cell growth. There is also homology between radiation and other cellular stress agents, particularly hypoxia. Whereas traditionally, hypoxia was regarded mainly as an agent conferring resistance to radiation, there is now much evidence illustrating the cytokine-like properties of hypoxia as well as radiation. Stress phenomena are likely to be important in risks arising from low doses of radiation. Conversely, exploitation of the stress response in settings appropriate to therapy can be particularly beneficial not only in regard to radiation alone but in combinations of radiation and drugs. Similarly, tissue hypoxia can be exploited in novel ways of enhancing therapeutic efficacy. Bioreductive drugs, which are cytotoxically activated in hypoxic regions of tissue, can be rendered even more effective by hypoxia-induced increased expression of enzyme reductases. Nitric oxide pathways are influenced by hypoxia thereby offering possibilities for novel vascular based therapies. Other approaches are discussed.

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. Images Figure 4

Large phosphomonoester (PME) signals are detected in the phosphorus magnetic resonance spectra (31P MRS) of many neoplastic and rapidly dividing tissues. In addition, alterations in phosphodiester (PDE) signals are sometimes seen. The present study of a murine lymphoma growing in liver showed a positive correlation between the hepatic PME/PDE ratio measured in vivo by 31P MRS at 4.7 T and the degree of lymphomatous infiltration in the liver, quantified by histology. High-resolution 31P MRS of liver extracts at 9.7 T showed that the PME peak consists largely of phosphoethanolamine (PE) and to a lesser extent of phosphocholine (PC). The concentration of both PE and PC increased positively with lymphomatous infiltration of the liver. In vivo, the PDE peak contains signals from phospholipids (mostly phosphatidylethanolamine and phosphatidylcholine) and the phospholipid breakdown products glycerophosphoethanolamine (GPE) and glycerophosphocholine (GPC). Low levels of GPE and GPC were detected in the aqueous extracts of the control and infiltrated livers; their concentrations remained unchanged as the infiltration increased. The total concentration of phospholipids measured by 31P MRS of organic extracts decreased about 3-fold as the infiltration increased to 70%. Thus, our data showed that the increased PME/PDE ratio in vivo is due to both an increase in the PME metabolites and a decrease in the PDE metabolites. We propose that this ratio can be used as a non-invasive measure of the degree of lymphomatous infiltration in vivo. Images Figure 2

Prolonged hypoxia induced transient drug resistance in Chinese hamster lung fibroblasts. Previously hypoxic cells were resistant to adriamycin and resistant to etoposide. Complete recovery of etoposide sensitivity was observed following re-aeration for 24 hr. A change in P-glycoprotein expression was unlikely to contribute to the resistance caused by hypoxia, since adriamycin resistance was not reversed by verapamil. However, alteration in the plasma membrane structure may be involved, since previously hypoxic cells were resistant to extracellular superoxide radical generated by the addition of xanthine/ xanthine oxidase. In contrast, adriamycin sensitivity was not altered by hypoxia in 3 human breast-cancer cell lines. MDA-468 and MCF-7/Adr differed in their response to EGF, independent of the presence of hypoxia. These results suggest that hypoxic-stress-induced drug resistance is not generalized.

For part I see ibid., vol. 8, no.2, pp. 251-256 (1993). The axisymmetric analysis of the magnetic field and the finite element method (FEM) formulation of the tubular linear induction motor (TLIM) for application to hydraulic capsule pipelines was discussed in part I. In these studies a three-phase sinusoidal current source supplies the stationary windings to produce a traveling magnetic field inducing currents in the cylindrical capsule conducting wall to produce thrust on the capsule. The results of the different studies using the developed FEM tool show that by introducing iron elements in the pipe wall, the thrust can be increased. Five cases are discussed here, and the magnetic field contours for each case are presented. FEM computation of the thrust for all cases is compared to choose the design which produces the maximum thrust. >

An axisymmetric model of the magnetic field of the tubular linear induction motor (TLIM) for application to hydraulic capsule pipelines is developed using finite element method (FEM) analysis. The FEM model is used to analyze a specific TLIM design at standstill for a given supply current. The finite element formulation of the field equations is discussed and the magnetic field contours at different instants of time are presented. FEM computation of the thrust compares very well with experimental data. >

The effect of combining photodynamic therapy (PDT) and bioreductive drugs has been investigated using the RIF-1 experimental murine tumour. Light was delivered interstially to the tumour at 675 nm using a single optical fibre attached to an argon-ion dye laser. The photosensitizer was disulphonated aluminium phthalocyanine (AlS2Pc) and the bioreductive drugs were the dual function nitroimidazole RSU1069 and its pro-drug RB6145. Varying the time between administration of the photosensitizer and light delivery (TL) from 30 min to 24 h had little influence on the extent of the anti-tumour effect of PDT alone, as measured by the regrowth delay endpoint. When the bioreductive drug was included in the treatment, administered 20 min before light irradiation, regrowth delay was greatly increased. The effectiveness of the combined treatment was optimum for short values of TL (about 1 h). Fluorescence microscopy was used to investigate the distribution of the photosensitizer within the tumours. This showed that the compound was mainly confined to the tumour vasculature over the first few hours post-treatment. The high efficacy of the combined treatment of PDT and bioreductive drugs for short values of TL suggest that photodynamic action, during the period when the photosensitizer AlS2Pc is confined to the vasculature, enhances the severity of tumour hypoxia which is sufficient to induce activation of the bioreductive drugs. Images Figure 1

A series of 2-nitroimidazoles bearing side chains terminating in or containing aziridinyl and oxiranyl groups has been prepared, and the compounds were evaluated in vitro as hypoxia-selective bioreductively-activated cytotoxins and selected compounds tested for their radiosensitizing properties toward hypoxic mammalian cells. Compounds were either the regioisomers of analogues of the potent dual-functional 2-nitroimidiazole alpha-[(1-aziridinyl)-methyl]-2-nitro-1H-imidazole-1- ethanol (RSU-1069, 1) with additional methyl groups or related oxiranes of varying side-chain length and type. Oxiranyl derivatives showed little differential toxicity, and those tested were less effective as radiosensitizers, and although these properties were influenced by side-chain length, differences were not great. Aziridinyl compounds related to 1 but with increased side-chain lengths were unstable. Methylation of 1 in various regions had little effect on radiosensitization and no clear advantages over 1 as differential cytotoxic drugs. Progressive methylation at C-3 was found to increase toxicity but decrease hypoxia selectivity. Incorporation of a cyclohexane side chain in 1,2-cis-2,3-trans-3-aziridin-1-yl-2-hydroxy-1-(2-nitroimidazol+ ++-1- yl)cyclohexane (26) abolished hypoxia-selective toxicity and unexpectedly reduced radiosensitizing efficiency. Of the aziridines, 1-(2-nitro-1-imidazolyl)-2-methyl-3-(1-aziridinyl)-2-propanol (20) was comparable in efficacy to 1 as a bioreductively-activated cytotoxin with slightly lower aerobic toxicity; however, the prodrugs of 1 remain as preferred candidates for clinical evaluation.

RB 6145 is a pro-drug of the hypoxic cell radiosensitizer RSU 1069 with reduced systemic toxicity. The maximum tolerated dose (MTD) of RSU 1069 for C3H/He mice was 80 mg/kg (0.38 mmol/kg) ip but 320 mg/kg (1.5 mmol/kg) following po administration. The MTD values of RB 6145 were 350 mg/kg (0.94 mmol/kg) ip and 1 g/kg (2.67 mmol/kg) po. Toxicity of RSU 1069 toward bone marrow stem cells was also less after po administration than after ip administration; 0.1 mmol/kg ip RSU 1069 and 0.38 mmol/kg po RSU 1069 both reduced the surviving fraction of clonogenic CFU-A cells by 50%. Oral administration of RSU 1069 resulted in lower spermatogenic toxicity. No loss of intestinal crypts was detected after ip or po administration of RSU 1069. Some nephrotoxicity was observed in half of the mice given the highest po dose of 1.5 mmol/kg of RSU 1069; this was not observed following the highest ip dose of drug. For RSU 1069 and RB 6145, administered by either route, the maximum hypoxic cell radiosensitization in murine KHT sarcomas, occurred when the drugs were given 45-60 min before 10 Gy of X rays. The degree of radiosensitization produced by a particular dose of either compound was largely independent of the route of administration. Preliminary pharmacokinetic studies, using 3H-RSU 1069, suggested that anti-tumor efficacy correlated with peak blood level of label and concentration in the tumor at the time of irradiation, which were not reduced by po compared with ip administration. Normal tissue toxicity tended to correlate with total exposure over time, which was reduced approximately two-fold by po administration. Oral administration of RSU 1069 or RB 6145, as well as being convenient, may give therapeutic benefit since dose-limiting toxicity in mice was reduced compared with parenteral administration, whereas radiosensitizing activity was less affected.

This paper presents a finite element method (FEM) analysis of a commercially available switched reluctance motor (SRM) supplied from its associated power electronic drive. The instantaneous current and torque waveforms are computed using the results of the FEM analysis with the numerical solution of the nonlinear differential equations describing the drive. The effects of different machine parameters, speeds, applied voltages and stator winding voltage switching on and off instants are given. Experimental results compare very well with the results calculated by the method of analysis presented in the paper.

A comparison of computed and measured performances of a 10 hp Oulton switched reluctance motor (SRM) and a comparison with the performance of induction motors are presented. The instantaneous current and torque waveforms are computed for the SRM using the results of finite element method (FEM) analysis. The SRM calculated and experimental locked rotor torque, instantaneous current, efficiency, and losses when pulsed voltage excitation is applied to the motor windings were compared. In all cases, there is quite good agreement between the theoretical and experimental results. In addition, the measured total losses, efficiency, and temperature rise for a 10 hp SRM and different induction motors of the same rating are presented. The SRM has a higher efficiency and a lower temperature rise than the induction motors tested. >

alpha-[(1-Aziridinyl)methyl]-2-nitro-1H-imidazole-1-ethanols, of general formula ImCH2CH(OH)CH2NCR1R2CR3R4, where Im = 2-nitroimidazole and R1, R2, R3, R4 = H, Me, are radiosensitizers and selective bioreductively activated cytotoxins toward hypoxic tumor cells in vitro and in vivo. Treatment of the aziridines with hydrogen halide in acetone or aqueous acetone gave the corresponding 2-haloethylamines of general formula ImCH2CH(OH)CH2(+)-NH2CR1R2CR3R4X X-, where R1, R2, R3, R4 = H, Me, and X = F, Cl, Br, I. These 2-haloethylamines were evaluated as prodrugs of the parent aziridines. The rates of ring closure in aqueous solution at pH approximately 6 were found to increase with increasing methyl substitution and to depend on the nature of the leaving group (I approximately Br greater than Cl much greater than F). A competing reaction of ImCH2CH(OH)CH2+NH2CH2CH2X X- (X = Cl, Br) with aqueous HCO3- ions gives 3-[2-hyroxy-3-(2-nitro-1H-imidazol-1-yl)propyl]-2-oxazolidinone. The activities of these prodrugs as radiosensitizers or as bioreductively activated cytotoxins were consistent with the proportion converted to the parent aziridine during the course of the experiment. alpha-[[(2-Bromoethyl)amino]methyl]-2-nitro-1H-imidazole-1- ethanol (RB 6145, 10), the prodrug of alpha-[(1-aziridinyl)methyl]-2-nitro-1H-imidazole-1-ethanol (RSU-1069, 3), is identified as the most useful compound in terms of biological activity and rate of ring closure under physiological conditions.

In this work tumour hypoxia is induced by physically occluding the tumour vascular supply by clamping, or by giving mice 5 mg kg-1 hydralazine. These methods have previously been shown to increase the radiobiological hypoxic fraction in tumours close to 100%. Their effectiveness in potentiating the bioreductive toxicity of: misonidazole (800 mg kg-1), RSU1069 (80 mg kg-1), mitomycin C (5 mg kg-1) and SR4233 (50 mg kg-1) is assessed in the RIF-1 and KHT tumours using regrowth delay as an assay. Clamping alone for 120 min gives little or no response, but when RSU1069 is administered 15 min before clamping, large growth delays result. RIF-1 tumours clamped for 90 or 120 min with RSU1069 give cure rates of 12.5% and 37.5% respectively. Less effect with clamping is seen for the other bioreductive agents. The effect of hydralazine with RSU1069 although significant in the RIF-1 tumour, is modest compared to that for clamping. Small enhancements of toxicity are seen with hydralazine in combination with misonidazole in the RIF-1 tumour and mitomycin C in both tumours. The varying effectiveness of these treatments is attributed to several factors which include the level and duration of hypoxia, concentration and contact time of the bioreductive drugs, the microenvironment of the tumour and the nature of the reductive metabolic pathways available in the different tumour cell types.

We have investigated the cross-sensitivity of a number of cell lines to three different classes of bioreductive drugs under both aerobic and hypoxic conditions. The cell lines used were selected for their sensitivity to DNA-damaging agents and fall into two groups. One group, MMC cells derived from CHO-K1 cells (Robson et al., 1985), show a range of sensitivities to mitomycin C in air. The second group, irs cells were cloned from V79 Chinese hamster fibroblasts (Jones et al., 1987) and exhibit sensitivity to ionising radiation. The sensitivity of both groups of cells to mitomycin C (MMC), RSU-1069 and SR4233 was assessed under aerobic and hypoxic conditions. No difference in aerobic or hypoxic toxicity of MMC was observed for CHO-K1 or MMC sensitive cell lines (MMC-2 and MMC-3). However, the MMC-resistant cell line (MMCr) was 10 times more sensitive under hypoxic than aerobic conditions. This suggests that MMCr cells lack or are deficient in the enzymes responsible for activating MMC under aerobic conditions compared to other MMC cells. In contrast, differential toxicities of between 3 and 30 have been observed for all CHO cells treated with RSU-1069 and SR4233. Treatment of V79 and irs cells with RSU-1069 and SR4233 also resulted in selective toxicity towards hypoxic cells. Differential toxicities between 50 and 100 were observed for V79 cells. For both RSU-1069 and SR4233, the hypoxic toxicities were similar in V79 and irs cells but in air, the radiation sensitive cells were up to 10 times more sensitive than wild type cells.

The authors present a comparison of the computed and measured performances of a 10 hp Oulton switched reluctance motor (SRM). The derivatives of the flux linkages with respect to rotor position angle and with respect to current excitation as a function of time are calculated. Using these results, calculated current waveforms of the SRM for different loads and speeds are compared with experimental results. The calculated and experimental instantaneous current, average torque, input power, efficiency, losses and temperature rise of the SRM are compared when pulsed voltage excitation is applied to the motor windings. In all cases there is quite good agreement between the theoretical and experimental results. In addition, the measured total losses, efficiencies and temperature rises for a 10 hp SRM and different induction motors of the same ratings are presented. >

The locked rotor torque and instantaneous current and torque waveforms for a switched-reluctance motor (SRM) have been calculated using a two-dimensional finite-element method. The effects of switching the stator winding excitation on and off at different positions of the rotor are included. Experimental results for a 10-hp Oulton switched-reluctance motor are shown. In addition, the authors compare the SRM with a Century electric induction motor of the same rating. >

The effects of acute hypoxia on integrin expression and adhesion to extracellular matrix proteins were investigated in two human melanoma cell lines, HMB-2 and DX3, and a human adenocarcinoma cell line, HT29. Exposure to hypoxia caused a significant down-regulation of cell surface integrins and an associated decrease in cell adhesion. Loss of cell adhesion and integrin expression were transient and levels returned to normal within 24 h of reoxygenation. Other cell adhesion molecules, such as CD44 and N-CAM, were also down-regulated after exposure of cells to hypoxia. Acute exposure to hypoxia of cells at confluence caused rapid cell detachment. Cell detachment preceded loss of viability. Detached HMB-2 and DX3 cells completely recovered upon reoxygenation, and floating cells re-attached and continued to grow irrespective of whether they were left in the original glass dishes or transferred to new culture vessels, while detached HT29 cells partly recovered upon reoxygenation. Cell detachment after decreased adhesion appears to be a stress response, which may be a factor enabling malignant cells to escape hypoxia in vivo, with the potential to form new foci of tumour growth.

One-electron oxidation of a series of C(2)- and C(3)-methyl substituted analogues of 5,6-dihydroxyindole (DHI), in the pH range 5-9, was studied using the technique of pulse radiolysis with spectrophotometric detection. This investigation was undertaken to further our understanding of the involvement of free radical species in the polymerization processes leading to melanin formation. The optical absorption spectra of the protonated indole semiquinone radicals resulting from the one-electron oxidation of C(2)- and C(3)-methyl substituted indoles were similar to those of their corresponding hydroxylated indoles. From this similarity, it is inferred that methylation at C(2) and C(3) of DHI has little or no effect upon the initial radicals. The semiquinone radicals of these analogues subsequently decay to yield the corresponding quinone methide/imine. However, methyl substitution at C(2) and C(3) of the quinone methide derived from these analogues results in their stabilization. This stabilization contracts with the reactivity of the corresponding quinone methide from DHI. Further, these stabilized quinone methides do not interact with the azide ion (N3-), in contrast to the reaction of N3- with the quinone methide of DHI. It is concluded that methylation at C(2) and C(3) of DHI will modify the pathways so that the polymerization processes are less effective than those with DHI.

Magnetic resonance spectroscopy (MRS) in situ was used to study changes in 31P metabolism occurring during and after treatment of murine RIF-1 tumours with photodynamic therapy (PDT). Tumours were irradiated using a fibreoptic light delivery system while the mice were in position within the magnet. Changes in 31P-MRS were observable during and immediately after treatments of several minutes' duration. Both the extent and duration of the increase in the Pi/total ratio were light dose dependent. The effect on the metabolism was also affected by the time interval (TL) between administering the photosensitiser disulphonated phthalocyanine, (A1S2Pc) and the light. With a dose of 50 J the increase in Pi/total was much faster when TL was 1 h than when TL was 24 h. This difference in rate probably reflects differences in the distribution of A1S2Pc within the tumour. Significant decreases in pH were only seen after a light dose of 50 J when TL was 1 h. Blood flow measurements using deuterium uptake were also carried out using MRS. These experiments showed that for a dose of 50 J the level of blood flow was reduced by approximately 90% of the control value within 10 min from the end of the 8 min light treatment. This occurred irrespective of the value of TL. The data indicate that it is possible to observe very early changes in 31P metabolism that can be attributed to direct cellular damage as opposed to the later changes indicative of overall tumour hypoxia caused by vascular damage.

We describe aspects of the tumour microenvironment that are available as targets for manipulation. In particular, the question asked is whether hypoxia in tumours is a problem to be overcome, or a physiological abnormality to be exploited? Bioreductive drugs require metabolic reduction to generate cytotoxic metabolites. This process is facilitated by appropriate reductases and the lower oxygen conditions present in solid tumours compared with normal tissues. Because of their specificity, bioreductive drugs are used to help answer this question. Other aspects of tumour physiology and biochemistry that may be exploited include tissue dependent reductase expression, pH and angiogenesis.

Twenty-three human tumour cell lines (lung, breast, and colon) and eight rodent cell lines were evaluated for their sensitivity to the quinone-based anticancer drug EO9 [3-hydroxymethyl-5-aziridinyl-1-methyl-2-(1H indole-4,7-dione)prop-beta-en-alpha-o1]. Sensitivity was compared with the intracellular levels of DT-diaphorase, and cell lines showing highest enzyme activity tended to be the most sensitive to EO9. The role of DT-diaphorase in determining drug sensitivity was confirmed by using the enzyme inhibitor dicoumarol, which protects cells containing high levels of DT-diaphorase from the cytotoxic action of EO9. Hypoxia increased the cytotoxicity of cells containing low but not high levels of DT-diaphorase, implying that both 1- and 2-electron reductive activation processes can be important for expression of EO9 toxicity. It is concluded that EO9 is a potentially useful agent in the enzyme directed approach to the use of bioreductive drugs in cancer therapy.

Phospholipid metabolism of human breast cancer was studied by 31P magnetic resonance spectroscopy (MRS). In vivo localised 31P MR spectra were obtained from the tumour alone using phase modulated rotating frame imaging. For 31 tumours, median (range) phosphomonoester (PME) to ATP ratio was 1.48 (0.57-3.78) and phosphodiester (PDE) to ATP ratio was 1.65 (0.44-3.89). DNA index and S phase fraction (SPF) were measured by flow cytometry of paraffin embedded tissue. Twelve (39%) tumours were diploid and 19 aneuploid. Median (range) SPF for 29 assessable tumours was 5.3% (0.6-28%), with significantly greater median SPF for aneuploid tumours (9.3%) than diploid (3.8%, P = 0.007). There was a significant association between PME/ATP and SPF (P = 0.03) due to a significant correlation for aneuploid tumours (P = 0.01). High resolution 31P MRS of extracts from 18 tumours (including seven studied in vivo) demonstrated that the PME peak consists predominantly of phosphoethanolamine (PE) with a smaller contribution from phosphocholine (PC) (median (range) PE/PC: 3.02 (1.13-5.09)). Changes in PME/ATP were observed for two tumours where tamoxifen stablized disease and may be consistent with the cytostatic effects of this drug.

The nitric oxide donor, SIN-1 and nitric oxide synthase inhibitor, nitro-L-arginine were examined for their effects on energy metabolism using 31P magnetic resonance spectroscopy, and on radiation sensitivity in the transplantable murine tumour, SCCVII/Ha. SIN-1 at 2 mg/kg i.v. reduced Pi/total by 40-50% within 10 min and increased X-ray sensitivity 3 fold, consistent with increased tumour oxygenation. Nitro-L-arginine at 10 mg/kg i.v. increased Pi/total by 250% at 45 min and was maintained for at least 2 hr. Nitro-L-arginine also increased radiation resistance 3-5 fold, consistent with the induction of tumour hypoxia. The results indicate that tumour energy metabolism may be altered through drug induced modification of nitric oxide availability, and that these changes are sufficient to modify tumour sensitivity to X-rays.

15 human tumour cell lines (lung, breast and colon) have been evaluated for their sensitivity to the quinone based anti-cancer drugs Mitomycin C, Porfiromycin, and E09 (3-hydroxymethyl-5-aziridinyl-l-methyl-2-(IH-indole-4,7-dione)prop-β-en-α-ol). Sensitivity has been compared with the intra-cellular levels of DT-diaphorase, an enzyme thought to be important in the reductive activation of these quinones. No correlation exists between levels of DT-diaphorase and sensitivity to Mitomycin C or Porfiromycin. However, for E09 those cell lines showing highest levels of DT-diaphorase activity tend to be the most sensitive.

In a directed search for the best compounds for clinical evaluation, some 150 selected nitroaromatic compounds, representing 6 distinct types, namely, furans, thiophenes, imidazoles, pyrazoles, pyrroles, and triazoles, have been synthesized and tested as hypoxic cell radiosensitisers and bioreductive drugs. These compounds have a wide range of one-electron redox potentials, ranging from −700 mV for 3-nitropyrroles to −250 mV for 5-nitrofurans. Within each series, those agents bearing alkylating moieties on the side chain are generally the more effective radiosensitisers in vitro . Studies in vivo demonstrated that the bifunctional nitroimidazoles were superior to the other nitroarenes tested. In terms of bioreductive cell killing, the best differential between oxic and hypoxic cell toxicity was shown for the bifunctional 2-nitroimidazoles, which had values greater than 20. In contrast, the other classes of nitroarines generally showed differential toxicities of less than 10.

Various bioreductive drugs that are potent hypoxic cell cytotoxins can also function as effective potentiators of radiation action when administered in vivo post irradiation. There is evidence that a contributory mechanism to this potentiation is enhanced sensitivity to the bioreductive drugs exhibited by cells that are damaged sublethally by radiation.

The effect of hydralazine on tumours appears to vary depending on tumour type. Blood flow and radiation sensitivity decrease more in murine tumours than human tumour xenografts. In this study a comparison between various tumour types has been made using in vivo 31P nuclear magnetic resonance spectroscopy (NMRS) to follow the metabolic responses occurring after clamping or intravenous administration of hydralazine (5 mg kg-1). Large increases in the Pi/total phosphate ratio were found with the murine sarcomas, KHT and RIF-1 implanted into C3H/He mice. However little or no effect was seen for the two human xenografted tumours, HX118 and HT29 implanted in MFI nu/nu/01a mice. An intermediate response was observed for KHT tumours grown in nu/nu mice. All tumours showed a large response to clamping. The anaesthetic Hypnorm/Hypnovel has a great influence on the response of the tumour metabolism to hydralazine appearing to both prolong and increase the changes induced. There is evidence to support the theory that the changes in 31P spectra are related to the oxygen status of the tumours.

A series of 2- and 3-nitrothiophene-5-carboxamides bearing N-(omega-aminoalkyl) side chains has been prepared by treatment of the thiophenecarbonyl chloride with the appropriate (protected) omega-aminoalkylamine. Analogous N-(oxiranylmethyl)nitrothiophene-5-carboxamides have been synthesized by epoxidation of the corresponding N-allylamide. Compounds in both classes were evaluated in vitro both as radiosensitizers of hypoxic mammalian cells and as selective bioreductively activated cytotoxins. The most potent radiosensitizers were those agents with strong tertiary amine bases or oxiranes in the side chain. Studies in vivo showed that 2-methyl-N-[2-(dimethyl-amino)ethyl]-3-nitrothiophene-5- carboxamide caused slight radiosensitization of the KHT sarcoma in mice given 0.34 mmol kg-1. However, administration of this and related tertiary amines at higher doses was precluded by systemic toxicity.

Hypoxic cells are radiation-resistant relative to oxic cells and it is now well established, both in experimental murine tumour systems, and in some clinical situations, that this resistance can adversely influence local tumour control by radiation. There is also evidence to suggest that oxygen-deficient tumour cells can be refractory to some anti-cancer agents1 – 3. Hypoxic cells are likely to be quiescent thereby limiting their sensitivity to cycle-selective agents and, because of their location, poorly accessible to cytotoxic drugs. Hypoxia will also affect the activity of a drug if oxygen- dependent processes are required for its cytotoxic effect. Further, it has been shown that hypoxia can induce gene amplification resulting in drug resistance4.