Inf. 1. Radiation
Oncology
Bid.
Phys.,
1977, Vol. 2, pp. 521-530.
Pergamon
Press.
Printed
in the U.S.A.
0 Rapid Communication
Ro-07-0582
AS A RADIOSENSITIZER ERIC
Radiological
Research
J.
AND CYTOTOXIC
AGENT?
M.A., D.Phil.
HALL,
Laboratory, College of Physicians University, New York, U.S.A.
and Surgeons
of Columbia
and JOHN Division
of Radiation
M.S., Ph.D.
BIAGLOW,
Biology, Department of Radiology, Case Western versity, Cleveland, Ohio, U.S.A.
Reserve
Uni-
The electron a5nic drugs, including the nltroimidazoles and the nltrofurans, were initially identlfled on the basis of their abllity to mhnic oxygen and dlfferentially sensitize hypoxic cells. More recently, these drugs have been found to exbibit selective toxicity toward hypoxic cells. The radiosensitizing effect of the 2-nitrohnidazole Ro-07-0582 is greatly enhanced if cells are pre-incubated in the presence of the drug for a period of hours prior to irradiation. In addition, the cytutoxicity of the drug is greatly increased at elevated temperatures. Both of these effects may be related to the metabolism of the drug, and it is clear that there is considerable room for maneuver ln the time sequence of administering the drug and delivering tbe radiation dose as these potent and useful sensitizers begin to be used in the human. Radiosensitlzer, Hypuxic cells, Electron affinic drugs.
INTRODUCTION The idea that foei of hypoxic cells in some tumors may limit their radiocurability by Xrays was a consequente of the pioneering work of Gray, Read, Thomlinson and their the colleagues.‘4.3”36 In the case of X-rays, fraction of cells killed by a given dose depends strongly on the presence of molecular oxygen; since some tumor cells may be hypoxic thcy would be spared from the effects of the radiation compared with wel1
oxygenated
normal tissues.
IThis investigation was supported by Contract EY-76-C-02-3243 from the Energy Research and Development Administration and by Grant NOS. CA-12536, CA-18506 and CA-13747 by the National Cancer Institute, DHEW. Acknowledgements-It is with pleasure that we acknowledge that Sir Oliver Scott, of the Royal
Because many of the early radiobiologists had their initial training in physics, they naturally and logically sought a physical solution for this problem, and came up with the suggestion that X-rays should be replaced by a more densely ionizing radiation, such as neutrons, for which the cell killing effect is less dependent on the presence of oxygen. Gray was largely responsible for conceiving the cyclotron to generate neutrons at the Hammersmith Hospital; this has led directly to the worldwide effort to test the efficacy of Marsden Hospita1 in London, suggested the experimentj involving the preincubation with the drug which led to a significantly greater sensitization. The compound Ro-07-0582 was generously supplied by Hoffman-La Roche. We thank Dr. Ged Adams of the Gray Laboratory for much useful discussion and continued stimulation. 521
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Radiation Oncology 0 Biology 0 Physics
neutrons and other high LET radiations as a potential alternative for X-rays in radiotherapy. In more recent years radiation chemists have played an increasingly prominent role in radiation research, and have brought with them a new and different outlook. When they were faced with the problem of countering hypoxic cells, instead of seeking new types of radiation, they fel1 back on their training as chemists and sought to overcome the lack of oxygen in tumor cells by using a suitable drug which could act as a substitute for oxygen.’ In this way the electron affinic nitroimidazoles were discovered and/or developed;‘” they are able to diffuse to cells which oxygen cannot reach because they are metabolized more slowly than oxygen in respiring tissue. Attention has focussed on the nitroimidazole known commonly by its code name Ro-070582.=” This drug now has been used extensively in radiobiological experiments, both in vitro and in vivo ; testing also has begun in a few patients. In the fhst instance, these hypoxic cel1 sensitizers were sought and chosen on the basis of their ability to mimic oxygen and differentially sensitize hypoxic cells to the effects of X-rays, without altering the response of the aerated cells. However, in the course of laboratory studies, it has become apparent that nitro derivatives such as the nitroimidazoles “Flagyl” and Ro-07-0582, as wel1 as the nitrofuran derivative nitrofurazone, are cytotoxic toward hypoxic cells. This toxic effect has been demonstrated with cells cultured in vitro17326 with multicell spheroids33,34 and also with transplanted tumors in animals.’ The action of Ro-07-0582 in any in vivo situation is likely to reflect a combination of its radiosensitizing properties coupled with its cytotoxicity. In both regards it acts selectively against hypoxic cells, at least at low concentrations. This means, in general, that the presence of the drug wil1 lead to an increase in the number of cells killed in tumors which contain hypoxic cells. The present paper describes experiments intended to further the understanding of Ro07-0582 as a sensitizer, and also to investigate
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1977, Volume 2, No. 5 and No. 6
the cytotoxic properties of this drug as a function of temperature. In addition, a property of this sensitizer, to alter cellular oxygen utilization in the presence of potassium cyanide (KCN),““” is used as an indicator of increased metabolic activity as a function of temperature. METHODS
AND MATERIALS
V79 Chinese hamster cells were used throughout these investigations, cultured by standard techniques, and grown in Gibco’s FlO medium supplemented with 10% fetal calf serum. Cells received their treatments, with radiation or with hyperthermia, with or without the addition of the nitroimidazoles, while held in suspension in smal1 glass ampules. For each experiment, cells were harvested by trypsinization from twenty actively growing, partly confluent stock bottles, spun down and washed to remove excess trypsin, and the concentration of cells counted with a Coulter electronic cell counter. This cell suspension then was divided into several parts and the cel1 concentration in each adjusted to 2 x 106cellslml, adding Ro-07-0582 at an appropriate concentration according to the plan of the particular experiment. From each of these suspensions, containing various concentrations of drug or no drug at all, series of glass ampules were filled by pipetting 1 ml of the suspension into each. After dilution by a factor of 200, another series of glass ampules were filled with 10” cells each. The ampules containing cells at the higher concentration then were handled in the following way in order to make the cells hypoxic. The ampules were flushed with high purity nitrogen, plus 5% CO1, through a long cannula, in order to displace the air above the cel1 suspension. Each ampule was heatsealed before it was transferred to a water bath at 37S”C where it was continuously shaken and rotated end-for-end to keep the cells in suspension. This procedure was maintained for a period of 1 hr to allow the cells to “scavenge” the oxygen dissolved in the medium by normal metabolism. The ampules containing the lower concentration of cells were gassed with a mixture of air plus 5%
Ro-07-0582 as a radiosensitizer
and cytotoxic agent 0 E. J. HALL and J. BIAGLOW
COZ and maintained at room temperature, so that they retained a fully aerated status. Following this procedure, the cells were irradiated with y-rays, or subjected to periods of heat treatment at various temperatures. The source of y-rays was a cobalt teletherapy unit, directed vertically upwards so that the cells were irradiated through the bottom of the ampules. At a treatment distante of 40 cm, the dose-rate was computed to be 350rad/min. Heat treatments were carried out by immersing the ampules in a water bath. Four different temperatures were used, namely 24’, 37.5”, 42.5” and 45”C, and each of which was controlled to within + O.l”C. At the conclusion of al1 radiation or heating procedures, each ampule was agitated on a vortex mixer to resuspend the cells, after which it was opened and various aliquots of the cell suspension were plated into Falcon tissue culture flasks containing fresh growth medium. The cells then were incubated for 8 days before they were fixed and stained; then, the number of colonies per flask was counted using a projection technique. By comparison with appropriate controls, the fraction of cells surviving each treatment schedule could be computed readily. For metabolic studies, V79 cells were grown as monolayer cultures in 2 ft long bottles on a New Brunswick roller drum apparatus. Gibco’s FlO medium, supplemented with 10% fetal calf serum, was used as the growth medium. The cells were harvested, before confluency, by scraping with a rubber policeman. After centrifugation the cells were suspended in complete growth medium at approximately 1 x 108 cells per ml and placed in an icebath. For each measurement, a fresh 0.1 ml aliquot of the cel1 suspension was added to 2.9 ml of the growth medium in the reaction chamber buffered with 0.05 M phosphate, pH 7.2. The mixture was allowed to equilibrate at the desired temperature for 5 min. The Clark electrode was immersed in the continuously stirred cel1 suspension, which was closed to the atmosphere, the change in concentration of dissolved oxygen was recorded. KCN, followed by Ro-07-0582 was added through the side groove of the oxygen electrode holder with the aid of a
523
Hamilton syringe. Stock solutions of 1 M Ro07-0582 were prepared in dimethylsulfoxide (DMSO). Fifteen microliters of Ro-07-0582 added to 3 ml growth medium produced a 5 mM drug concentration. DMSO at the concentrations introduced with Ro-07-0582 did not affect oxygen utilization. The initial rates of oxygen utilization for Ro-07-0582 in the presence of KCN were corrected for cyanide insensitive respiration by subtracting the rates of oxygen utilization in the presence of KCN from the rate obtained in the presence of KCN + Ro-07-0582. REXJLTS In the first series of experiments the effect of Ro-07-0582 as a radiosensitizer was studied as a function of the time that the drug was in contact with the cells before irradiation. Figure 1 shows the result of one DOSE
(rad)
Fig. 1. The closed triangles and circles represent the survival of Chinese hamster cells irradiated with ?Zo y-rays under aerated and hypoxic conditions. The survival of hypoxic cells irradiated in the presence of Ro-07-0582 at a concentration of 0.2 mM is represented by open and closed squares. Open squares refer to irradiations carried out immediately after the drug was added and the ceils made hypoxic. Closed squares refer to irradiations carried out after the cells were stored at room temperature for 24 hr following the addition of the drug, a treatment which killed about 80% of the cells.
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such experiment. The storage period kills about 80% of the cells, and it is evident from Fig. 1 that the cells which survive are sensitized to the @‘Co y-rays to a much greater extent than those irradiated at 0 hr. Figure 2 DOSE
May-June 1977, Volume 2, No. 5 and No. 6
erentially killed cells in S more than in other phases of the cycle, this would explain why the survivors of a prolonged drug treatment were more sensitive to radiation. An experiment was performed to test this hypothesis; the results are shown in Fig. 3. Actively
(rad)
Hypaxic
cells + 5 mM “0582”
3 hours
0
I TIME AFTER
Fig. 2. The closed triangles and circles represented the survival of Chinese hamster cells irradiated with “Co y-rays under aerated and hypoxic conditions. The survival of hypoxic cells irradiated in the presence of Ro-07-0582 at a concentration of 2mM is represented by open and closed squares. Open squares refer to irradiations carried out immediately after the drug was added and the cells made hypoxic. Closed squares refer to irradiations carried out after cells were stored at room temperature for 5 hr following the addition of the drug, a treatment which killed 80% of the cells.
shows data from a similar experiment except that a higher drug concentration for a shorter storage time was used, namely 2 mM for 5 hr. This treatment also leads to the killing of 80% of the cel1 population, but those that survive are greatly sensitized by the drug to the subsequent effects of radiation. Indeed, hypoxic cells treated with 2 mM of Ro-07-0582 for 5 hr are almost as sensitive as aerated cells. In the course of attempting to explain the data in Figs. 1 and 2, one obvious possibility was that Ro-07-0582 might be cel1 cycle specifìc in its toxicity. If, for example, it pref-
2
at 370C
3 REMOVAL
4 5 6 OF HYDROXYUREA
7
Fig. 3. Response of Chinese hamster cells to Xrays and Ro-07-0582 as a function of age in the mitotic cycle. Upper curve: Response of aerated cells to a single dose of 8OOrad of X-rays at various times after synchrony with hydroxyurea. Lower curve: Cells were made hypoxic and treatments with Ro-07-0582 (5 mM for 3 hr at 37S”C) started at various times after synchrony with hydroxyurea.
growing cells in a large number of parallel flasks were synchronized by adding hydroxyurea to the growth medium to a fìnal concentration of 2 mM for a period of 3.5 hr; this arrests cells at the GJS interface. Following this treatment, the hydroxyurea was removed and replaced by fresh growth medium, and the cells returned to the incubator to allow the surviving cohort of cells to move synchronously through the mitotic cycle. At hourly intervals, a pair of flasks was removed and irradiated with 800 rad of X-rays; the results are shown in the top panel of Fig. 3. Cel1 survival to this fixed dose rises sharply over the f?rst few hours, reaching a peak at about
525
Ro-07-0582 as a radiosensitizer and cytotoxic agent 0 E. J. HALL and J. BIAGLOW
4 hr which corresponds to late& At three times in the cycle, the toxicity of Ro-07-0582 was tested. The three times chosen were 0, 3 and 7 hr after the removal of the hydroxyurea, corresponding to the GJS interface, mid-S and GJM. At each of these times, 6 flasks were removed from the incubator, the cells harvested by trypsinization and made hypoxic in glass ampules by the methods described above, with or without the addition of Ro-07-0582 at a concentration of 5 mM. After they were made hypoxic, they were maintained at 37S”C for a period of 3 hr before they were opened and various aliquots of the cel1 suspension plated into tissue culture flasks containing fresh growth medium. The fraction of cells surviving this treatment delivered (or at least commenced) at the three positions in the cel1 cycle is shown in the lower half of Fig. 3. Cell sensitivity to X-rays varies dramatically through the cycle, but by comparison the response to Ro-07-0582 is almost independent of the position in the cel1 cycle. The fraction of cells surviving a dose of 800 rad varies by a factor of approximately ten according to the phase of the cycle in which the radiation is delivered, while for the cytotoxic effect of the drug the fraction of cells surviving the 3 hr tratement varies by a factor of less than two with the phase of the cycle. The next series of experiments was designed to investigate the effect of hyperthermia combined with the hypoxic sensitizer Ro-070582. Figure 4 shows the result of an experiment in which cells were exposed to 45°C for various periods of time under aerated and hypoxic conditions, with and without the drug at a concentration of 5 mM. It has been shown previously that hypoxia does not protect from the effects of hyperthermia as it does from X-rays;12,19,22indeed hypoxic cells tend to be slightly more sensitive to hyperthermia than aerated cells, although this may result primarily from the change of pH and/or nutritional trauma associated with methods of producing hypoxia.13,15,29The addition of Ro-07-0582 at a concentrathon of 5 mM slightly increases the cel1 killing effect of hyperthermia on aerated cells, but has a dramatic effect on hypoxic
TIME AT 45°C ( min.) 20
25
I 0-3
10* Fig. 4. The effect of various time periods at 45°C on aerated and hypoxic cells in the presence and absente of Ro-07-0582 at a concentration of 5 mM.
and The combination of hyperthermia the hypoxic sensitizer Ro-07-0582 is extremely toxic to hypoxic cells. This observation led to a systematic study of the temperature dependence of the toxicity of this drug on hypoxic cells. Figure 5 shows survival curves for cells exposed to Ro-070582 at a concentration of 5 mM for various times at four different temperatures. It is evident that the toxicity of this hypoxic sensitizer is strongly temperature dependent. More extensive data on this subject, with a range of temperatures and drug concentrations wil1 be published.‘6.32 The increased killing caused by Ro-07-0582 occurs under hypoxic conditions. Presumably this may occur because the metabolic reduction of the drug produces reactive intermediates that are toxic under anaerobic conditions but are detoxified in air.‘*5*6,23V25,38*39 The first step in the cellular reduction of nitroimidazoles, and other nitro compounds involves the transfer of an electron from the microsomes to produce the nitro radic~*4,5,6,2s.38 In the presence of oxygen this radicells.
Radiation Oncology 0 Biology 0 Physics
526
; 10 \
37 50 c
\ 10 ch 8 t
May-June
1977, Volume 2, No. 5 and No. 6
conveniently as oxygen dismeasured appearance.“ The CN- inhibited cells provide a simple system for studying the first step in the metabolic reduction of nitrocompounds.4 Figure 6 shows the effects of different concentrations of nitrofurazone and “Flagyl” (both known to be cytotoxic)26 and Ro-070582 on the initial rate of oxygen consumption for V79 lung cells, incubated with 1 mM KCN. For simplicity we have plotted the initial rate versus the concentration of drug. Because of the production of oxygen reactive nitro radical anions, the oxygen con-
F '5 '5 b v)
50
10’ .E
40
\
HYFOXIA t 5 mm ‘0582’
$ s c
30
s 10
5 A 8
20
P is c 10 10
Ï
2 Th
3
4
(hourn)
Fig. 5. Illustrating the cytotoxic effect of Ro-070582 on hypoxic cells exposed to the drug for various periods of time at four different temperatures. cal is deactivated by transferring its electron, at an extremely high rate, to oxygen. Cellular
0.02 Drug
0.04
0.06
(molar)
Fig. 6. The effect of “Flagyl”, nitrofurazone and Ro-07-0582 on oxygen consumption by V79 cells under the influence of KCN induced anaerobic metabolism. The initial rate of oxidation was determined 1 min after the addition of drug through the side port of the oxygen electrode apparatus. The reaction medium consisted of growth medium buffered with 20 mM Hepes, pH 7.2, and 37°C. The cel1 density was 2 X 106/ml and the final volume 3 ml.
enzymes such as superoxide dismutase and catalase continue the deactivation by reducing the oxygen radical to water.4’6 However, in the absente of oxygen the nitro radical anion may become further reduced to other reactive intermediates such as nitroso and hydroxylamine radical.4 The reduced in- sumption continues uninterrupted as long as a termediates are believed to be responsible for supply of reducing equivalents in the form of the drug reactivity with DNA.23339 In our glucose are present.4P6 The metabolic producdetailed studies we have found that CN- in- tion of nitro radicals, as measured by oxygen hibited cells, in the presence of oxygen, consumption, is greatest with nitrofurazone, produce nitro-radical anions that react with followed by Ro-07-0582 and “Flagyl”, and oxygen to produce oxygen superoxide.4S6*25S38correlates wel1 with the one electron redox The oxygen superoxide radicals react with value? as wel1 as with the rate of reaction of themselves to produce peroxide.4’6 Oxygen the nitroimidazole and nitrofuran radicals is the most consumed in formation of peroxide can be with oxygen.6.38 Nitrofurazone
Ro-07-0582 as a radiosensitizer and cytotoxic agent 0 E. J. HALL and J. BIAGLOW
active metabolically at concentrations lower than either Ro-07-0582 or “Flagyl”. A nearly linear relationship was obtained between Ro07-0582 concentration and oxygen utilization. However, with “Flagyl” little increase in oxygen consumption was seen after 20mM. These results predict that the observed cytotoxicity for Ro-07-0582 should lie between nitrofurazone and “Flagyl”. In a similar experiment, employing a slightly higher cel1 density, the effect of temperature was determined on the rate of oxygen utilization alone and in the presence of 1 mM KCN Ro-07-0582 (Fig. 7). The rate of oxygen utilization was seen to increase in a linear marmer with increasing temperature. However, in the presence of 1 mM KCN and 5 mM Ro-07-0582 there is a sharp increase in the initial rate of oxygen utilization between 37.5”C and 45°C. A slight inhibition of oxygen utilization in the absente of Ro-07-0582 occurs at 45°C but is not enough to offset the increase caused by the rise in temperature.
52
KCN + 5mM “0582”
t
Temperature(CY)
Fig. 7. The effect of temperature on the rate of oxygen consumption by cells incubated in the absence (curve on left) and presence of KCN plus 5 mM Ro-07-0582 (curve on right). The initial rate of oxidation was determined after addition of cells to the thermally equilibrated reaction medium. Ro07-0582 was added to thermally equilibrated cells inhibited with KCN and the rate of oxygen utilization was determined one minute after drug addition. The reaction conditions are the same as those listed in Fig. 6. The cel1 density was 3 x 106/ml. ?NADP : picotineadenine dinucleotide phosphate; NADPH: reduced nicotineadenine dinucleo-
527
DISCUSSION The electron-tinic drugs, including the nitroimidazoles19’0 as wel1 as the nitrofurans,’ were identilìed initially as sensitizers of hypoxic cells. More recently, both the nitroimidazoles and nitrofurans have been found to exhibit selective toxicity towards hypoxic cells.7,17,26,33,34 Our studies with Ro-070582 suggest an increasing cytotoxicity with increase in temperature. In addition, preenhances with Ro-07-0582 incubation radiosensitivity. Presumably, both of these effects are related to the metabolism of the drug. For metabolic reduction of Ro-07-0582 both NADPH and GSH are required, similar to the reduction of other nitro drugs.4,6.25The reduction of the nitro group, in the absente of oxygen, produces a short lived nitro radinitrosoamine, cal anion, hydroxylamine, NADP’ and GSSG.“.23’25”8X39 The oxidation of NADPH and GSH to NADP’ and GSSGt may result in a depletion of intracellular reducing species, which are capable of hydrogen repair of target radicals.18’26 Such a depletion may have occurred when Ro-07-0582 was preincubated with cells (Figs. 1 and 2). Another product of the nitro reduction, most likely the short lived nitroso intermediate, also could inIluence the radiation response if it reacted with protein sulfhydryls necessary for repair of radiation damage.’ The anaerobic metabolism of Ro-07-0582 also may have been important in the effect of temperature on cytotoxicity (Fig. 5). The cytotoxicity, similar to nitrofuran? and “Flagyl” is more pronounced under anaerobic conditions in vitro. Previously, Olive and McCal1a28demonstrated that anaerobic metabolism of nitrofurans leads to DNA breaks in mammalian cells, and that such breaks were oxygen dependent. Presumably, both the lack of toxicity (Fig. 5) and decreased DNA breaksZ in air result from an inability for partially reduced intermediates to accumulate in the presence of oxygen. 4s~6~23.25~38~39 Under anaerobic conditions either the nitro radical or the nitroso intermediate might be extremely reactive with tide phosphate; glutathione.
GSH: glutathione;
GSSG: oxidized
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Radiation Oncology 0 Biology 0 Physics
It has been demonstrated that chemical reduction of Flagyl, in the absente of oxygen, produces intermediates that are reactive with calf thymus DNA.23 In addition, we have found“’ that the sulfhydryl reagent cysteamine protects against the cytotoxicity of Ro-07-0582 in vitro. Protection by cysteamine suggests that reactive intermediates, such as the nitro radical anion or the nitroso intermediate, were produced during the incubation period. Cysteamine would chemically inactivate these intermediates preventing their reaction with DNA and subsequent cytotoxicity.‘6 As a cytotoxic agent and in vitro radiosensitizer Ro-07-0582 is more potent than “Flagyl” but less than nitrofurazone.% Nitrofurazone is more electron affinic than either “Flagyl” or Ro-07-0582.2’38The greater ability of nitrofurazone to accept electrons was seen metabolically by the increase in oxygen reactive nitro radical anions (Fig. 6) at lower concentrations than either Flagyl or Ro-070582. The greater metabolic activity of nitrofurazone limits its use to in vitro conditions where metabolism may be greatly reduced by the appropriate choice of cel1 density and reaction conditions.” The increased effect of temperature on radical production appears to parallel an increased killing in the presence of Ro-070582. The elevated temperatures would increase the rate of metabolic reduction of Ro-07-0582 to reactive intermediates which would decrease the intracellular concentration of NADPH and GSH. These intracellular species most likely are needed for the transfer of electrons to the drug.4,6.25 Of additional interest is the finding that the rate of cellular oxidation is increased (Fig. 7). In vivo such an increase in oxidative metabolism of a tumor would slightly increase the hypoxic fraction of cells within the tumor area. An increase in oxidative capacity would promote the hypoxic cytotoxicity of Ro-070582.
DNA.*=’
MayJune
1977, Volume 2, No. 5 and No. 6
The testing of the nitroimidazoles in human cancer patients3s.37 represents one of the most significant developments of the past decade, though it is not certain how much of the effect results from a true radiosensitizing, and how much results from killing of hypoxic cells by the specific cytotoxic action of the drug against these cells. Obviously, the mode of action of Ro-07-0582 in vivo may be quite complex. Some of these complexities, such as metabolic effects and cytotoxicity, can best be identified and sorted out with in vitro cel1 systems. There are two principal conclusions to be drawn from the in vitro experiments described in this paper: (1) Ro-07-0582 is an extremely cytotoxic agent; at concentrations that lead to an appreciable radiosensitization, a significant proportion of hypoxic cells are killed by the drug. Furthermore, cellular oxidative rate, cytotoxicity and metabolic reduction of the drug increase dramatically with temperature, to such an extent that the combination of a hypoxic sensitizer with some form of hyperthermia is an attractive possibility. More extensive investigations along these lines are in progress.‘6332 (2) The sensitizing effect of a given concentration of drug increases appreciably with time. This is evident as an empirical fact from the data presented in this paper, but the work of others and our own metabolic studies suggest that this effect probably results from both the accumulation with time of reduced products as wel1 as alterations in cellular redox, i.e. GSH or NADPH resulting in an improved radiosensitization by Ro-07-0582. In the clinical application of the nitroimidazole there may be room for considerable maneuvers in the time sequence of administering the drug and delivering the radiation fractions.
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Ro-07-0582 as a radiosensitizer and cytotoxic agent 0 E. J. HALL and J. BIAGLOW
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with selective toxicity for hypoxic cells. Proc. 66 Meet. Cancer Res. 17: 221, 1976. 35. Thomlinson, R.H., Dische, S., Gray, A.J.: Clinical testing of the radiosensitizer Ro-07“0582”. Clin. Radiol. 27: 151-157, 1976. 36. Thomlinson, R.H., Gray, L.H.: The histological structure of some human lung cancers and the possible implications for radiotherapy. Br. J. Cancer 9: 539-549, 1955. 37. Urtasun, R., Band, P., Chapman,
D., Feldstein, M., Mielke, B., Fryer, C.: Radiation and high-
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dose metronidazole in supratentorial Glioblastomas. N. Engl. J. Med. 294: 1364-1367, 1976. 38. Wardman, P., Clark, E.D.: Oxygen inhibition of nitroreductase: Electron transfer from nitro radical-anions to oxygen. Biochem. Biophys. Res. Commun. 69: 942-949, 1976. 39. Willson, R.L., Cramp, W.A., Ings,
R.M.J.: Metronidazole (“Flagyl”) mechanisms of radiosensitization. Int. J. Radiat. Biol. 26: 557569, 1974.
Oncology
Bid.
Phys.,
1977, Vol. 2, pp. 521-530.
Pergamon
Press.
Printed
in the U.S.A.
0 Rapid Communication
Ro-07-0582
AS A RADIOSENSITIZER ERIC
Radiological
Research
J.
AND CYTOTOXIC
AGENT?
M.A., D.Phil.
HALL,
Laboratory, College of Physicians University, New York, U.S.A.
and Surgeons
of Columbia
and JOHN Division
of Radiation
M.S., Ph.D.
BIAGLOW,
Biology, Department of Radiology, Case Western versity, Cleveland, Ohio, U.S.A.
Reserve
Uni-
The electron a5nic drugs, including the nltroimidazoles and the nltrofurans, were initially identlfled on the basis of their abllity to mhnic oxygen and dlfferentially sensitize hypoxic cells. More recently, these drugs have been found to exbibit selective toxicity toward hypoxic cells. The radiosensitizing effect of the 2-nitrohnidazole Ro-07-0582 is greatly enhanced if cells are pre-incubated in the presence of the drug for a period of hours prior to irradiation. In addition, the cytutoxicity of the drug is greatly increased at elevated temperatures. Both of these effects may be related to the metabolism of the drug, and it is clear that there is considerable room for maneuver ln the time sequence of administering the drug and delivering tbe radiation dose as these potent and useful sensitizers begin to be used in the human. Radiosensitlzer, Hypuxic cells, Electron affinic drugs.
INTRODUCTION The idea that foei of hypoxic cells in some tumors may limit their radiocurability by Xrays was a consequente of the pioneering work of Gray, Read, Thomlinson and their the colleagues.‘4.3”36 In the case of X-rays, fraction of cells killed by a given dose depends strongly on the presence of molecular oxygen; since some tumor cells may be hypoxic thcy would be spared from the effects of the radiation compared with wel1
oxygenated
normal tissues.
IThis investigation was supported by Contract EY-76-C-02-3243 from the Energy Research and Development Administration and by Grant NOS. CA-12536, CA-18506 and CA-13747 by the National Cancer Institute, DHEW. Acknowledgements-It is with pleasure that we acknowledge that Sir Oliver Scott, of the Royal
Because many of the early radiobiologists had their initial training in physics, they naturally and logically sought a physical solution for this problem, and came up with the suggestion that X-rays should be replaced by a more densely ionizing radiation, such as neutrons, for which the cell killing effect is less dependent on the presence of oxygen. Gray was largely responsible for conceiving the cyclotron to generate neutrons at the Hammersmith Hospital; this has led directly to the worldwide effort to test the efficacy of Marsden Hospita1 in London, suggested the experimentj involving the preincubation with the drug which led to a significantly greater sensitization. The compound Ro-07-0582 was generously supplied by Hoffman-La Roche. We thank Dr. Ged Adams of the Gray Laboratory for much useful discussion and continued stimulation. 521
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neutrons and other high LET radiations as a potential alternative for X-rays in radiotherapy. In more recent years radiation chemists have played an increasingly prominent role in radiation research, and have brought with them a new and different outlook. When they were faced with the problem of countering hypoxic cells, instead of seeking new types of radiation, they fel1 back on their training as chemists and sought to overcome the lack of oxygen in tumor cells by using a suitable drug which could act as a substitute for oxygen.’ In this way the electron affinic nitroimidazoles were discovered and/or developed;‘” they are able to diffuse to cells which oxygen cannot reach because they are metabolized more slowly than oxygen in respiring tissue. Attention has focussed on the nitroimidazole known commonly by its code name Ro-070582.=” This drug now has been used extensively in radiobiological experiments, both in vitro and in vivo ; testing also has begun in a few patients. In the fhst instance, these hypoxic cel1 sensitizers were sought and chosen on the basis of their ability to mimic oxygen and differentially sensitize hypoxic cells to the effects of X-rays, without altering the response of the aerated cells. However, in the course of laboratory studies, it has become apparent that nitro derivatives such as the nitroimidazoles “Flagyl” and Ro-07-0582, as wel1 as the nitrofuran derivative nitrofurazone, are cytotoxic toward hypoxic cells. This toxic effect has been demonstrated with cells cultured in vitro17326 with multicell spheroids33,34 and also with transplanted tumors in animals.’ The action of Ro-07-0582 in any in vivo situation is likely to reflect a combination of its radiosensitizing properties coupled with its cytotoxicity. In both regards it acts selectively against hypoxic cells, at least at low concentrations. This means, in general, that the presence of the drug wil1 lead to an increase in the number of cells killed in tumors which contain hypoxic cells. The present paper describes experiments intended to further the understanding of Ro07-0582 as a sensitizer, and also to investigate
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1977, Volume 2, No. 5 and No. 6
the cytotoxic properties of this drug as a function of temperature. In addition, a property of this sensitizer, to alter cellular oxygen utilization in the presence of potassium cyanide (KCN),““” is used as an indicator of increased metabolic activity as a function of temperature. METHODS
AND MATERIALS
V79 Chinese hamster cells were used throughout these investigations, cultured by standard techniques, and grown in Gibco’s FlO medium supplemented with 10% fetal calf serum. Cells received their treatments, with radiation or with hyperthermia, with or without the addition of the nitroimidazoles, while held in suspension in smal1 glass ampules. For each experiment, cells were harvested by trypsinization from twenty actively growing, partly confluent stock bottles, spun down and washed to remove excess trypsin, and the concentration of cells counted with a Coulter electronic cell counter. This cell suspension then was divided into several parts and the cel1 concentration in each adjusted to 2 x 106cellslml, adding Ro-07-0582 at an appropriate concentration according to the plan of the particular experiment. From each of these suspensions, containing various concentrations of drug or no drug at all, series of glass ampules were filled by pipetting 1 ml of the suspension into each. After dilution by a factor of 200, another series of glass ampules were filled with 10” cells each. The ampules containing cells at the higher concentration then were handled in the following way in order to make the cells hypoxic. The ampules were flushed with high purity nitrogen, plus 5% CO1, through a long cannula, in order to displace the air above the cel1 suspension. Each ampule was heatsealed before it was transferred to a water bath at 37S”C where it was continuously shaken and rotated end-for-end to keep the cells in suspension. This procedure was maintained for a period of 1 hr to allow the cells to “scavenge” the oxygen dissolved in the medium by normal metabolism. The ampules containing the lower concentration of cells were gassed with a mixture of air plus 5%
Ro-07-0582 as a radiosensitizer
and cytotoxic agent 0 E. J. HALL and J. BIAGLOW
COZ and maintained at room temperature, so that they retained a fully aerated status. Following this procedure, the cells were irradiated with y-rays, or subjected to periods of heat treatment at various temperatures. The source of y-rays was a cobalt teletherapy unit, directed vertically upwards so that the cells were irradiated through the bottom of the ampules. At a treatment distante of 40 cm, the dose-rate was computed to be 350rad/min. Heat treatments were carried out by immersing the ampules in a water bath. Four different temperatures were used, namely 24’, 37.5”, 42.5” and 45”C, and each of which was controlled to within + O.l”C. At the conclusion of al1 radiation or heating procedures, each ampule was agitated on a vortex mixer to resuspend the cells, after which it was opened and various aliquots of the cell suspension were plated into Falcon tissue culture flasks containing fresh growth medium. The cells then were incubated for 8 days before they were fixed and stained; then, the number of colonies per flask was counted using a projection technique. By comparison with appropriate controls, the fraction of cells surviving each treatment schedule could be computed readily. For metabolic studies, V79 cells were grown as monolayer cultures in 2 ft long bottles on a New Brunswick roller drum apparatus. Gibco’s FlO medium, supplemented with 10% fetal calf serum, was used as the growth medium. The cells were harvested, before confluency, by scraping with a rubber policeman. After centrifugation the cells were suspended in complete growth medium at approximately 1 x 108 cells per ml and placed in an icebath. For each measurement, a fresh 0.1 ml aliquot of the cel1 suspension was added to 2.9 ml of the growth medium in the reaction chamber buffered with 0.05 M phosphate, pH 7.2. The mixture was allowed to equilibrate at the desired temperature for 5 min. The Clark electrode was immersed in the continuously stirred cel1 suspension, which was closed to the atmosphere, the change in concentration of dissolved oxygen was recorded. KCN, followed by Ro-07-0582 was added through the side groove of the oxygen electrode holder with the aid of a
523
Hamilton syringe. Stock solutions of 1 M Ro07-0582 were prepared in dimethylsulfoxide (DMSO). Fifteen microliters of Ro-07-0582 added to 3 ml growth medium produced a 5 mM drug concentration. DMSO at the concentrations introduced with Ro-07-0582 did not affect oxygen utilization. The initial rates of oxygen utilization for Ro-07-0582 in the presence of KCN were corrected for cyanide insensitive respiration by subtracting the rates of oxygen utilization in the presence of KCN from the rate obtained in the presence of KCN + Ro-07-0582. REXJLTS In the first series of experiments the effect of Ro-07-0582 as a radiosensitizer was studied as a function of the time that the drug was in contact with the cells before irradiation. Figure 1 shows the result of one DOSE
(rad)
Fig. 1. The closed triangles and circles represent the survival of Chinese hamster cells irradiated with ?Zo y-rays under aerated and hypoxic conditions. The survival of hypoxic cells irradiated in the presence of Ro-07-0582 at a concentration of 0.2 mM is represented by open and closed squares. Open squares refer to irradiations carried out immediately after the drug was added and the ceils made hypoxic. Closed squares refer to irradiations carried out after the cells were stored at room temperature for 24 hr following the addition of the drug, a treatment which killed about 80% of the cells.
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such experiment. The storage period kills about 80% of the cells, and it is evident from Fig. 1 that the cells which survive are sensitized to the @‘Co y-rays to a much greater extent than those irradiated at 0 hr. Figure 2 DOSE
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erentially killed cells in S more than in other phases of the cycle, this would explain why the survivors of a prolonged drug treatment were more sensitive to radiation. An experiment was performed to test this hypothesis; the results are shown in Fig. 3. Actively
(rad)
Hypaxic
cells + 5 mM “0582”
3 hours
0
I TIME AFTER
Fig. 2. The closed triangles and circles represented the survival of Chinese hamster cells irradiated with “Co y-rays under aerated and hypoxic conditions. The survival of hypoxic cells irradiated in the presence of Ro-07-0582 at a concentration of 2mM is represented by open and closed squares. Open squares refer to irradiations carried out immediately after the drug was added and the cells made hypoxic. Closed squares refer to irradiations carried out after cells were stored at room temperature for 5 hr following the addition of the drug, a treatment which killed 80% of the cells.
shows data from a similar experiment except that a higher drug concentration for a shorter storage time was used, namely 2 mM for 5 hr. This treatment also leads to the killing of 80% of the cel1 population, but those that survive are greatly sensitized by the drug to the subsequent effects of radiation. Indeed, hypoxic cells treated with 2 mM of Ro-07-0582 for 5 hr are almost as sensitive as aerated cells. In the course of attempting to explain the data in Figs. 1 and 2, one obvious possibility was that Ro-07-0582 might be cel1 cycle specifìc in its toxicity. If, for example, it pref-
2
at 370C
3 REMOVAL
4 5 6 OF HYDROXYUREA
7
Fig. 3. Response of Chinese hamster cells to Xrays and Ro-07-0582 as a function of age in the mitotic cycle. Upper curve: Response of aerated cells to a single dose of 8OOrad of X-rays at various times after synchrony with hydroxyurea. Lower curve: Cells were made hypoxic and treatments with Ro-07-0582 (5 mM for 3 hr at 37S”C) started at various times after synchrony with hydroxyurea.
growing cells in a large number of parallel flasks were synchronized by adding hydroxyurea to the growth medium to a fìnal concentration of 2 mM for a period of 3.5 hr; this arrests cells at the GJS interface. Following this treatment, the hydroxyurea was removed and replaced by fresh growth medium, and the cells returned to the incubator to allow the surviving cohort of cells to move synchronously through the mitotic cycle. At hourly intervals, a pair of flasks was removed and irradiated with 800 rad of X-rays; the results are shown in the top panel of Fig. 3. Cel1 survival to this fixed dose rises sharply over the f?rst few hours, reaching a peak at about
525
Ro-07-0582 as a radiosensitizer and cytotoxic agent 0 E. J. HALL and J. BIAGLOW
4 hr which corresponds to late& At three times in the cycle, the toxicity of Ro-07-0582 was tested. The three times chosen were 0, 3 and 7 hr after the removal of the hydroxyurea, corresponding to the GJS interface, mid-S and GJM. At each of these times, 6 flasks were removed from the incubator, the cells harvested by trypsinization and made hypoxic in glass ampules by the methods described above, with or without the addition of Ro-07-0582 at a concentration of 5 mM. After they were made hypoxic, they were maintained at 37S”C for a period of 3 hr before they were opened and various aliquots of the cel1 suspension plated into tissue culture flasks containing fresh growth medium. The fraction of cells surviving this treatment delivered (or at least commenced) at the three positions in the cel1 cycle is shown in the lower half of Fig. 3. Cell sensitivity to X-rays varies dramatically through the cycle, but by comparison the response to Ro-07-0582 is almost independent of the position in the cel1 cycle. The fraction of cells surviving a dose of 800 rad varies by a factor of approximately ten according to the phase of the cycle in which the radiation is delivered, while for the cytotoxic effect of the drug the fraction of cells surviving the 3 hr tratement varies by a factor of less than two with the phase of the cycle. The next series of experiments was designed to investigate the effect of hyperthermia combined with the hypoxic sensitizer Ro-070582. Figure 4 shows the result of an experiment in which cells were exposed to 45°C for various periods of time under aerated and hypoxic conditions, with and without the drug at a concentration of 5 mM. It has been shown previously that hypoxia does not protect from the effects of hyperthermia as it does from X-rays;12,19,22indeed hypoxic cells tend to be slightly more sensitive to hyperthermia than aerated cells, although this may result primarily from the change of pH and/or nutritional trauma associated with methods of producing hypoxia.13,15,29The addition of Ro-07-0582 at a concentrathon of 5 mM slightly increases the cel1 killing effect of hyperthermia on aerated cells, but has a dramatic effect on hypoxic
TIME AT 45°C ( min.) 20
25
I 0-3
10* Fig. 4. The effect of various time periods at 45°C on aerated and hypoxic cells in the presence and absente of Ro-07-0582 at a concentration of 5 mM.
and The combination of hyperthermia the hypoxic sensitizer Ro-07-0582 is extremely toxic to hypoxic cells. This observation led to a systematic study of the temperature dependence of the toxicity of this drug on hypoxic cells. Figure 5 shows survival curves for cells exposed to Ro-070582 at a concentration of 5 mM for various times at four different temperatures. It is evident that the toxicity of this hypoxic sensitizer is strongly temperature dependent. More extensive data on this subject, with a range of temperatures and drug concentrations wil1 be published.‘6.32 The increased killing caused by Ro-07-0582 occurs under hypoxic conditions. Presumably this may occur because the metabolic reduction of the drug produces reactive intermediates that are toxic under anaerobic conditions but are detoxified in air.‘*5*6,23V25,38*39 The first step in the cellular reduction of nitroimidazoles, and other nitro compounds involves the transfer of an electron from the microsomes to produce the nitro radic~*4,5,6,2s.38 In the presence of oxygen this radicells.
Radiation Oncology 0 Biology 0 Physics
526
; 10 \
37 50 c
\ 10 ch 8 t
May-June
1977, Volume 2, No. 5 and No. 6
conveniently as oxygen dismeasured appearance.“ The CN- inhibited cells provide a simple system for studying the first step in the metabolic reduction of nitrocompounds.4 Figure 6 shows the effects of different concentrations of nitrofurazone and “Flagyl” (both known to be cytotoxic)26 and Ro-070582 on the initial rate of oxygen consumption for V79 lung cells, incubated with 1 mM KCN. For simplicity we have plotted the initial rate versus the concentration of drug. Because of the production of oxygen reactive nitro radical anions, the oxygen con-
F '5 '5 b v)
50
10’ .E
40
\
HYFOXIA t 5 mm ‘0582’
$ s c
30
s 10
5 A 8
20
P is c 10 10
Ï
2 Th
3
4
(hourn)
Fig. 5. Illustrating the cytotoxic effect of Ro-070582 on hypoxic cells exposed to the drug for various periods of time at four different temperatures. cal is deactivated by transferring its electron, at an extremely high rate, to oxygen. Cellular
0.02 Drug
0.04
0.06
(molar)
Fig. 6. The effect of “Flagyl”, nitrofurazone and Ro-07-0582 on oxygen consumption by V79 cells under the influence of KCN induced anaerobic metabolism. The initial rate of oxidation was determined 1 min after the addition of drug through the side port of the oxygen electrode apparatus. The reaction medium consisted of growth medium buffered with 20 mM Hepes, pH 7.2, and 37°C. The cel1 density was 2 X 106/ml and the final volume 3 ml.
enzymes such as superoxide dismutase and catalase continue the deactivation by reducing the oxygen radical to water.4’6 However, in the absente of oxygen the nitro radical anion may become further reduced to other reactive intermediates such as nitroso and hydroxylamine radical.4 The reduced in- sumption continues uninterrupted as long as a termediates are believed to be responsible for supply of reducing equivalents in the form of the drug reactivity with DNA.23339 In our glucose are present.4P6 The metabolic producdetailed studies we have found that CN- in- tion of nitro radicals, as measured by oxygen hibited cells, in the presence of oxygen, consumption, is greatest with nitrofurazone, produce nitro-radical anions that react with followed by Ro-07-0582 and “Flagyl”, and oxygen to produce oxygen superoxide.4S6*25S38correlates wel1 with the one electron redox The oxygen superoxide radicals react with value? as wel1 as with the rate of reaction of themselves to produce peroxide.4’6 Oxygen the nitroimidazole and nitrofuran radicals is the most consumed in formation of peroxide can be with oxygen.6.38 Nitrofurazone
Ro-07-0582 as a radiosensitizer and cytotoxic agent 0 E. J. HALL and J. BIAGLOW
active metabolically at concentrations lower than either Ro-07-0582 or “Flagyl”. A nearly linear relationship was obtained between Ro07-0582 concentration and oxygen utilization. However, with “Flagyl” little increase in oxygen consumption was seen after 20mM. These results predict that the observed cytotoxicity for Ro-07-0582 should lie between nitrofurazone and “Flagyl”. In a similar experiment, employing a slightly higher cel1 density, the effect of temperature was determined on the rate of oxygen utilization alone and in the presence of 1 mM KCN Ro-07-0582 (Fig. 7). The rate of oxygen utilization was seen to increase in a linear marmer with increasing temperature. However, in the presence of 1 mM KCN and 5 mM Ro-07-0582 there is a sharp increase in the initial rate of oxygen utilization between 37.5”C and 45°C. A slight inhibition of oxygen utilization in the absente of Ro-07-0582 occurs at 45°C but is not enough to offset the increase caused by the rise in temperature.
52
KCN + 5mM “0582”
t
Temperature(CY)
Fig. 7. The effect of temperature on the rate of oxygen consumption by cells incubated in the absence (curve on left) and presence of KCN plus 5 mM Ro-07-0582 (curve on right). The initial rate of oxidation was determined after addition of cells to the thermally equilibrated reaction medium. Ro07-0582 was added to thermally equilibrated cells inhibited with KCN and the rate of oxygen utilization was determined one minute after drug addition. The reaction conditions are the same as those listed in Fig. 6. The cel1 density was 3 x 106/ml. ?NADP : picotineadenine dinucleotide phosphate; NADPH: reduced nicotineadenine dinucleo-
527
DISCUSSION The electron-tinic drugs, including the nitroimidazoles19’0 as wel1 as the nitrofurans,’ were identilìed initially as sensitizers of hypoxic cells. More recently, both the nitroimidazoles and nitrofurans have been found to exhibit selective toxicity towards hypoxic cells.7,17,26,33,34 Our studies with Ro-070582 suggest an increasing cytotoxicity with increase in temperature. In addition, preenhances with Ro-07-0582 incubation radiosensitivity. Presumably, both of these effects are related to the metabolism of the drug. For metabolic reduction of Ro-07-0582 both NADPH and GSH are required, similar to the reduction of other nitro drugs.4,6.25The reduction of the nitro group, in the absente of oxygen, produces a short lived nitro radinitrosoamine, cal anion, hydroxylamine, NADP’ and GSSG.“.23’25”8X39 The oxidation of NADPH and GSH to NADP’ and GSSGt may result in a depletion of intracellular reducing species, which are capable of hydrogen repair of target radicals.18’26 Such a depletion may have occurred when Ro-07-0582 was preincubated with cells (Figs. 1 and 2). Another product of the nitro reduction, most likely the short lived nitroso intermediate, also could inIluence the radiation response if it reacted with protein sulfhydryls necessary for repair of radiation damage.’ The anaerobic metabolism of Ro-07-0582 also may have been important in the effect of temperature on cytotoxicity (Fig. 5). The cytotoxicity, similar to nitrofuran? and “Flagyl” is more pronounced under anaerobic conditions in vitro. Previously, Olive and McCal1a28demonstrated that anaerobic metabolism of nitrofurans leads to DNA breaks in mammalian cells, and that such breaks were oxygen dependent. Presumably, both the lack of toxicity (Fig. 5) and decreased DNA breaksZ in air result from an inability for partially reduced intermediates to accumulate in the presence of oxygen. 4s~6~23.25~38~39 Under anaerobic conditions either the nitro radical or the nitroso intermediate might be extremely reactive with tide phosphate; glutathione.
GSH: glutathione;
GSSG: oxidized
528
Radiation Oncology 0 Biology 0 Physics
It has been demonstrated that chemical reduction of Flagyl, in the absente of oxygen, produces intermediates that are reactive with calf thymus DNA.23 In addition, we have found“’ that the sulfhydryl reagent cysteamine protects against the cytotoxicity of Ro-07-0582 in vitro. Protection by cysteamine suggests that reactive intermediates, such as the nitro radical anion or the nitroso intermediate, were produced during the incubation period. Cysteamine would chemically inactivate these intermediates preventing their reaction with DNA and subsequent cytotoxicity.‘6 As a cytotoxic agent and in vitro radiosensitizer Ro-07-0582 is more potent than “Flagyl” but less than nitrofurazone.% Nitrofurazone is more electron affinic than either “Flagyl” or Ro-07-0582.2’38The greater ability of nitrofurazone to accept electrons was seen metabolically by the increase in oxygen reactive nitro radical anions (Fig. 6) at lower concentrations than either Flagyl or Ro-070582. The greater metabolic activity of nitrofurazone limits its use to in vitro conditions where metabolism may be greatly reduced by the appropriate choice of cel1 density and reaction conditions.” The increased effect of temperature on radical production appears to parallel an increased killing in the presence of Ro-070582. The elevated temperatures would increase the rate of metabolic reduction of Ro-07-0582 to reactive intermediates which would decrease the intracellular concentration of NADPH and GSH. These intracellular species most likely are needed for the transfer of electrons to the drug.4,6.25 Of additional interest is the finding that the rate of cellular oxidation is increased (Fig. 7). In vivo such an increase in oxidative metabolism of a tumor would slightly increase the hypoxic fraction of cells within the tumor area. An increase in oxidative capacity would promote the hypoxic cytotoxicity of Ro-070582.
DNA.*=’
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1977, Volume 2, No. 5 and No. 6
The testing of the nitroimidazoles in human cancer patients3s.37 represents one of the most significant developments of the past decade, though it is not certain how much of the effect results from a true radiosensitizing, and how much results from killing of hypoxic cells by the specific cytotoxic action of the drug against these cells. Obviously, the mode of action of Ro-07-0582 in vivo may be quite complex. Some of these complexities, such as metabolic effects and cytotoxicity, can best be identified and sorted out with in vitro cel1 systems. There are two principal conclusions to be drawn from the in vitro experiments described in this paper: (1) Ro-07-0582 is an extremely cytotoxic agent; at concentrations that lead to an appreciable radiosensitization, a significant proportion of hypoxic cells are killed by the drug. Furthermore, cellular oxidative rate, cytotoxicity and metabolic reduction of the drug increase dramatically with temperature, to such an extent that the combination of a hypoxic sensitizer with some form of hyperthermia is an attractive possibility. More extensive investigations along these lines are in progress.‘6332 (2) The sensitizing effect of a given concentration of drug increases appreciably with time. This is evident as an empirical fact from the data presented in this paper, but the work of others and our own metabolic studies suggest that this effect probably results from both the accumulation with time of reduced products as wel1 as alterations in cellular redox, i.e. GSH or NADPH resulting in an improved radiosensitization by Ro-07-0582. In the clinical application of the nitroimidazole there may be room for considerable maneuvers in the time sequence of administering the drug and delivering the radiation fractions.
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Ro-07-0582 as a radiosensitizer and cytotoxic agent 0 E. J. HALL and J. BIAGLOW
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18. Han, A., Sinclair, W.K., Kimler, B.F.: The effect of N-ethylmaleimide on the response of X-rays of synchronized HeLa cells. Radiat. Res. 65: 337-350, 1976. 19. Harisiadis, L., Hall, E.J., Kraljevic, U., Borek, c.: Hyperthermia: Biological studies at the cellular level. Radiology 117: 447-452, 1975. 20. Harris, J.W., Koch, C.J., Power, J.A., Biaglow, J.E.: Radiosensitization of hypoxic mammalian cells by Diamide 11 studies of mechanism. Radiat. Res. in press, 1977. 21. Harris, J.W., Power, J.A., Koch, C.J.: Radiosensitization of hypoxic mammalian cells by diamide-1. Effects of experimental conditions on survival. Radiat. Res. 64: 270-280, 1975. 22. Kim, S.H., Kim, J.H., Hahn, E.W.: Enhanced killing of hypoxic tumor cells by hyperthermia. Br. J. Radiol. 48: 872-874, 1975. 23. LaRusso, N.F., Tomasz, M., Muller, M.: In vitro interaction of metronidazole with nucleic acids. Gastroenterology 71: 917, 1976. 24. Lehninger, A.O.: Biochemistry, The molecular basis of cel1 structure and function. Worth Publishers, 1974, p. 379. 25. Mason, R.P., Holtzman, J.: The mechanism of microsomal and mitochondrial nitroreductase. ESR evidente for nitroaromatic free radical intermediates. Biochemistry 14: 1626-1631, 1975. 26. Mohindra, J.E., Rauth, A.M.: Increased cel1 killing by metronidazole and nitrofurazone of hypoxic compared to aerobic mammalian cells. Cancer Res. 36: 930-936, 1976. 27. Nygaard, O.F., Biaglow, J.E.: Metabolic
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