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Inhibition of flocculation by sugars was studied in 41 strains of Saccharomyces cerevisiae. Two distinct groupings were found. The first type, including all strains containing FLO 1, FLO 4, FLO 5, FLO 8 and TUP 1, were partially inhibited by mannose only. The second type (NewFLO) were completely inhibited by mannose, maltose, glucose and sucrose. Sugar inhibitions were reversible and were exacerbated by increased agitation. NewFLO strains were more sensitive to inhibition by inorganic salts than the FLO 1 type strains. This inhibition is probably chaotropic in nature. Most FLO 1 type strains are constitutive, expressing flocculation throughout growth, whereas, NewFLO strains are completely repressed by the presence of excess ammonium ions. It is proposed that this indicates two entirely distinct mechanisms of flocculation, probably involving a mannose-specific lectin in the FLO 1 type and a broad specificity lectin in the NewFLO type.

Ro 03-8799, a lipophilic nitroimidazole with a basic side chain, has now been administered intravenously to 69 patients. The elimination half-life in plasma was 5.1 hr and the plasma concentration at 30 min was 14.8 micrograms/ml standardized to a dose of 1 g per square meter of surface area. Immediate symptoms of malaise, heat, sweating and disorientation limit the amount of the drug which may be given on any one occasion. However, a dose of 750 mg per square meter of surface area may be given combined with daily radiotherapy. Our data suggest that when given with a 20 fraction course of radiotherapy, sensitization of hypoxic cells may be achieved equal to a 10-fold increase in the dose of misonidazole above that presently permitted.

The concentration of desmethylmisonidazole (DESMISO) was determined in 60 biopsy samples taken from 13 human tumours and in cerebrospinal fluid (CSF) from 8 patients after oral administration. In comparison with misonidazole (MISO), peak concentrations in plasma were reached at earlier times and half-lives were shorter, so that the area under the curve of plasma concentration with time (AUC) was reduced by 45%; the AUC of CSF concentration with time was reduced by 67%. Between 1 and 2 h after administration of DESMISO, concentrations in tumours were generally 85-90% of those of MISO estimated approximately 4 h after it was given. The two drugs when tested in equimolar concentrations have been found in laboratory experiment to be equally potent as hypoxic cell radiosensitizers. Recognizing the lower mol. wt of DESMISO and the trend to higher concentrations in the more necrotic areas of the tumours studied equal doses by weight of the two drugs given orally may give equal radiosensitization of hypoxic cells in human tumours.

Femulen, a progestogen only oral contraceptive (ethynodiol diacetate 0.5mg), was evaluated for its contraceptive efficacy and safety in 425 women aged between 16 and 47 years. This was a multicentre open study carried out in General Practice and Family Planning clinics. Five pregnancies were reported, three of which were a result of patient failure. The net pregnancy rate at one year for method failure was 0.5%. No ectopic pregnancy was reported. The median length of the menstrual period was between four and five days and the average length of the non-bleeding interval remained between 24 and 25 days throughout the study. Blood pressure on the whole remained within normal limits. However, there was a small decrease in both systolic and diastolic blood pressure which did not reach significant levels. Body weight was unaltered and no abnormality was found in cervical smears. Femulen was shown to be an effective and acceptable contraceptive in women of varying ages.

We have studied the intracellular uptake of a number of neutral, acidic, and basic radiosensitizers. For neutral sensitizers, we observed a correlation between the measured intracellular concentration and sensitization, but for bases, a large change in average intracellular concentration results in only a small change in sensitization. In addition, by modifying the intralysosomal pH, we have altered the measured average intracellular concentration of the weak base pimonidazole by a factor of two, although this had no detectable effect upon sensitization. Using spin filtration of solutions of sensitizers with naked calf thymus DNA or chromatin we have assessed the affinity of DNA for sensitizers with different prototropic and lipophilic properties. We have also shown that this anomalous behavior of the basic sensitizers could be partly explained on the basis of intracellular localization adjacent to the DNA due to ionic interactions. Thus, intracellular localization needs to be considered when interpreting average intracellular uptake data.

The increase in the degree of radiosensitization of Escherichia coli cells following prolonged pre-irradiation incubation with nitroimidazoles is not correlated with the loss of intracellular non-protein thiols (NPSH) alone. The rates of reduction of the nitro compounds and the NPSH removal do not show strong dependencies on the lipophilicities of the nitroimidazoles whereas the highly lipophilic compound RGW-609 effects an increase in radiosensitization in a much shorter incubation time than the other nitroimidazoles. Exogenous dithiothreitol (DTT) increased the rate of reduction of misonidazole in the cells but did not alter the fraction converted to the amine. Added DTT (0.15 mmol dm-3) completely protected against the pre-irradiation incubation effect of misonidazole (2.5 mmol dm-3) when added at the start of the incubation but only partially protected when added before irradiation. It is suggested that NPSH can intercept metabolite(s) (or their precursors) of nitroimidazoles which can potentiate cell killing by radiation.

Cellular reduction of nitroaryl compounds is efficiently inhibited by oxygen, and detection of products characteristic of reduction could form the basis for diagnostic tests for the presence of hypoxic cells in tumors. The criteria for suitable compounds include a high sensitivity and selectivity of detection response between oxic and hypoxic cells, which can be provided using fluorescence detection and suitable nitroaryl compounds which have very low fluorescence until reduced. Examples described include a nitroacridine and nitronaphthalimides. Although the intercalating ability of these ring systems lead to high sensitivity for detection of reduced metabolites in vitro by flow cytometry, poor bioavailability is an unwanted consequence of intercalation. The application of several model reducing systems for reduction of potential fluorescent probes for hypoxia is described, and the absorption and fluorescence spectral characteristics of other examples of structures which could form the basis for useful probes are outlined.

The effect of chronic anemia on tumor radiosensitivity in a murine tumor has been investigated. Anemia was induced by bilateral kidney irradiation given several months before tumor implantation. Anemic, anemic transfused, and normal non-anemic age-matched tumor bearing animals were irradiated with X rays (2 F/24 hr) either in air, air plus misonidazole, or under hyperbaric oxygen. The most resistant response was that of tumors grown in normal mice treated in air. Anemia produced an increase in radiosensitivity which was further enhanced by red blood cell replacement. The most sensitive overall response was seen in the anemic-transfused group treated with HBO.

Flocculent yeast cells have an absolute requirement for mechanical energy input in order for flocculation to occur. Flocculation is arrested by cessation of energy input. The initial rate of flocculation increases as the square of the cell concentration. There is a minimum shaking speed to initiate flocculation and thereafter the initial rate of flocculation increases exponentially with the shaking speed. The minimum shaking speed for flocculation to occur increases with pH value. Activation energy for flocculation, derived from Arrhenius-like plots, varies with pH value. We propose that activation energy is required to overcome mutual repulsion between charged yeast cells and allow flocculent bonds to be formed.

Pharmacokinetic studies were carried out in mice after co-administration of 2-nitroimidazoles with differing prototropic properties: Ro 03-8799, misonidazole, SR 2508, and azomycin. In addition, the distribution of sensitizers in different areas of individual tumors, as well as several normal tissues were measured after intravenous dosing of 0.5 mmol kg-1 (145-56 mg/kg). Although there appeared to be no significant pharmacokinetic interaction between sensitizers after co-administration, there was a significant difference in the distribution of sensitizers within tumors of differing type and size. In small, fast growing SaFa and large, slow growing CaRH tumors, the levels of all sensitizers was relatively constant throughout. However, large fast growing SaFa tumors demonstrated the high degree of variability in the Ro 03-8799 concentration that is seen in the clinic. This may be important in the modelling of human tumors with regard to sensitizer distribution.

Dithiothreitol reduces cystine in tissue-culture medium, and the resulting cysteine is accumulated in Chinese hamster V79 cells to an extent which should be taken into account in evaluating models of radiosensitivity and the effects of thiol radioprotectors.

The "extra" radiosensitization seen with GSH-reactive nitro compounds is too large to be accounted for by GSH-depletion acting independently--there must be competition. The GS-conjugate leaks out of cells slowly and is trapped at high concentrations. Its properties, such as concentration trapped and reduction potential, must be considered. Limited therapeutic exploitation of the glutathione conjugate trapping and concomitant GSH depletion may be possible if intratumor injection is permitted.

Misonidazole (MISO) has been shown to affect the pharmacokinetics of both cyclophosphamide (CY) and melphalan (MEL) in WHT mice resulting in increased plasma levels of the cytotoxic drugs. The effect is not solely due to the reduction in body temperature observed with large single doses of MISO, as a change in MEL pharmacokinetics was still observed when the mice were maintained at 37 degrees C. Inhibition of cytotoxic drug metabolism may also be a possible mechanism. Such a pharmacokinetic effect could account for part of the potentiation of MEL and CY action observed in tumours with large single doses of MISO. However, a chronic low dosing schedule of MISO did not affect the plasma half-life of either cytotoxic drug, although a significant potentiation of each drug in combination with a chronic MISO dose has been obtained in some tumours. These results suggest that potentiation of chemotherapeutic drug action by MISO in the clinical situation is unlikely to be due to changes in drug pharmacokinetics.

Depletion of glutathione by pretreatment with buthionine sulphoximine greatly enhances the radiosensitizing efficiency of misonidazole in mammalian cells in vitro, but a similar effect has not yet been observed in vivo. In thiol-depleted V79 Chinese hamster cells and human HT1080 fibrosarcoma cells, physiological concentrations of ascorbate greatly reduce misonidazole radiosensitization, although there is little effect of ascorbate on misonidazole sensitization in untreated cells. The effect of ascorbate on misonidazole radiosensitization is not markedly dependent on the extracellular concentration of ascorbate; this may be explained by the non-equilibrium uptake of ascorbate at different extracellular concentrations. Failure to obtain a large enhancement of misonidazole radiosensitization as a result of buthionine sulphoximine treatment in vivo may be due, in part at least, to the presence of ascorbate.

A prospective randomized trial is reported involving 97 patients with locally advanced cancer of the head and neck. Using six large fractions of radiation (3600 rad in 17 days) the addition of misonidazole (2.0gm per m2 body surface) with each fraction did not increase the local control rate at one year. It is thought that this is probably because of an inadequate tumor concentration of the drug.

The nitroimidazole Ro 03-8799 has been tested as a sensitizer of hypoxic tumour cells, using regrowth delay of a mouse mammary carcinoma. This drug has a short biological half-life and has previously proved to be less promising in tumour experiments than was predicted from in vitro studies and from artificially hypoxic skin. The postulate that this might result from the delay in penetrating to poorly vascularized tumour regions has been tested by maintaining constant blood and tumour levels for 2 hours before irradiation, using an infusion pump. For equivalent gross tumour concentrations at the time of irradiation there was no significant difference in the radiosensitization achieved with a single dose or with prolonged administration. This indicates that slow penetration of the drug is not a problem.

The toxic side-effects of misonidazole (MISO) have been studied in two strains of mice over a wide weight range. The sensitivity of the mice, determined as the LD50 within 7 days of administration, varied by almost a factor of two. Differences were seen in the same strain, with heavy mice being most sensitive, and from strain to strain, with CBA mice being more susceptible than WHT albino mice. The toxicity of the MISO was closely related to a change in the peak blood levels, but showed no correlation with biological half-life. The tissue volume available for drug distribution appears to be reduced in large, heavy mice at high drug doses, and cannot be explained simply on the basis of drug exclusion from adipose tissue. For radiobiological studies with MISO doses of more than 0.5 mg/g it is recommended that it should be administered in a dosage that will give a known blood level of the drug, rather than simply by giving a constant dose based on body weight.

The effect of melphalan alone or combined with various schedules of misonidazole (MISO) has been tested on a murine fibrosarcoma. The tumoricidal effect has been determined using the growth delay assay. Large single doses (500-1000 mgkg-1) of MISO enhanced the anti-tumour effect of melphalan, especially at high melphalan doses. This was accompanied by a drop in body and tumour temperature and an increase in the melphalan half-life. The MISO-induced hypothermia was prevented in one experiment by keeping the mice in an ambient temperature of 35 degrees C for 3 h. This reduced the exposure to melphalan but did not diminish the cytotoxic effect of the drug combination. Chronic administration of MISO for an 8 h period gave no enhancement of melphalan damage, whether melphalan was given half-way through or at the end of the period of dosing. It seems that a threshold tumour concentration of MISO, in excess of 70 micrograms g-1, is needed for enhancement of melphalan cytotoxicity; prolonged exposures to very low doses are ineffective.

The radiosensitizing efficiencies of 4 structural analogues of misonidazole (MISO) have been compared with that of the parent compound. Three of these were charged basic compounds, previously shown in vitro to be 10 times more efficient. Enhancement ratios were measured from pairs of tumour growth-delay curves for the mouse fibrosarcoma SA Fab. Two routes of administration and ranges of drug dose and intervals between injection and irradiation were tested. Drug concentrations in blood, brain and tumor were measured using high-performance liquid chromatography. The peak concentration in tumours coincided with the peak in radiosensitization: 20 min after i.v. injection and 40 min after i.p. injection. The concentration in tumours was similar for either route. Comparison of radiosensitizing efficiency on the basic of equal administered dose showed no difference between the 5 compounds, but after equimolar doses the charged compounds achieved lower tumour concentrations. Comparison of sensitizing efficiency on the basis of tumour concentration showed that they were 3 times more potent than MISO, as predicted from their higher electron-affinity. The resultant improvement in radiosensitization at low, clinically relevant, concentrations is so slight that any therapeutic benefit would depend on reduced drug toxicity in man.

The toxicity to cultured cells of the cancer chemotherapeutic agent methylglyoxal-bis[guanylhydrazone] (MGBG) varies considerably between different cell lines and is always more toxic in the absence of exogenous polyamine. We have looked at the relative MGBG toxicity of two different murine melanoma tumour cell lines (Harding-Passey and Cloudman) and normal murine fibroblasts, and found wide variation with no correlation in sensitivity to MGBG between tumour and normal cells. High sensitivity to MGBG may be associated with high transport across the cell membrane.

It has been shown that the propanolamine 2-nitroimidazole Ro 03-8799, is about an order of magnitude more efficient than misonidazole as a radiosensitizer in vitro, which is more than its electron affinity would predict. However, in vivo studies have so far shown only a minimal improvement compared to misonidazole. One reason for this could be that diffusion to the site of action in the hypoxic cell is restricted by the short half-life in the mouse and the net positive charge carried by the drug at physiological pH. Ways of maintaining a constant blood or tumor concentration over a period of 1--2 hours were investigated so that the tumor response could be assessed after this time. Initial experiments used multiple intraperitoneal or intravenous injections, for which the amount of drug required can be calculated theoretically provided sufficient pharmacokinetic data are available. Later experiments using continuous infusion of the drug were aimed at maintaining the gross tumor concentration equal to that found at the time of peak sensitization following a single administration. Results will be presented using each of these approaches. The technical feasibility and advantages of each method, together with general problems associated with interpreting any radiobiological results obtained using this approach will be discussed.

The effect of varying octanol: water partition coefficients, P, (range 0.026-260) on the uptake of uncharged 2-nitroimidazoles into Chinese hamster V79 379A cells has been studied. Average intracellular concentrations were measured by high performance liquid chromatography after centrifuging cells through oil or an aqueous medium. The ratio of intracellular concentration of radiosensitizer to extracellular concentration (Ci/Ce) for misonidazole (P = 0.43) was 0.85 for the oil method and 0.68 for the aqueous method. For values of P less than about 0.05 uptake was initially very slow and Ci was always less than Ce. When P greater than or equal to 0.1 uptake was rapid and then remained unchanged for times up to 3 h; for P greater than or equal to 10, Ci/Ce increased rapidly as P increased. Ro 31-1405 (P = 260) concentrated by a factor of 7 inside the cell. Although uptake was identical for cells suspended in full growth medium and PBS, radiosensitization was greater for cells in PBS: 1 mmol dm-3 misonidazole produced an enhancement ratio of 1.6 in full growth medium and 1.9 in PBS. This increase in radiosensitization could not be accounted for by protein binding. However, measurements on cellular non-protein sulphydryl (NPSH) demonstrated the levels to be reduced to about 60 per cent for cells in PBS. Similar reductions in NPSH levels have previously been shown not to increase the radiosensitivity of control cells but to increase greatly the effectiveness of nitroimidazole radiosensitizers.

Glutathione peroxidase (GSH-Px) has been implicated in mediating the radioprotective effects of glutathione (GSH). This hypothesis was tested in vivo by determining the effect of GSH-Px depletion on the radiation response of murine kidneys. Renal GSH-Px levels were depleted to 17% of control values by feeding animals a selenium deficient diet for 6 weeks; this had no significant effect on renal levels of GSH or GSH-S-transferase (GST). However, we also tested the effect of direct depletion of GSH to 3-4% of control values, using a combination of DL-buthionine sulphoximine (BSO) and diethyl maleate (DEM). Kidneys with normal or depleted levels of GSH-Px and/or GSH were irradiated with 240kVp X rays (2 fractions, 7 days apart to minimize intestinal injury). Mice breathed 7% oxygen during irradiation. Renal damage was assessed at 20, 25, and 32 weeks after the first fraction of X rays, in terms of reduced hematocrit and renal clearance of 51Cr-EDTA. Depletion of GSH-Px levels to 17% of control did not alter renal radiosensitivity, but depletion of GSH to 3-4% of control values radiosensitized the kidney by a factor of 1.4. Depletion of both GSH and GSH-Px together did not radiosensitize the kidney any more than was achieved by GSH depletion alone.

Misonidazole, SR-2508 and Ro 03-8799 have been given in sequence to patients before tumor sampling. Tumor concentrations of the three drugs have been measured and it has been possible to make prediction as to the likely advantage of the newer drugs over misonidazole. Based upon the three cases described here and 13 others given two drug combination, we suggest that in multifraction radiotherapy, both are likely to prove more than five times more efficient than misonidazole.

The nitroimidazole, Ro 03-8799, has proved unique among the drugs tested as chemical hypoxic cell radiosensitizers because of the preferential concentration which has been observed in tumors. Our accumulation of experience has allowed new analyses to be performed upon 127 samples from 39 patients; 47 samples of normal tissue were also obtained from 26 of these patients. Tissue sampling was performed usually between 20 and 30 minutes after initiation of infusion of Ro 03-8799. By expressing results as tumor: plasma ratios, difficulties in comparison because of differing doses and body sizes, together with a variation in the actual time of sampling, have been avoided. A small portion of each specimen which was analyzed for drug concentration was also examined histologically to give an impression of the percentage of the specimen occupied by tumor cells. Analyses have shown that the average tumor concentration is approximately twice that of normal tissues which have been sampled and four times that in plasma. In 38 breast tumor samples, the concentration of drug varied directly as the proportion occupied by tumor cells. The highest tumor: plasma ratios were seen in samples taken from some samples of malignant melanoma. These findings confirm that a greater potency can be expected for this drug as a radiosensitizer because of its ability to enter tumor cells in high concentration. In drug development programs for chemical sensitizing and cytotoxic agents, drugs which show this phenomenon should be explored.