The new generation of hospital-based neutron therapy facilities involve cyclotrons using protons on beryllium. The spectrum of neutrons produced includes a large and variable proportion of low-energy neutrons that are poorly penetrating but biologically effective. Cells cultured in vitro were used to compare the three US facilities at Seattle, M.D. Anderson and UCLA, together with the UK facility at Clatterbridge. Cyclotrons were compared within a given experiment on the same day using cells from a common suspension. Among the three US facilities, the relative potency factor at a depth of 25 mm differs by about 11%, with Seattle the least and UCLA the most biologically effective. Clatterbridge was compared directly with M.D. Anderson and found to be less effective by about 5%; it has a slightly lower biological effectiveness than any of the US facilities. There is evidence for an increased biological effectiveness in the build-up region, which reduces the effective skin sparing potential. There is not much difference in build-up between the three US facilities. Using the proton-on-beryllium neutron production process results in a wide spectrum of neutrons with a large but variable low-energy component. The biological effectiveness of the beam depends on target design and thickness as well as the design of the collimating system. Consequently the biological effectiveness of neutron beams generated by this process must be assessed on an individual basis. It cannot be assumed that because cyclotrons have similar accelerating energies that the relative biological effectiveness will be the same.

Chinese hamster V79 cells have been used to assess changes in RBE of the p(62)Be neutron beam at the Clatterbridge Hospital with depth in a phantom and with use of a hydrogenous filter. The cells were exposed at depths of 2 and 12 cm and at a depth of 2 cm with a hydrogenous filter. Two groups of experimenters each conducted two experiments. The ratios of relative biological effectiveness (RBE) at a depth of 12 cm to that at 2 cm were found by the two groups to be 0.99 +/- 0.04 and 0.96 +/- 0.02 (standard errors). The effect of a polythene filter 4.5 cm thick was measured at a depth of 2 cm and the ratio of RBE with and without the filter was found by both groups to be 0.99 +/- 0.02. All the experiments suggest that there may be small effects of beam hardening by depth and filtration but these results are in marked contrast with those obtained using an in vivo system.

The effect of pretreating the C3H/He mouse MBT-2 tumor with diethyl maleate (DEM), buthionine-S R-sulfoximine (BSO), or misonidazole (MISO) before administration of cyclophosphamide (CTX) was studied with the use of tumor volume-doubling time delay as an endpoint. The kinetics of glutathione (GSH) depletion and regeneration in the tumor and in the host liver were determined after treatment with the thiol-depleting agents. CTX was administered at appropriate time points. MISO was the most effective chemosensitizer at a time point at which tumor GSH content was 80-85% of the control value. Both BSO and DEM were chemosensitizers in relation to the degree they had reduced tumor GSH levels. This chemosensitization was significant at 50% GSH reduction. By combining MISO and BSO at doses lower than previously used for each agent alone, highly effective sensitization of subsequent CTX was obtained.

We have measured the rate of GSH resynthesis in plateau phase cultures of A549 human lung carcinoma cells subjected to a fresh medium change. Buthionine sulfoximine (BSO) blocks this resynthesis. Diethyl maleate (DEM) causes a decrease in accumulation of GSH. If DEM is added concurrently with BSO there is a rapid decline in GSH that is maximal in the presence of 0.5 mM DEM. GSH depletion rapidly occurs when BSO is added to log phase cultures which initially are higher in GSH content. Twenty-four hr treatment of A549 cells with BSO results in cells that are more radiosensitive in air and show a slight hypoxic radiation response. A 2 hr treatment with either 0.25 mM or 0.5 mM DEM results in some hypoxic sensitization and little increase in the aerobic radiation response. The 24 hr BSO + 2 hr DEM treatment sensitizes hypoxic cells to a greater degree than either agent alone but does not increase the aerobic response more than is seen with BSO alone. Cells treated simultaneously with BSO + DEM show little increase in the hypoxic radiation response, compared to DEM alone, but are more sensitive under aerobic conditions. Decreased cell survival for aerobically irradiated log phase A549 cells occurs within minutes after addition of a mixture of BSO + DEM. The decreased cell survival following aerobic irradiation, after prolonged treatment with BSO or acute exposure to BSO + DEM, may be in part due to inhibition of glutathione peroxidases. For example, glutathione-S-transferase, known to have glutathione peroxidase activity (non-selenium), is nearly completely inhibited by the BSO treatments. In addition, cellular capacity to react with peroxide (glutathione peroxidase, selenium containing) was also inhibited. We suggest that the enhanced aerobic radiation response is related to an inability of GSH depleted cells to inactivate either peroxy radicals or hydroperoxides that may be produced during irradiation of BSO treated cells. Furthermore, enhancement of the aerobic radiation response may be useful In vivo if normal tissue responses are not also increased.

Optimizing the interval between administration of misonidazole and irradiation: an in vitro study

Chinese hamster cells in culture were used to compare the radiosensitizing efficiency and cytotoxicity of misonidazole with several 2, 4 and 5 substituted nitroimidazoles. The two substituted compounds (SR 2508 and SR 2555) are similar to misonidazole in radiosensitizing effectiveness, but are significantly less toxic to hypoxic cells. This reduced cytotoxicity may result from either slower drug penetration or slower removal of non-protein sulfhydryl compounds (NPSH) The compound MJLr1-191-VII∗ (a 4-nitroimidazole) is a much more effective radiosensitizer than would be predicted from its electron affinity. It appears to sensitize by two mechanisms, the first resulting from its electron affinity and the second a consequence of its rapid removal of endogeneous cellular NPSH; which are naturally occurring radioprotective substances.

Electron affinic compounds, such as misonidazole, preferentially sensitize hypoxic cells to killing by X rays, and are also preferentially cytotoxic to cells deficient in oxygen. Prolonged exposure of cells to misonidazole prior to irradiation results in an increased radiosensitization. This is expressed as the Extra Enhancement Ratio (EER), defined as the ratio of the doses delivered immediately after the addition of the sensitizer or after prolonged incubation, that produce a given biological effect. Chinese hamster V79 cells have been used to investigate this prolonged incubation effect for a variety of 2-nitroimidazoles including misonidazole, desmethylmisonidazole and SR-2508 and as well as two ortho-substituted-4-nitroimidazoles with a bromine or sulfonamide group substituted in the 5-position. A considerable variation was observed in the magnitude of the Extra Enhancement Ratio (EER) produced by pre-incubation with different compounds at concentrations that produce the same sensitizing effect. There is a good correlation between the EER and the measured rate at which the various sensitizers deplete cells of non-protein sulfhydryl compounds. There is also a good correlation between the EER and the fraction of cells killed by the pre-incubation period in the drug.

Chinese hamster (V79) cells were used to investigate the radiosensitizing efficiency and chemical reactivity with non-protein sulfhydryl (NPSH) compounds such as glutathione, for a series of a-substituted 4 and 5 nitroimidazoles. When added to cells 5 minutes prior to irradiation, MJL-1–191-VII (1-methyl-5-sulfonamide-4-nitroimidazole) sensitizes hypoxic cells commensurate with its electron affinity, while not affecting the radiosensitivity of aerated cells. If the drug is incubated with the cells for a period of time at 37°C prior to irradiation, both aerated and hypoxic cells show an increase in radiosensitivity. Under these conditions the radiosensitizing effectiveness towards hypoxic cells appears to be particularly anomalous, inasmuch as enhancement ratios similar to misonidazole can be obtained at concentrations 50 to 100 times lower. The isomer SK-21981 (1-methyl-4-sulfonamide-5-nitroimidazole) does not behave in this anomalous way, but sensitizes to an extent predictable from its electron affinity. These compounds differ in the rate at which they react spontaneously and intracellularly with NPSH compounds such as glutathione. It is suggested that MJL sensitizes by two mechanisms; the first a function of its electron affinity and the second a function of its rapid reaction with endogenous radioprotective and chemotherapeutic compounds in the cell.

Our results have enabled us to make a number of observations concerning the reactivity of nitrocompounds and non-nitrocompounds with glutathione (GSH) and cellular nonprotein thiols (NPSH), which may be as much as 90 % GSH. We have summarized our observations as follows with respect to the different types of reactions responsible for part or all of the cellular NPSH depletion by hypoxic cell radiosensitizing drugs. (A) Some nitrocompounds, such as 4-nitroimidazoles containing a 5-sulfonamide group, react spontaneously with GSH; (B) A number of hypoxic cell radiosensitizing drugs such as chlorodinitrobenzene (CDNB), dimethylfumarate (DMF) and diethylmaleate (DEM) are substrates for the enzyme glutathione-S-transferase and form covalent bonds with GSH; (C) NPSH may be oxidized by diamide; (D) form covalent bonds with N-ethylmaleimide; (E) or be converted by thiol-reactive drug intermediates formed under anaerobic conditions. The latter reaction occurs with misonidazole, Ro-05–9963, SR 2508 and SR 255:5; its mechanism is still unknown. It is obvious from the above that there are a variety of means by which radiosensitiizing drugs can alter cellular metabolism as reflected by changes in the NPSH. It remains to be determined whether a relationship exists between altered NPSH, metabolism and the radiosensitizing capacity of nitrocompounds when used alone or in combination with other drugs. Our studies strongly suggest that potential new sensitizers be routinely examined for their capacity to react spontaneously with GSH or to remove cellular NPSH under aerobic as well as anaerobic conditions. This is especially true for radiosensitizing drugs showing anomalous behavior, i.e., better sensitization than predicted by their one-electron reduction potentials. Such screening would pay dividends insofar as drugs that are too reactive could be excluded from further in vivo study.

Mammalian cells cultured in vitro were used to study the radiobiological characteristics of neutron beams generated by 43 MeV protons on beryllium or 25 MeV deutrons on beryllium. For an unfiltered beam of neutrons generated by 43 MeV p+ leads to Be the relative biological effectiveness was found to be 8-12% higher at a depth of 2 cm than at a depth of 12 cm due to the presence of a large component of low-energy neutrons. The addition of a hydrogenous filter 4 cm thick preferentially removed the low-energy neutrons from the beam and, as a result, the neutron RBE was independent of depth. There was no significant difference in the oxygen enhancement ratio between the filtered neutrons produced by 43 Mev p+ leads to Be and neutrons produced by 25 MeV d+ leads to Be; for both beams the OER value was about 1.6.

The effectiveness of misonidazole as a hypoxic radiosensitizer of mammalian cells is increased by prolonged exposure of hypoxic cells to the drug. Increased radiosensitization occurs even after the cells are washed free of excess Hall and Biaglow, 1977; Whitmore et al., 1978; Varnes et al., 1981). Hall et al. (1977) suggested and Biaglow et al. 1978) found that drug intermediates, produced during hypoxic incubation of cells with misonidazole, might react with endogenous non-protein thiols (NPSH). These thiols function to protect the cell against deleterious intermediates that could otherwise attack and modify critical macromolecules such as DNA, RNA and protein. This hypothesis is in agreement with the cytotoxic mechanisms proposed earlier for many different nitro compounds (Biaglow, 1981). With respect to the effects of preincubation with misonidazole on the radiation response, alterations in both NPSH and protein thiols (Varnes et al., 1981) would be expected to alter the shoulder of the radiation response curve if hydrogen-donating species such as NPSH are involved in chemical repair of radiation damage (Chapman et al., 1973) or if thiol-containing enzymes are necessary for biochemical repair of damaged macromolecules (Han et al., 1976).

The search for electron affinic compounds that exhibit the properties needed for a clinically useful hypoxic cell radiosensitizer has led to the setting up of clinical trials with misonidazole, which is a 2-nitroimidazole. Recently, MTDQ a new drug of novel design, a dihydroquinoline (6,6-methylene-bis-2,2,4-trimethyl-1,2-dihydroquinoline)has been synthesized and suggested as a radiosensitizer. The radiosensitizing and cytotoxic properties of misonidazole are compared with MTDQ and a second 2-nitroimadazole Ro-07-0741, which has warranted attention as a possible alternative to misonidazole for clinical use.

A combination of misonidazole and MTDQ (6,6'-methylene-bis-2,2,4 trimethyl-1,2-dihydroquinoline) has been tested for its radiation-sensitizing properties and cytotoxicity, using Chinese hamster V79 cells cultured in vitro. Both compounds sensitize hypoxic cells to the effects of X-rays, and when used in combination their sensitizing properties are additive. By contrast, the presence of MTDQ completely inhibits the cytotoxicity that misonidazole exhibits towards hypoxic cells. These experiments shed some light on the mechanism of action of electron-affinic hypoxic cell sensitizers, and the combination of radiosensitizers suggested may have an application in human cancer radiotherapy by eliminating the neurotoxicity experienced by patients receiving misonidazole during radiotherapy.

Experiments were performed at the Los Alamos Meson Physics Facility (LAMPF) to determine the oxygen enhancement ratio (OER) for the clinically used beam of negative pi mesons. V79 Chinese hamster cells, cultured in vitro, were used as the biological test system; hypoxia was produced by metabolic depletion as a result of sealing 2 million cells in 1 ml glass ampules. The Bragg peak of the pion depth dose curve was spread out to cover 10 cm by using a dynamic range shifter. Cells were irradiated at the center of the spread out Bragg peak, where the dose/rate was 0.1 Gy/min over a 6 × 6 cm field. The OER obtained was 2.2, compared with 3.8 obtained for γ-rays under the same conditions.