Potentiation in vivo of melphalan activity by nitroimidazole compounds.

Many nitroimidazole compounds have been shown to potentiate the activity of melphalan against the murine anaplastic MT tumor. The degree of potentiation achieved by these compounds probably depends on their octanol-water partition coefficient and electron affinity: for the greater the partition coefficient of electron affinity, the greater the potentiation. The mechanism of this potentiation remains uncertain. However, it is not due to either nitroimidazole-induced hypothermia, or to the elimination of the recovery from melphalan-induced potentially lethal damage (PLD).

RSU 1069, a 2-nitroimidazole containing an alkylating group: high efficiency as a radio- and chemosensitizer in vitro and in vivo.

Electron affinity as measured by the one-electron reduction potentian, E7(1), is the major factor influencing radiosensitizing efficiency in vitro. RSU 1069 has an electron affinity (E7(1) = 398 mV) similar to misonidazole; however, the ability of this compound to sensitize hypoxic cells is considerably greater in vitro than that of misonidazole, e.g., 0.2 mM RSU 1069 gives an enhancement ratio of greater than 2.0 compared to 1.4 for the same concentration of misonidazole. Radiosensitization studies with the MT tumor in vivo also showed RSU 1069 to be a more efficient sensitizer than misonidazole. An administered dose of 0.08 mg/g RSU 1069 yielded an enhancement of 1.8 to 1.9 using tumor cell survival and tumor cure as end-points. At least a 10-fold higher dose of misonidazole is required for a similar degree of sensitization. Low doses of RSU 1069 also radiosensitize the Lewis lung and B16 experimental tumors. The ability of RSU 1069 to potentiate the cytotoxic action of melphalan and other cytotoxic drugs towards the MT tumor was also examined. RSU 1069 (0.08 mg/g) given to mice 1 hour before melphalan gave an enhancement of 2.8.

Potentiation of cyclophosphamide cytotoxicity in vivo: a study with misonidazole and fifteen other 1-substituted 2-nitroimidazoles.

It has recently been reported that compounds more lipophilic than misonidazole are better potentiators of CCNU tumor cytotoxicity in vivo. There is now a need to extend these studies to include other tumors and cytotoxic drugs. In the present study we have shown that misonidazole (MISO) can potentiate cyclophosphamide cytotoxicity in the Lewis lung carcinoma but not in the B16 melanoma. Further studies in the Lewis lung carcinoma, using 15 1-substituted 2-nitroimidazoles possessing a range of octanol:water partition coefficient (P) (from 0.18- greater than 100) have shown that potentiation increases with increasing lipophilicity. The most efficient compounds at administered dose levels of 1.0 and 2.0 mumol/g were Ro-07-1902, benznidazole (Ro-07-1051) and RSU 1050 which possess octanol:water partition coefficients in the range of 2.5-10. However, on the basis of an equitoxic administered dose (1/2LD50/2d), little difference in potentiation is seen over the range of P studied.

Evaluation of novel radiation sensitizers in vitro and in vivo.

In principle, radiation sensitizers with therapeutic ratios greater than that of misonidazole can be obtained either by increasing sensitizing efficiency, decreasing toxicity or preferably both. This paper illustrates, firstly that a 5-nitroimidazole (S73-0662) with an electron affinity close to that of metronidazole shows sensitizing efficiency similar to misonidazole both in vitro and in vivo. The suggestion is made that this compound should receive a detailed toxicological study to ascertain if its toxicity is lower than misonidazole. Secondly, Imuran, a 5-substituted 4-nitroimidazole and one of a series of compounds which show sensitizing efficiencies in vitro much greater than would be predicted from electron affinity considerations, also shows good sensitization in vivo. Compounds in this series are generally metabolically unstable and the positive results with Imuran in vivo provide a direction for future synthesis of novel sensitizers.

Neurotoxicity of radiation sensitizers in the mouse.

The neurotoxicity of a homologous series of 1-substituted, 2-nitroimidazole compounds, synthesized in this laboratory, has been studied in mice. This involves measurement of the enzyme beta-glucuronidase in the distal sciatic, tibial and common peroneal nerves. The amount of compound required to give a known neurotoxic response, in terms of elevated beta-glucuronidase (arbitrarily set at 60% increase) has been determined. A correlation between increased number of methylene groups (N) in the side chain and neurotoxicity has been shown. A correlation between increased neurotoxicity and effective octanol; water coefficient, at pH 7.4, was also established. A more soluble version of the n = 4 member of the homologous series, that is the compound RSU 1047 (NSC 328897), and misonidazole also fit this correlation of partition coefficient and dose.

Intestinal microflora as potential modifiers of sensitizer activity in vivo.

Treatment of mice (some bearing Lewis Lung tumors), with penicillin (PEN) at 500 mg/l drinking water for one week prior to treatment with misonidazole (MIS), resulted in: the elimination of their anaerobic cecal flora; a decrease in MIS-induced neurotoxicity; an increase in pharmacological exposure to MIS; a decrease in MIS chemopotentiation; a probable increase in MIS radiosensitization; an increase in MIS induced hypothermia. Assuming no chemical interaction between PEN and MIS, these observations indicate that the intestinal microflora can influence the activity of MIS in vivo. Therefore their influence should be considered in all sensitizer-related studies in vivo. The observed reduction in the neurotoxic but not the radiosensitizing potential of MIS following PEN treatment indicates a therapeutic benefit.

Analogues of RSU-1069: radiosensitization and toxicity in vitro and in vivo.

A series of nitroimidazoles containing aziridine and alkyl-substituted aziridine functions has been synthesized. The 2-nitroimidazole compounds examined all show greater radiosensitizing efficiency in vitro than misonidazole. The 4- and 5-nitroimidazole analogues are also more efficient than equivalent compounds which do not contain the alkylating aziridine moiety. All the compounds show increased toxicity towards hypoxic cells relative to aerobic cells, but this toxicity is reduced by alkyl-substitution of the aziridine ring. In vivo toxicity can also be reduced by modification of the aziridine function, but such alterations appear to have less effect upon the high radiosensitizing efficiency of these compounds in vivo. As a consequence of this study, compounds of potentially improved therapeutic utility compared to RSU-1069, the first reported member of this class, have been identified.

Induction of hypoxia in normal and malignant tissues by changing the oxygen affinity of hemoglobin--implications for therapy.

The drug BW12C, which increases the oxygen affinity of hemoglobin, reduces oxygen availability to tissues. This results in protection against radiation damage to the hemopoietic system and epidermal Langerhans cells in CBA mice. The drug also protects against beta-irradiation damage in pig epidermis. BW12C increases the hypoxic cell fraction in tumors and histological examination of an experimental T cell lymphoma shows that the induced hypoxia leads to tumor necrosis.

In vitro microdialysis: a novel technique for stimulated neurotransmitter release measurements.

A novel microdialysis technique suitable for parallel measurements of neurotransmitter release and single unit recordings from brain slices is presented. The effects of 3,4-methylenedioxymethamphetamine (MDMA) on slices of dorsal raphe nucleus and frontal cortex in a perfusion chamber for electrophysiological measures were studied. MDMA caused measurable release of serotonin which, in the case of the dorsal raphe, was of similar duration as the period of reduced cell firing induced by MDMA. Tryptophan potentiated the action of MDMA. Possible additional applications of this technique are discussed.

Serotonin (5-HT) induces IPSPs in pyramidal layer cells of rat piriform cortex: evidence for the involvement of a 5-HT2-activated interneuron.

In a slice preparation of rat piriform cortex, both intracellular and extracellular techniques were used to examine the pharmacological and electrophysiological actions of serotonin (5-HT). Bath application of 5-HT resulted in either depolarization (57%), hyperpolarization (34%) or no change (9%) in membrane potential of cells in the pyramidal cell layer (layer II) of piriform cortex. Additionally, when KCl-containing electrodes were used, 5-HT induced an increase in depolarizing synaptic potentials in 41% of these cells. It was concluded that these potentials were reverse inhibitory post-synaptic potentials (IPSPs) because they were blocked by bicuculline and tetrodotoxin. The induction of IPSPs by 5-HT was blocked by the 5-HT2-selective antagonist ritanserin. By recording extracellularly in the presence of 5-HT, a group of 5-HT-activated, putative interneurons was found at the border of layers II and III of piriform cortex, 5-HT but not norepinephrine activation was blocked by ritanserin. The actions of 5-HT were mimicked by the 5-HT2 agonist alpha-methyl-5-HT; the 5-HT2 partial agonist, 2,5-dimethoxy-4-methyl-amphetamine had a small agonist action of its own and blunted the effect of 5-HT. Activation of a larger group of putative interneurons by the more universal excitant N-methyl-D-aspartate showed that the 5-HT-activated interneurons represented 23% of the interneurons located on the border between layers II and III. We conclude that 5-HT induces IPSPs in layer II pyramidal cells by activating a subpopulation of interneurons at the border of layers II and III of piriform cortex.

Acetylcholinesterase and cholinesterase activities in the mouse cerebellum following misonidazole treatment.

The acetylcholinesterase and cholinesterase activities in the mouse cerebellum were unchanged following misonidazole treatment. Inhibition of acetylcholinesterase activity only occurred at concentrations of misonidazole which were in excess of those obtained clinically. These findings indicate that the clinical neurotoxicity of the drug cannot be attributed to an effect on cholinesterases.

The effect of radiation on tumour growth delay, cell survival and cure of the animal using a single tumour system.

The response of a murine tumour to single doses of X rays has been measured using three different assays--animal cure, cell survival in vitro after irradiation in vivo, and tumour growth delay. The dose to cure 50% of the animals, the TCD50, was 79.0 Gy. This was not affected by clamping the tumours to render all the cells hypoxic at the time of irradiation, implying that most of the cells in the tumour were hypoxic already. The enhancement ratio for the hypoxic cell sensitizer Ro-07-0582 was 2.1. The cell survival assay gave an enhancement ratio of 1.6 and an hypoxic fraction of 5%. The discrepancy in the estimates of the hypoxic fraction can be explained by the ability of the naturally hypoxic cells, but not the oxic ones, to recover from potentially lethal damage in vivo. Neither the cell survival assay nor the growth delay assay agreed with the TCD50 assay as to the effect of the hypoxic cell sensitizer, even allowing for recovery from potentially lethal damage. It is doubtful whether the measured survival curve would predict the measured TCD50.

The effect of recovery from potentially lethal damage on the determination of reoxygenation in a murine tumour.

The pattern of reoxygenation in the murine anaplastic MT tumour was investigated using the established method of determining the hypoxic fraction, at intervals after a priming X-ray dose, from test doses given either to unclamped or clamped-off tumours. Little reoxygenation was apparent whilst the tumour was increasing in size for 12--72 hours after a single dose of 20.3 Gy, but extensive reoxygenation was evident whilst the tumour was shrinking at nine days after a dose of 50 Gy. However, the degree of reoxygenation may have been underestimated, especially after the smaller priming dose. This is because only the chronically hypoxic cells in this tumour have the ability to recover from potentially lethal damage (PLD) and so are more radioresistant than cells rendered acutely hypoxic by clamping. Because of this, even when tumours are clamped off during irradiation, the resulting survival curve is biphasic and the apparent effect of the clamp becomes a function of the X-ray dose used. The larger the dose, the smaller the observed effect of the clamp, so the greater the apparent hypoxic fraction and hence the smaller the apparent degree of reoxygenation.

A randomized blind trial of Iopamidol and meglumine calcium metrizoate (Triosil 280, Isopaque Cerebral) in cerebral angiography.

A randomized blind trial of iopamidol and meglumine calcium metrizoate (Triosil 280, Isopaque Cerebral) for cerebral angiography was performed in 20 patients. The Iopamidol was better tolerated and caused significantly less discomfort in patients than metrizoate. There was no difference in the quality of the radiographs. A further 13 patients also received Iopamidol for cerebral angiography with similar results. No adverse effects were observed in any of the patients. Our evidence suggests that Iopamidol has significant advantages over currently available contrast media for cerebral angiography.

The effect of recovery from potentially lethal damage on the determination of repair and repopulation in a murine tumour.

Repair and repopulation following X irradiation of clamped-off murine anaplastic MT tumours was investigated using the established method of (Dn-D1)/(n-1). Repair was complete in 4 h, similar in extent to that reported in other tumours, and within the range of that reported for normal tissues. Subsequent repopulation commenced after 4 days and was equivalent to 1.8 Gy/day recovered dose, corresponding to a clonogenic cell number doubling time of 1.8 days. However, estimates of repair and repopulation may have been in error because the chronically hypoxic cells in this tumour alone have the ability to recover from potentially lethal damage (PLD) and so are more radioresistant than cells rendered acutely hypoxic by clamping. Because of this, even clamping off tumours at irradiation does not render all cell populations equally radioresistant, and so reoxygenation between fractions could result in an underestimate of repair and repopulation. Further, the differing sensitivity between acutely and chronically hypoxic cells renders the apparent OER a function of dose (i.e., oxygen not truly dose-modifying to chronically hypoxic cells). Consequently it is incorrect to assume a constant OER in order to compare repair in tumours irradiated under hypoxic conditions with that in normal tissues irradiated under aerobic conditions. It will be argued here that in the case of the present tumour neither reoxygenation nor the choice of OER will have qualitatively altered the conclusion reached from the conventional method.

A comparative trial of sodium meglumine ioxaglate (Hexabrix) and iopamidol (Niopam) for cerebral angiography.

We have carried out a randomized blind trial comparing iopamidol (Niopam) and sodium meglumine ioxaglate (Hexabrix) in cerebral angiography in 50 patients. There was no significant difference in the degree of pain or movement produced by the contrast media. The degree of heat felt was slightly greater with iopamidol than with ioxaglate. Both were very well tolerated by patients, producing only a mild or moderate sensation of heat, and in only a few instances slight pain during the injections. No difference in radiographic quality was observed. We conclude that both media are a significant advance over the conventional ionic media and the choice between them will be determined by factors of neurotoxicity, ease of use and cost.

Deformity of the labyrinth and internal auditory meatus in congenital deafness.

In congenital deafness it is important that the radiological examination should exclude any deformities of the bony labyrinth or internal auditory meatus as well as demonstrating lesions of the middle and external ears. Radiological assessment of inner ear abnormalities should be used as a guide both to the feasibility of reconstructive surgery to the middle and external ears and for the future training of the child. Inner ear abnormalities demonstrated by tomography in 56 patients are discussed in relation to the cochlea function. The importance of demonstrating the central bony spiral of the cochlea and of assessing the size and shape the internal auditory meatus is stressed.

Influence of in vitro assay conditions on the assessment of radiobiological parameters of the MT tumour.

We have examined the survival parameters of anaplastic "MT" tumour cells following irradiation with 60Co gamma rays in vitro acd in vivo, comparing colony formation in soft agar and monolayer. Following in vitro irradiation under oxic or hypoxic conditions, or in vivo hypoxic irradiation, we found that the D0 values were about 30% greater when measured in soft agar compared with monolayer (soft agar, oxic D0 = 1.6 Gy, hypoxic D0 = 3.7 Gy; monolayer, oxic D0 = 1.2 Gy, hypoxic D0 = 2.8 Gy). However, other parameters were similar for each assay (oxic and hypoxic n approximately 11; hypoxic fraction in vivo approximately 0.07; PLD repair in naturally hypoxic cells increased the D0 by a factor of approximately 1.3). Recently, McNally and Sheldon (1977) measured cell-survival parameters for this tumour using only a monolayer colony assay, and from these estimated TCD50 for the tumour. However, their estimate was lower than the directly measured value of 79 Gy. We have also estimated TCD50 from our cell-survival data after correction for RBE (we used gamma rays while McNally and Sheldon used 250 kV X rays). Like McNally and Sheldon we were unable to predict the measured TCD50 from our monolayer data, even when PLD repair was taken into account. However, the soft agar data with PLD repair could predict the observed TCD50.

Congenital deformities of the middle and external ear.

Congenital deformities of the external and middle ears were shown by tomography in 246 patients. Surgical exploration has been undertaken at some time in many of these ears. The appearance of the tomograms and their significance were reviewed in the light of the operative findings and the authors' experience of congenital ear lesions. In nearly every case a middle ear cavity could be demonstrated although this varied from a normally aerated middle ear and mastoid in association with an isolated unilateral atresia of the external auditory meatus to a small slit-like hypotympanum in patients with craniofacial abnormalities. The ossicles were nearly always present but deformed. The typical appearances of the ossicles and the frequently aberrant pathways of the facial nerve are described.

Relative effectiveness of 12-hourly fractionation and a non-uniform x-ray schedule in the optimum fractionation of C3H mouse mammary tumours.

In previous experiments the highest proportions of tumours controlled for 150 days at a particular level of skin reaction were obtained with five or nine fractions of X rays in nine or ten days respectively. Poor results were obtained for the same numbers of fractions given in 4 or 18 days respectively. The present work reports results of three "non-standard" fractionation schedules: two with equal doses given at 12-hour intervals over four or nine days and one with eight decreasing doses given at decreasing intervals over 11 days. The two 12-hour interval schedules gave results which were equal to the best obtained by any other schedule tested. The 8F/11d non-uniform schedule, however, gave mediocre results in spite of being close to the previously optimum overall time. When these results are compared with previously published results from the same tumour system two, phases of optimum fractionation are demonstrated. At short overall times, up to four days, the situation is finely balanced, so that fraction size and interval matter greatly and results of the schedules vary from good to bad. At longer overall times, of nine days or more in these tumours, the response is no longer so variable. Eighteen days (two to three times the average volume doubling time) is too long for any successful treatment with this system, presumably because of proliferation in the tumour.