- Email: [email protected]
HEJ mice
ht. J. Radiation Oncology Bid.
Phys.,
1976, Vol.
I, pp. 1125-l 131.
Perg’dmon Press.
Printed
in the U3.A
SOME PHARMACOLOGICAL ASPECTS OF MULTIPLE DOSE METRONIDAZOLE IN C3H/HeJ MICE? M. J. HAYNES, M.Sc Department
of Biophysics,
The University of Western SC 1, Canada
Ontario,
London,
Ontario
N6A
and W. R. INCH, Ph.D. Ontario Cancer Experimental
Treatment Oncology,
and Research Foundation, London Clinic, Division Victoria Hospital, London, Ontario N6A 4G5, Canada
of
Metronidazole is one of several electron affinic chemicals currently being tested as a sensitizer of radio-resistant hypoxic cells. Little is known, however, about its pharmacological effects in man or experimental animals. The half-life of metroufdazole in the C3H mouse was found to be 1.7 hr following a single intraperitoneal injection of 0.6 mg/g body weight. The same dose caused a decrease in heart rate of approximately 35% and rectal temperature fell by almost 6°C. Anhnals maintained on twice daffy doses of 0.6 mg/g for 9 days showed a smaller drop in both heart rate and rectal temperature by the third day. Blood sample measurements taken 1 hr after injection during the 9 day period suggested that the decreased pharmacological responses were related to lower blood concentrations of the drug. Metronidazofe, Hypoxia, Pharmacology.
INTRODUCTION Radiobiology has firmly established that mammalian cells are more sensitive to ionizing radiation in the presence of oxygen.‘5*‘7,‘9As a
tumor mass develops, it tends to outgrow its blood supply, and cells at a distance from the vessels become hypoxic and thus radioresistant. The existence of such cells has been demonstrated in several animal tumors;22*29.33 it has been suggested that some human tumors also contain hypoxic areas which may be a major cause of tumor recurrence following radiotherapy.‘3+“2 One of the methods being employed to combat the problem of radio-resistant, hypoxic cells has been the use of chemicals which selectively sensitize hypoxic cells to radiation. Many of these chemicals are electron affinic, as .is oxygen, and were first recognized as sensitizers by Adams and Dewey’ who demonstrated an increased sentSupported
by: The Ontario Cancer Treatment
sitivity to X-rays in serratia marcescens when irradiated in the presence of N-ethylmalemide, benzophenone or diacetyl. Many of the electron affinic sensitizers studied to date are nitroheterocyclic compounds including some nitrofurans, nitrobenzenes and nitroimidazoles. Although many of these drugs are effective in mammalian cell cultures,2*3.9.2’they must satisfy at least the following four requirements to be effective in animal tumor systems: they must sensitize at concentrations which are not toxic to the animal; they must be able to diffuse from the relatively poor blood supply of the tumor into the hypoxic areas; they must not be metabolized or excreted rapidly; and they must sensitize hypoxic cells selectively so as not to increase radiation damage to normal tissues. The most successful class of the electron affinic sensitizers tested to date in uiuo are the nitroimidazoles. Several investigators have and Research
1125
Foundation.
1126
Radiation Oncology 0 Biology ??Physics
demonstrated radiosensitization of animal tumors with metronidazole. tinidazole and the Roche chemical 07-0582.5~8~‘6~23~27 One of the advantages of the nitroimidazoles over other sensitizers is their relatively favorable toxicity in animals. Asquith et a1.4 have reported a lethal dose for 50% in mice of between 4 and 5 mg/g of body weight for metronidazole which is well above the amount of drug required to sensitize tumors. Metronidazole is used clinically at low doses in the treatment of the vaginal protozoa1 infection, trichomoniasis.” Little is known, however, about the pharmacological effects of high dose metronidazole although it has been reported not to affect the cardiovascular system in animals.*’ Evidence taken from measurements of the effect of metronidazole on heart rate and rectal temperature in C3HIHeJ mice will be presented that clearly disputes this claim. It will be shown that the heart rate and temperature responses to the drug change with time in animals who are maintained on multiple doses of it for several days. Measurements of blood levels of metronidazole from animals who are maintained on the same multiple dose regime support the pharmacological findings. The implications of these results to the clinical potential of metronidazole will be discussed in view of conventional radiotherapy schedules. METHODS AND MATERIALS Female C3HIHeJt mice, age 6-10 weeks and weighing between 20 and 25 g, were used in all experiments. The number of animals used in each experiment is indicated in brackets on the corresponding figure. The plasma half-life of metronidazole was determined using a spectrophotometric assay. Metronidazole absorbs ultraviolet light maximally at 320 nm. The drug was prepared in solution at a concentration of 20 mglml (117 mM) of bacteriostatic water containing 0.9% benzyl alcohol. The solution had to be heated to 40°C and agitated for 15 min to achieve this concentration; it was injected intraperitoneally at this temperature at a dose of 0.6 mglg of tobtained
from
Roscoe
B. Jackson
Laboratories,
November-December
1976, Vol. I. No. 11 and No. 12
body weight (0.6 ml for a 20g mouse) into recipient mice. Blood samples were taken from the tails of the animals at 15, 30, 60, 120. 180 and 300 min after injection. Each animal was used for three different times since additional samples could not be taken reliably from one mouse. Control samples, used as blanks in the spectrophotometer, were taken from each mouse prior to drug injection. Blood was drawn in heparinized hematocrit tubes which were then sealed and centrifuged to eliminate cellular elements. Known volumes of plasma were diluted with phosphate buffered saline. Absorbances were read on an ultraviolet spectrophotometer and plasma concentration determined from a calibration of concentration versus absorbance. The heart rate and rectal temperature measurements were made with the animals immobilized on perspex rods with elastoplast tape. The apparatus was contained within an electrically shielded cage and the two parameters measured were recorded on a fast time response multichannel recorder. Three steel pin electrodes were inserted under the skin to measure heart rate, one in each front leg and one in the right hind leg. A cardiotachometer coupler in the recorder permitted display of both electrocardiogram and heart rate. Rectal temperature was measured with a glass covered thermistor inserted approximately 2 cm into the rectum. All animals received a light ether anesthetic to assist in the immobilization procedure and during the placing of electrodes. A polyethylene catheter was inserted into the peritoneal cavity also under ether anesthetic. It is important to note that the animals were not anesthetized while the responses to metronidazole were being recorded. The parameters were monitored continuously from 30 min before to 90 min after intraperitoneal injection of 0.6 mg/g of body weight of metronidazole; controls were given equivalent volumes of saline containing 0.9% benzyl alcohol. Again the drug was prepared in solution at 40°C so that it was injected at approximately normal body temperature. Injections were made through the polyethylene Bar Harbor,
Maine.
1127
Pharmacological aspects of metronidazole in mice 0 M, J. HAYNE~ andW.R. INCH
catheter so as not to disturb the animal or the positioning of the electrodes. Another group of animals received 0.6 mglg of metronidazole intraperitoneally twice a day at 12 hr intervals for a period of 9 days. The heart rate and temperature responses were measured in the usual manner four times throughout the 9 day period, after the first injection on days 0,3,7 and 9. Control animals were given equivalent volumes of saline. An additional group of animals maintained on the same schedule had blood samples taken 1 hr after injections of drug at the corresponding times over the 9 day period, that is 1 hr following the first injection on days 0, 3,7 and 9. This was done to determine whether changes seen in the heart rate and rectal temperature responses were related to the plasma concentration of metronidazole. RESULTS The peak concentration of metronidazole of roughly 4mM (Fig. 1) was reached within 15 min of intraperitoneal injection and the level was still high at 60 min. The concentration dropped off exponentially with a half-life of 1.7 hr..
An intrapertoneal injection of 0.6 mglg of metronidazole caused the C3H mouse to become generally unresponsive and lethargic within 5 min and remain so for approximately 10-15 min. Although the animals appeared normal again 30 min after injection, their heart rates and rectal temperatures still were falling at this time (Figs. 2 and 3). Heart rate was expressed as a percentage of its value before drug injection and the change in rectal temperature was shown as the drop in “C from the average preinjection value. The average heart rate and rectal temperature prior to the drug injection were 700 beats per minute (bpm) and 365°C. There was an initial rapid drop in heart rate (Fig. 2) during the first 5 min followed by a short plateau in response and
\
g
b
2 k 360 I
---o-
I
t
WINE (lo) METRONIMZOLE (IO) MEAN i SEM
\l
Ei N
a
B60
t
p OO
20
40
60 TIME
80
loo
(MINI
Fig. 2. Effect of 0.6 mg/g of metronidazole on heart rate of the C3H mouse. Time is relative to time of injection. Number of animals used in brackets.
-.-
SALINE (9) METRONIDAZOLE (9) MEAN t SEM
--o--
t,,= 1.70hours
TIME (HOURS)
c
0
Fig. 1. Determination of half-life of metronidazole in plasma of C3H/HeJ mice. Animals injected with a single dose of 0.6 mglg of metronidazole intraperitoneally.
20
40
60 TIME
60
100
(MINI
Fig. 3. Effect of 0.6mg/g of metronidazole rectal temperature of C3H mouse.
on
1128
Radiation Oncology 0 Biology 0 Physics
then a gradual decrease until levelling off at about 80 min after injection. By this time, the rate had dropped to between 65 and 70% of the value before administration of the drug. The saline controls showed a decrease of only 5% which levelled off after 15 min. The drop inrectal temperature (Fig. 3) did not show the initial rapid decline seen in the heart rate response but followed a smooth pattern resulting in a drop of almost 6°C by 90 min. The saline controls showed a decrease of about 0S”C which was constant 15 min after injection. Animals maintained on 1.2 mg/g/day of metronidazole given in two equal fractions for 9 days appeared to build up a tolerance to the drug (Fig. 4). Although the same qualitative pattern was seen in both the heart rate and temperature responses over the 9 days, there was a decrease in the magnitude of the response by the third day. The maximum change in heart rate and temperature was plotted for the 90 min period during which the responses were measured, on days 0.3, 7 and 9 (Fig. 4). There was no significant change in
November-December
1976, Vol. 1, No. I1 and No. 12
the responses after day 3. Plasma level determinations (Fig. 5) at 1 hr after injection on another group of animals who were maintained on the same schedule suggested that the decreased responses are related to the drug concentration in the blood. Although the day 3 concentration of drug does not differ significantly from the day 0 concentration, both day 7 and 9 values were different from day 0, p < 0.05. Again, however, there was essentially no change in the values from day 3 on.
01
0
2
4 TIME
A. HEART RATE
6
8
IO
(DAYS)
Fig. 5. Plasma levels of metronidazole 1 hr after injection of 0.6 mg/g. Levels measured on days 0,3, 7 and 9. DISCUSSION
TIME (DlrvsI
8. RECTAL TEMPERATURE
-6.0
.
-.-
!3ALINE
r-o--
METRONICMOLE 1
MEAN
(5) (6)
f SEM
Fig. 4. Maximum effect of metronidazole observed within 90 min of injection on heart rate and rectal temperature of C3H mouse over a 9 day period. Measurements were taken on days 0,3,7 and 9.
One of the advantages of metronidazole in the mouse over other hypoxic cell radiosensitizers is its relatively long biological half-life. Many other drugs which were effective sensitizers in vitro have been unsuccessful in uivb because they are eliminated rapidly’0.23 and thus not present in sufficient concentrations at the time of irradiation. In addition to being present in the cell, the effective sensitizer must be in its original state, that is, not bound to proteins or metabolized, unless the altered forms are capable of sensitizing to the same extent. Taylor et ~1.~’have found that approximately 20% of the metronidazole in the blood of humans was bound to plasma proteins. Plasma level determinations based upon ultraviolet absorption depend on the NO2 group in the 5 position of the ring structure of metronidazole.“’ This group remains intact in the breakdown products of the
Pharmacological aspects of metronidazole in mice0 M. J. HAYNES and W. R. INCH
drug. 1*18Therefore, what is actually measured by the assay is the total plasma content of the drug in both its original and metabolized forms. However, this may not result in a loss of radiosensitization as this property is associated with the compound’s electron affinity which is conferred by the NO2 group. It is possible that the metabolites may be equally potent as sensitizers and more or less toxic to the animal. On the basis of the present data, it would appear that the time at which animals should be irradiated following injection of metronidazole is between 15 and 60 min, assuming that the tumor levels closely follow those in the blood. In vivo studies with metronidazole generally suggest that 30 min is probably the minimum time that should be chosen between drug injection and irradiation to allow for distribution and diffusion into tumor tissue. “JO’~ In addition, the results of Sutherland and Richardsot?’ in the spheroid model tumor system indicate that metronidazole is more effective at intervals longer than 30 min. Mice, in general, have poor temperature regulation mainly resulting from their large surface to volume ratio;6 this results in considerable heat loss from convection and radiation. Therefore, it is necessary for the mouse to have a high metabolic rate to produce the much needed heat. Any reduction in metabolism would contribute to a lowering of body temperature. Metronidazole inhibits cellular oxygen utilization7.‘2 which could lead to less energy and heat production. Heart rate and cardiac output are coupled strongly to metabolic rate; thus it is not surprising that the drops in heart rate and rectal temperature are fairly well correlated. Richards et ~1.‘~ have found that rectal temperature in the mouse dropped from 37°C to 23°C because of exposure to cold with a corresponding drop in heart rate from 615 to 240 bpm. We have also shown in this laboratory that the same dose, 0.6 mglg, of metronidazole causes an increase in oxygenation of the C3HBA tumor by a factor of 2.5 times as measured polarographically with a platinum microelectrode.’ This may result from a peripheral vasodilating effect of metronidazole which also would result in in-
1129
creased heat loss and hence a fall in core temperature. The initial sharp decline in heart rate may be caused by vagal stimulation resulting from irritation of the peritoneum by metronidazole or from a direct effect of the drug on the peritoneum with a resulting drop in heart rate. In an attempt to determine whether vagal responses were involved, 6 animals were injected with 0.25 mg/kg of atropine 20 min prior to the injection of 0.6 mg/g metronidazole. Atropine acts as an antagonist to acetylcholine and other muscarinic agents and thus would be expected to block any reduction in heart rate resulting from increased parasympathetic tone via the vagus nerve. It did not block the initial drop in heart rate resulting from metronidazole although on the average the drop was slightly less in the animals that received the atropine. However, the normal response to atropine alone (a characteristic tachycardia) was not observed. Therefore, these experiments did not establish clearly whether or not vagal stimulation is involved in the initial rapid drop in heart rate. The decrease observed from day 3 to day 9, in both physiological responses and plasma concentration of metronidazole 1 hr after injection, probably resulted from either faster breakdown because of stimulation of enzymes or faster excretion of the drug. The results of McCalla et ~1.~ confirm the presence of enzymes in mammalian tissues which reduce various nitrofurans to compounds that react with cellular constituents. Their work suggests that these compounds are responsible for the carcinogenic properties of the nitrofurans. If similar enzymes are present which reduce metronidazole, then one would expect that lower levels of the drug would be found since the spectrophotometric assay depends on the NO2 group which would be reduced. This assumes that a certain period of time is required for enzyme induction. It is also conceivable that reduced forms of metronidazole could be responsible for its reported carcinogenic activity.26 Since conventional radiotherapy is given in many fractions over a period of weeks, a lowering of the sensitizer level resulting from the’induction of reducing enzymes might be expected and
1130
Radiation Oncology 0 Biology 0 Physics
could hinder seriously the clinical potential of the drug. It is generally felt that the levels that can be obtained in man (because of problems of nausea and vomiting) may be a limiting factor in its success.4.‘.34 Direct experiments to determine the presence of reducing enzymes have not been reported yet. However, the preliminary results by Urtasun et af.34 in humans do not indicate a reduction in blood levels of metronidazole with successive daily
1.
November-December
1976, Vol. I. No. 11and No. 12
doses but rather a slight increase. This probably results from the much longer halflife of the drug in man (approximately 12 hr), so that appreciable concentrations may have been present from previous drug doses. Fewer larger fractions of radiation may be required to increase tumor cure by radiosensitizers. Such a change in fractionation would also help to overcome the problem of reduced levels following multiple doses.
REFERENCES Adams, G.E., Dewey, D.L.: Hydrated electrons Hypoxic radiosensitizers and
radiobiological
Biochem. It: 473477, 1%3. J.C., Dewey, D.L.,
sensitization.
Biophys. Res. Commun. 2. Adams, G.E., Asquith,
Foster, J.L., Michael, B.D., Willson, R.L.: Electron affinic sensitization: II. Paranitroacetophenone, a radiosensitizer for anoxic bacterial and mammalian cells. Int. J. Radiat. Biol. 19: 575-585, 1971. 3. Ashwood-Smith, M.J., Robinson, D.M., Barnes, J.H., Bridges, B.A.: Radiosensitization of bacterial and mammalian cells by substituted glyoxals. Nature 216: 137-139, 1%7. 4. Asquith, J.C., Foster, J.L., Willson, R.L., Ings, R., McFadzean, J.A.: Metronidazole (Flagyl). A radiosensitizer of hypoxic cells. Br. J. Radiol. 47: 474-481, 1974. 5. Begg, A.C., Sheldon, P.W.,
Foster, J.L.: Demonstration of hypoxic cells in solid tumors by metronidazole. Br. J. Radiol. 47: 399-404, 1974.
6. Bernstein,
S.E.: Physiological characteristics. In Biology of the Laboratory Mouse. 2nd. Bdn, ed. by Green, E.L. Toronto, McGraw-Hill, 1966, pp. 337-350. 7. Biaglow, J.E., Nygaard, O.F., Greenstock, C.L.: Redox reactions of anoxic radiosensitizers (Abstr). Radiat. Res. 59: 158, 1974. 8. Brown, J.M.: Selective radiosensitization of the hypoxic cells of mouse tumors with the nitroimidazoles metronidazole and Ro 07-0582. Radiat. Res. 64: 633-647,
1975.
9. Chapman,
J.D., Reuvers, A.P., Borsa, J., Petkau, A., McCalla, D.R.: Nitrofurans as radiosensitizers of hypoxic mammalian cells. Cancer Res. 32: 2616-2624, 1972. 10. Chapman, J.D., Reuvers, A.P., Borsa, J., Henderson, J.S., Migliore, R.D.: Nitroheterocyclic drugs as selective radiosensitizers of hypoxic mammalian cells. Cancer Chemother.
Rep. 1.58: 559-570,
1974.
11. Cosar,
C., Julou, L.: Activity of 1 - (2’ hydroxyethyl) - 2 - methyl - 5 - nitroimidazole in experimental trichomonas vaginalis infections. Ann. Inst. Pasteur. 96: 238-245,
1959.
12. Durand, R.E., Biaglow, J.E., Sutherland.
R.M.:
tion (Letter to Editor).
blood
and cellular respiraBr. J. Radiol. 49, 568-
569, 1976.
13. Evans, N.T.S., Naylor, B.F.D.: The effect of O2 breathing and radiotherapy upon the tissue oxygen tension of some human tumors. Br. .I. Radiol. 36: 41&-425, 1%3.
14. Gray, L.H., Conger, A.D., Ebert, M., Hornsey, S., Scott, O.C.A.: The concentration of O2 dissolved in tissues at the time of irradiation as a factor in radiotherapy. Br. J. Radiol. 26: 638-648,
1953.
15. Gray, L.H.: Radiobiologic basis of oxygen as a modifying factor in radiation therapy. Am. J, Roentgenol. 85: 803-815, 1961. 16. Haynes, M.J., Inch, W.R.: Effect of metronidazole on the radiocurability and oxygen tension of the C3HBA tumor. Radiat. Res. in press. 17. Hewitt, H.B., Wilson, C.W.: Survival curves for tumor cells irradiated in vivo. Ann. NY Acad. Sci. 95: 818-827, 1961. 18. Ings, R.M.J., Law, C.L., Parnell, E-W.: The metabolism of metronidazole. Biochem. Pharmacol. 15: 515-519, 1966. 19. Mottram, J.C.: Factor of importance in radiosensitivity of tumors. Br. J. Radiol. 9: 606-614, 1936. 20. McCalla, D.R., Reuvers, A., Kaiser, C.: Breakage of bacterial DNA by nitrofuran derivatives. Cancer Res. 31: 2184-2188, 1971. 21. Parker, L., Skarsgard, L., Emmerson, P.T.: Sensitization of anoxic mammalian cells to X-rays by TAN. Survival and toxicity studies. Radiat. Res. 38: 493-500,
1968.
22. Powers, W.E., Tolmach, L.J.: Demonstration of an anoxic component in a mouse tumor-cell population by in vivo assay of survival following irradiation. Radiology 83: 328-336, 19%
23. Rauth, A.M., Kaufmann, K.: In vivo testing of hypoxic radiosensitizers using the KHT murine tumor assayed by the lung-colony technique. Br. J. Radiol. 48: 209-220,
1975.
24. Richards, A.C., Simonson, E., Visscher, M.B.: Electrocardiogram and phonogram of adult and
Pharmacological
25.
26.
27. 28.
29.
aspects of metroniclazole in mice ?? M. J. HAYNESand W. R. INCH
newborn mice in normal conditions and under the effect of cooling, hypoxia and potassium. Am. J. Physiol. 174: 293-298, 1953. Rollo, I.M.: Miscellaneous drugs used in the treatment of protozoa1 infections. In The Pharmacological Basis of Therapeutics, 3rd Edn. ed. by Goodman, L.S., Gilman, A. Toronto, Collier-Macmillan, 1%5, pp. 11351143. Rustia, M., Shubik, P.: Induction of lung tumors and malignant lymphomas in mice by metronidazole. J. Nat1 Cancer Inst. 48: 721729, 1972. Stone, H., Withers, H.R.: Tumor and normal tissue response to metronidazole and irradiation in mice. Radiology 113: 441-444, 1974. Stambaugh, J.E., Feo, L.C., Manthei, R.W.: Isolation and identification of the major urinary metabolites of metronidazole. Life Sci. 6: 1811-1819, 1967. Suit, H-D., Maeda, M.: Hyperbaric oxygen and radiobiology of a C3H mouse mammary carcinoma. J. Nat1 Cancer Inst. 39: 639-650,1%7.
1131
30. Sutherland, R.M., Richardson, J.: Radiosensitization of chronically hypoxic cells in multicellular spheroids (Abstr). Radial. Res. 59: 159, 1974 (appears on one page only). 31. Taylor, J.A.. Migliardi, J.R., Schach von Wittenau, M.: Tinidazole and metronidazole pharmacokinetics in man and mouse. Antimic rob. Agents Chemother. 267-270, 1%9. 32. 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. 33. Thomlinson, R.H., Craddock, E.A.: The gross response of an experimental animal tumor to single doses of X-rays. Br. J. Cancer 21: 108-123, 1%7. 34. Urtasun, R.C., Sturmwind, J., Rabin, H., Band, P.R., Chapman, J.D.: “High-Dose” metronidazole: A preliminary pharmacological study prior to its investigational use in clinical radiotherapy trials. Br. J. Radiol. 47: 297-299, 1974.
Phys.,
1976, Vol.
I, pp. 1125-l 131.
Perg’dmon Press.
Printed
in the U3.A
SOME PHARMACOLOGICAL ASPECTS OF MULTIPLE DOSE METRONIDAZOLE IN C3H/HeJ MICE? M. J. HAYNES, M.Sc Department
of Biophysics,
The University of Western SC 1, Canada
Ontario,
London,
Ontario
N6A
and W. R. INCH, Ph.D. Ontario Cancer Experimental
Treatment Oncology,
and Research Foundation, London Clinic, Division Victoria Hospital, London, Ontario N6A 4G5, Canada
of
Metronidazole is one of several electron affinic chemicals currently being tested as a sensitizer of radio-resistant hypoxic cells. Little is known, however, about its pharmacological effects in man or experimental animals. The half-life of metroufdazole in the C3H mouse was found to be 1.7 hr following a single intraperitoneal injection of 0.6 mg/g body weight. The same dose caused a decrease in heart rate of approximately 35% and rectal temperature fell by almost 6°C. Anhnals maintained on twice daffy doses of 0.6 mg/g for 9 days showed a smaller drop in both heart rate and rectal temperature by the third day. Blood sample measurements taken 1 hr after injection during the 9 day period suggested that the decreased pharmacological responses were related to lower blood concentrations of the drug. Metronidazofe, Hypoxia, Pharmacology.
INTRODUCTION Radiobiology has firmly established that mammalian cells are more sensitive to ionizing radiation in the presence of oxygen.‘5*‘7,‘9As a
tumor mass develops, it tends to outgrow its blood supply, and cells at a distance from the vessels become hypoxic and thus radioresistant. The existence of such cells has been demonstrated in several animal tumors;22*29.33 it has been suggested that some human tumors also contain hypoxic areas which may be a major cause of tumor recurrence following radiotherapy.‘3+“2 One of the methods being employed to combat the problem of radio-resistant, hypoxic cells has been the use of chemicals which selectively sensitize hypoxic cells to radiation. Many of these chemicals are electron affinic, as .is oxygen, and were first recognized as sensitizers by Adams and Dewey’ who demonstrated an increased sentSupported
by: The Ontario Cancer Treatment
sitivity to X-rays in serratia marcescens when irradiated in the presence of N-ethylmalemide, benzophenone or diacetyl. Many of the electron affinic sensitizers studied to date are nitroheterocyclic compounds including some nitrofurans, nitrobenzenes and nitroimidazoles. Although many of these drugs are effective in mammalian cell cultures,2*3.9.2’they must satisfy at least the following four requirements to be effective in animal tumor systems: they must sensitize at concentrations which are not toxic to the animal; they must be able to diffuse from the relatively poor blood supply of the tumor into the hypoxic areas; they must not be metabolized or excreted rapidly; and they must sensitize hypoxic cells selectively so as not to increase radiation damage to normal tissues. The most successful class of the electron affinic sensitizers tested to date in uiuo are the nitroimidazoles. Several investigators have and Research
1125
Foundation.
1126
Radiation Oncology 0 Biology ??Physics
demonstrated radiosensitization of animal tumors with metronidazole. tinidazole and the Roche chemical 07-0582.5~8~‘6~23~27 One of the advantages of the nitroimidazoles over other sensitizers is their relatively favorable toxicity in animals. Asquith et a1.4 have reported a lethal dose for 50% in mice of between 4 and 5 mg/g of body weight for metronidazole which is well above the amount of drug required to sensitize tumors. Metronidazole is used clinically at low doses in the treatment of the vaginal protozoa1 infection, trichomoniasis.” Little is known, however, about the pharmacological effects of high dose metronidazole although it has been reported not to affect the cardiovascular system in animals.*’ Evidence taken from measurements of the effect of metronidazole on heart rate and rectal temperature in C3HIHeJ mice will be presented that clearly disputes this claim. It will be shown that the heart rate and temperature responses to the drug change with time in animals who are maintained on multiple doses of it for several days. Measurements of blood levels of metronidazole from animals who are maintained on the same multiple dose regime support the pharmacological findings. The implications of these results to the clinical potential of metronidazole will be discussed in view of conventional radiotherapy schedules. METHODS AND MATERIALS Female C3HIHeJt mice, age 6-10 weeks and weighing between 20 and 25 g, were used in all experiments. The number of animals used in each experiment is indicated in brackets on the corresponding figure. The plasma half-life of metronidazole was determined using a spectrophotometric assay. Metronidazole absorbs ultraviolet light maximally at 320 nm. The drug was prepared in solution at a concentration of 20 mglml (117 mM) of bacteriostatic water containing 0.9% benzyl alcohol. The solution had to be heated to 40°C and agitated for 15 min to achieve this concentration; it was injected intraperitoneally at this temperature at a dose of 0.6 mglg of tobtained
from
Roscoe
B. Jackson
Laboratories,
November-December
1976, Vol. I. No. 11 and No. 12
body weight (0.6 ml for a 20g mouse) into recipient mice. Blood samples were taken from the tails of the animals at 15, 30, 60, 120. 180 and 300 min after injection. Each animal was used for three different times since additional samples could not be taken reliably from one mouse. Control samples, used as blanks in the spectrophotometer, were taken from each mouse prior to drug injection. Blood was drawn in heparinized hematocrit tubes which were then sealed and centrifuged to eliminate cellular elements. Known volumes of plasma were diluted with phosphate buffered saline. Absorbances were read on an ultraviolet spectrophotometer and plasma concentration determined from a calibration of concentration versus absorbance. The heart rate and rectal temperature measurements were made with the animals immobilized on perspex rods with elastoplast tape. The apparatus was contained within an electrically shielded cage and the two parameters measured were recorded on a fast time response multichannel recorder. Three steel pin electrodes were inserted under the skin to measure heart rate, one in each front leg and one in the right hind leg. A cardiotachometer coupler in the recorder permitted display of both electrocardiogram and heart rate. Rectal temperature was measured with a glass covered thermistor inserted approximately 2 cm into the rectum. All animals received a light ether anesthetic to assist in the immobilization procedure and during the placing of electrodes. A polyethylene catheter was inserted into the peritoneal cavity also under ether anesthetic. It is important to note that the animals were not anesthetized while the responses to metronidazole were being recorded. The parameters were monitored continuously from 30 min before to 90 min after intraperitoneal injection of 0.6 mg/g of body weight of metronidazole; controls were given equivalent volumes of saline containing 0.9% benzyl alcohol. Again the drug was prepared in solution at 40°C so that it was injected at approximately normal body temperature. Injections were made through the polyethylene Bar Harbor,
Maine.
1127
Pharmacological aspects of metronidazole in mice 0 M, J. HAYNE~ andW.R. INCH
catheter so as not to disturb the animal or the positioning of the electrodes. Another group of animals received 0.6 mglg of metronidazole intraperitoneally twice a day at 12 hr intervals for a period of 9 days. The heart rate and temperature responses were measured in the usual manner four times throughout the 9 day period, after the first injection on days 0,3,7 and 9. Control animals were given equivalent volumes of saline. An additional group of animals maintained on the same schedule had blood samples taken 1 hr after injections of drug at the corresponding times over the 9 day period, that is 1 hr following the first injection on days 0, 3,7 and 9. This was done to determine whether changes seen in the heart rate and rectal temperature responses were related to the plasma concentration of metronidazole. RESULTS The peak concentration of metronidazole of roughly 4mM (Fig. 1) was reached within 15 min of intraperitoneal injection and the level was still high at 60 min. The concentration dropped off exponentially with a half-life of 1.7 hr..
An intrapertoneal injection of 0.6 mglg of metronidazole caused the C3H mouse to become generally unresponsive and lethargic within 5 min and remain so for approximately 10-15 min. Although the animals appeared normal again 30 min after injection, their heart rates and rectal temperatures still were falling at this time (Figs. 2 and 3). Heart rate was expressed as a percentage of its value before drug injection and the change in rectal temperature was shown as the drop in “C from the average preinjection value. The average heart rate and rectal temperature prior to the drug injection were 700 beats per minute (bpm) and 365°C. There was an initial rapid drop in heart rate (Fig. 2) during the first 5 min followed by a short plateau in response and
\
g
b
2 k 360 I
---o-
I
t
WINE (lo) METRONIMZOLE (IO) MEAN i SEM
\l
Ei N
a
B60
t
p OO
20
40
60 TIME
80
loo
(MINI
Fig. 2. Effect of 0.6 mg/g of metronidazole on heart rate of the C3H mouse. Time is relative to time of injection. Number of animals used in brackets.
-.-
SALINE (9) METRONIDAZOLE (9) MEAN t SEM
--o--
t,,= 1.70hours
TIME (HOURS)
c
0
Fig. 1. Determination of half-life of metronidazole in plasma of C3H/HeJ mice. Animals injected with a single dose of 0.6 mglg of metronidazole intraperitoneally.
20
40
60 TIME
60
100
(MINI
Fig. 3. Effect of 0.6mg/g of metronidazole rectal temperature of C3H mouse.
on
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Radiation Oncology 0 Biology 0 Physics
then a gradual decrease until levelling off at about 80 min after injection. By this time, the rate had dropped to between 65 and 70% of the value before administration of the drug. The saline controls showed a decrease of only 5% which levelled off after 15 min. The drop inrectal temperature (Fig. 3) did not show the initial rapid decline seen in the heart rate response but followed a smooth pattern resulting in a drop of almost 6°C by 90 min. The saline controls showed a decrease of about 0S”C which was constant 15 min after injection. Animals maintained on 1.2 mg/g/day of metronidazole given in two equal fractions for 9 days appeared to build up a tolerance to the drug (Fig. 4). Although the same qualitative pattern was seen in both the heart rate and temperature responses over the 9 days, there was a decrease in the magnitude of the response by the third day. The maximum change in heart rate and temperature was plotted for the 90 min period during which the responses were measured, on days 0.3, 7 and 9 (Fig. 4). There was no significant change in
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1976, Vol. 1, No. I1 and No. 12
the responses after day 3. Plasma level determinations (Fig. 5) at 1 hr after injection on another group of animals who were maintained on the same schedule suggested that the decreased responses are related to the drug concentration in the blood. Although the day 3 concentration of drug does not differ significantly from the day 0 concentration, both day 7 and 9 values were different from day 0, p < 0.05. Again, however, there was essentially no change in the values from day 3 on.
01
0
2
4 TIME
A. HEART RATE
6
8
IO
(DAYS)
Fig. 5. Plasma levels of metronidazole 1 hr after injection of 0.6 mg/g. Levels measured on days 0,3, 7 and 9. DISCUSSION
TIME (DlrvsI
8. RECTAL TEMPERATURE
-6.0
.
-.-
!3ALINE
r-o--
METRONICMOLE 1
MEAN
(5) (6)
f SEM
Fig. 4. Maximum effect of metronidazole observed within 90 min of injection on heart rate and rectal temperature of C3H mouse over a 9 day period. Measurements were taken on days 0,3,7 and 9.
One of the advantages of metronidazole in the mouse over other hypoxic cell radiosensitizers is its relatively long biological half-life. Many other drugs which were effective sensitizers in vitro have been unsuccessful in uivb because they are eliminated rapidly’0.23 and thus not present in sufficient concentrations at the time of irradiation. In addition to being present in the cell, the effective sensitizer must be in its original state, that is, not bound to proteins or metabolized, unless the altered forms are capable of sensitizing to the same extent. Taylor et ~1.~’have found that approximately 20% of the metronidazole in the blood of humans was bound to plasma proteins. Plasma level determinations based upon ultraviolet absorption depend on the NO2 group in the 5 position of the ring structure of metronidazole.“’ This group remains intact in the breakdown products of the
Pharmacological aspects of metronidazole in mice0 M. J. HAYNES and W. R. INCH
drug. 1*18Therefore, what is actually measured by the assay is the total plasma content of the drug in both its original and metabolized forms. However, this may not result in a loss of radiosensitization as this property is associated with the compound’s electron affinity which is conferred by the NO2 group. It is possible that the metabolites may be equally potent as sensitizers and more or less toxic to the animal. On the basis of the present data, it would appear that the time at which animals should be irradiated following injection of metronidazole is between 15 and 60 min, assuming that the tumor levels closely follow those in the blood. In vivo studies with metronidazole generally suggest that 30 min is probably the minimum time that should be chosen between drug injection and irradiation to allow for distribution and diffusion into tumor tissue. “JO’~ In addition, the results of Sutherland and Richardsot?’ in the spheroid model tumor system indicate that metronidazole is more effective at intervals longer than 30 min. Mice, in general, have poor temperature regulation mainly resulting from their large surface to volume ratio;6 this results in considerable heat loss from convection and radiation. Therefore, it is necessary for the mouse to have a high metabolic rate to produce the much needed heat. Any reduction in metabolism would contribute to a lowering of body temperature. Metronidazole inhibits cellular oxygen utilization7.‘2 which could lead to less energy and heat production. Heart rate and cardiac output are coupled strongly to metabolic rate; thus it is not surprising that the drops in heart rate and rectal temperature are fairly well correlated. Richards et ~1.‘~ have found that rectal temperature in the mouse dropped from 37°C to 23°C because of exposure to cold with a corresponding drop in heart rate from 615 to 240 bpm. We have also shown in this laboratory that the same dose, 0.6 mglg, of metronidazole causes an increase in oxygenation of the C3HBA tumor by a factor of 2.5 times as measured polarographically with a platinum microelectrode.’ This may result from a peripheral vasodilating effect of metronidazole which also would result in in-
1129
creased heat loss and hence a fall in core temperature. The initial sharp decline in heart rate may be caused by vagal stimulation resulting from irritation of the peritoneum by metronidazole or from a direct effect of the drug on the peritoneum with a resulting drop in heart rate. In an attempt to determine whether vagal responses were involved, 6 animals were injected with 0.25 mg/kg of atropine 20 min prior to the injection of 0.6 mg/g metronidazole. Atropine acts as an antagonist to acetylcholine and other muscarinic agents and thus would be expected to block any reduction in heart rate resulting from increased parasympathetic tone via the vagus nerve. It did not block the initial drop in heart rate resulting from metronidazole although on the average the drop was slightly less in the animals that received the atropine. However, the normal response to atropine alone (a characteristic tachycardia) was not observed. Therefore, these experiments did not establish clearly whether or not vagal stimulation is involved in the initial rapid drop in heart rate. The decrease observed from day 3 to day 9, in both physiological responses and plasma concentration of metronidazole 1 hr after injection, probably resulted from either faster breakdown because of stimulation of enzymes or faster excretion of the drug. The results of McCalla et ~1.~ confirm the presence of enzymes in mammalian tissues which reduce various nitrofurans to compounds that react with cellular constituents. Their work suggests that these compounds are responsible for the carcinogenic properties of the nitrofurans. If similar enzymes are present which reduce metronidazole, then one would expect that lower levels of the drug would be found since the spectrophotometric assay depends on the NO2 group which would be reduced. This assumes that a certain period of time is required for enzyme induction. It is also conceivable that reduced forms of metronidazole could be responsible for its reported carcinogenic activity.26 Since conventional radiotherapy is given in many fractions over a period of weeks, a lowering of the sensitizer level resulting from the’induction of reducing enzymes might be expected and
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Radiation Oncology 0 Biology 0 Physics
could hinder seriously the clinical potential of the drug. It is generally felt that the levels that can be obtained in man (because of problems of nausea and vomiting) may be a limiting factor in its success.4.‘.34 Direct experiments to determine the presence of reducing enzymes have not been reported yet. However, the preliminary results by Urtasun et af.34 in humans do not indicate a reduction in blood levels of metronidazole with successive daily
1.
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doses but rather a slight increase. This probably results from the much longer halflife of the drug in man (approximately 12 hr), so that appreciable concentrations may have been present from previous drug doses. Fewer larger fractions of radiation may be required to increase tumor cure by radiosensitizers. Such a change in fractionation would also help to overcome the problem of reduced levels following multiple doses.
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15. Gray, L.H.: Radiobiologic basis of oxygen as a modifying factor in radiation therapy. Am. J, Roentgenol. 85: 803-815, 1961. 16. Haynes, M.J., Inch, W.R.: Effect of metronidazole on the radiocurability and oxygen tension of the C3HBA tumor. Radiat. Res. in press. 17. Hewitt, H.B., Wilson, C.W.: Survival curves for tumor cells irradiated in vivo. Ann. NY Acad. Sci. 95: 818-827, 1961. 18. Ings, R.M.J., Law, C.L., Parnell, E-W.: The metabolism of metronidazole. Biochem. Pharmacol. 15: 515-519, 1966. 19. Mottram, J.C.: Factor of importance in radiosensitivity of tumors. Br. J. Radiol. 9: 606-614, 1936. 20. McCalla, D.R., Reuvers, A., Kaiser, C.: Breakage of bacterial DNA by nitrofuran derivatives. Cancer Res. 31: 2184-2188, 1971. 21. Parker, L., Skarsgard, L., Emmerson, P.T.: Sensitization of anoxic mammalian cells to X-rays by TAN. Survival and toxicity studies. Radiat. Res. 38: 493-500,
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newborn mice in normal conditions and under the effect of cooling, hypoxia and potassium. Am. J. Physiol. 174: 293-298, 1953. Rollo, I.M.: Miscellaneous drugs used in the treatment of protozoa1 infections. In The Pharmacological Basis of Therapeutics, 3rd Edn. ed. by Goodman, L.S., Gilman, A. Toronto, Collier-Macmillan, 1%5, pp. 11351143. Rustia, M., Shubik, P.: Induction of lung tumors and malignant lymphomas in mice by metronidazole. J. Nat1 Cancer Inst. 48: 721729, 1972. Stone, H., Withers, H.R.: Tumor and normal tissue response to metronidazole and irradiation in mice. Radiology 113: 441-444, 1974. Stambaugh, J.E., Feo, L.C., Manthei, R.W.: Isolation and identification of the major urinary metabolites of metronidazole. Life Sci. 6: 1811-1819, 1967. Suit, H-D., Maeda, M.: Hyperbaric oxygen and radiobiology of a C3H mouse mammary carcinoma. J. Nat1 Cancer Inst. 39: 639-650,1%7.
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