1992, The British Journal of Radiology, 65, 1025-1029
Calcium antagonists protect mice against lethal doses of ionizing radiation By G. L. Floersheim Department of Research, University Hospitals, Hebelstrasse 20, 4031 Basel, Switzerland (Received 28 October 1991 and in final form 8 June 1992, accepted 29 June 1992) Keywords: Radioprotection, Calcium antagonists, Diltiazem, Nifedipine, Nimodipine
Abstract. Currently available radioprotectors are poorly tolerated in man and the general use of aminothiol radioprotectors is compromised by their side-effects. In a search for less toxic radioprotective agents, diltiazem, a calcium antagonist with a benzothiazepine structure, was found to protect mice against a lethal (LD100) y radiation dose allowing survival of up to 93%. Dihydropyridine calcium antagonists such as nifedipine, nimodipine, isradipine and nitrendipine also provided radioprotection. Calcium antagonists might attenuate radiation-induced injury by inhibiting cellular calcium overload, subsequent to cell: membrane damage caused by radiation-generated free radicals. In view of their good tolerance, calcium antagonists may be applied safely in situations of radiation exposure, including radiotherapy and internal radionuclide contamination. These calcium antagonists may also be viewed in other contexts where free radicals are implicated in pathological processes.
The search for chemical agents which protect against the tissue damage caused by exposure to ionizing radiation has led to the identification of thiol (sulfhydryl) compounds with marked activity in experimental models (Sweeney, 1979). However, the use of the most promising agent, WR 2721 (S-2-(3-aminopropylamino)ethylphosphorothioic acid), has been limited by its poor clinical tolerance (Cairnie, 1983; Turrisi et al, 1983). In this study a different approach has been initiated by testing thiol substances not designed for radioprotective activity but known from their use in other contexts to have a favourable therapeutic index. This included diltiazem, a benzothiazepine whose use as a calcium antagonist in cardiovascular pharmacology is based on extensive clinical experience. Since diltiazem protected against radiation-induced damage, the effects of other calcium antagonists were also investigated. Materials and methods
Mice and irradiation The experiments were carried out using C3H mice of both sexes (Gl. Bomholtgard, Ry, Denmark) weighing 22-24 g and fed Nafag pellets and water ad libitum. Groups of mice were irradiated in a perforated plexiglass chamber from a 60Co source (Gammatron). The target distance was 80 cm. Dosimetry was performed with a strontium-calibrated ionization chamber. For the experiments with diltiazem, the results of which are illustrated in Fig. 1, female mice {n — 173) were irradiated with 10.5 Gy (dose rate ~ 0.15 Gy/min). A smaller scale experiment (n = 18) was also peformed with a dose of 8.5 Gy from a source delivering 0.9 Gy/min. The doses delivered by the two radiation sources corresponded to the respective LD100 values and resulted in the same mean survival times. Accordingly, they were assumed to be biologically isoeffective and Vol. 65, No. 779
100
Figure 1. Survival of female C3H mice irradiated with 10.5 Gy (dose rate ~ 0.15 Gy/min). A small number of the mice (6 treated with HOmg/kg diltiazem at — 15min, 6 treated with HOmg/kg diltiazem at +10 min and 6 controls) were irradiated with 8.5 Gy at a dose rate of 0.9 Gy/min. The mice were treated subcutaneously with diltiazem before or after irradiation. The doses of diltiazem were as follows: HOmg/kg (n = 29) at - 15 min (O); 55 mg/kg (n = 24) at - 1 5 min ( • ) ; 27.5 mg/kg (n = 12) at - 1 5 min (A); 110 mg/kg (« = 12) at + 10 min (V)- Controls (n = 114) were injected with 0.1 ml distilled water per 10 g body weight ( # ) .
results obtained with both radiation dose rates were pooled. The female mice used for the experiments whose results are shown in Fig. 2 received 8.5 Gy at a rate of 0.9 Gy/min. In the experiments with male mice, the results of which are illustrated in Fig. 3, the irradiation doses were 8.1 and 9.0 Gy at a rate of 0.9 Gy/min. The efficacy of a compound was measured by its ability to protect mice against a lethal dose of whole1025
G. L. Floersheim
(b) Figure 2. Groups of 12 female C3H mice irradiated with 8.5 Gy (dose rate 0.9 Gy/min). Their radiation LD 50 was ~ 7.75 Gy. The mice were injected intraperitoneally 30 min before irradiation. For a better detection of the relative contributions, the agents were applied at only half their optimal dose, (a) The mice received 1.5 mg/kg body weight nifedipine in 0.15 ml/10 g standard solvent ( • ) , 0.15 ml/10 g standard solvent containing 18% ethanol (A), or 0.15 ml/10 g of an 18% ethanol solution in distilled water, corresponding to 2295 mg/kg ethanol (V)Controls received 0.15 ml/10 g distilled water ( # ) . (b) The mice were injected with 2.0 mg/kg nimodipine in 0.1 ml/10 g standard solvent ( • ) ; 0.1 ml/10 g standard solvent containing 23.7% ethanol (A), or 0.1 ml/10 g of a 23.7% ethanol solution in distilled water corresponding to 2015 mg/kg ethanol (V)Controls recevied 0.1 ml/10 g distilled water ( # ) .
body 7 radiation, protection being identified by animal survival at 30 days. The agents were first tested at 50% of the LD50 and, in case of efficacy, at progressively halved doses. The figures for survival after the respective treatments are presented in the Figures. Since, in general, the mice succumbed at approximately day 10 after irradiation, their death can be attributed to bone marrow damage. However, in the experiments in which a supralethal radiation dose was applied, the results of which are depicted in Figure 3b, gut-related deaths may have occurred. 1026
Figure 3. Survival in groups of 12 male C3H mice irradiated with 8.1 Gy (a) or 9.0 Gy (b) at the dose rate 0.9 Gy/min. The radiation LD 50 in these mice was ~ 6.35 Gy (dose rate 0.9 Gy/min). The mice were injected intraperitoneally 30 min before irradiation with either nifedipine or nimodipine in their solvents or with ethanol at the dose corresponding to that present in the solvent. Treatments were as follows: Controls: 0.2 ml distilled water/10 g body weight ( # ) ; nifedipine, 3 mg/kg in 0.3 ml solvent/10 g ( • ) ; 0.3 ml/10 g of an 18% ethanol solution in distilled water corresponding to 4590 mg/kg ethanol (A); nimodipine, 4 mg/kg in 0.2 ml solvent/10 g ( • ) ; 0.2 ml/10 g of an 23.7% ethanol solution (4050 mg ethanol/kg) in distilled water (A)-
Drugs Diltiazem was provided in ampoules containing 25 mg diltiazem hydrochloride and 150 mg mannitol (Warner-Lambert). The content of the ampoules was dissolved in distilled water and 0.1 ml/10 g body weight was administered by the subcutaneous route to the mice. In control experiments, it was ascertained that mannitol alone did not exert any radioprotection. Nifedipine and nimodipine were available as commercial solutions (Bayer) and applied by the intraperitoneal route. The solvents for nifedipine and nimodipine contained 18% and 23.7% ethanol, respectively. In addition, the solvent included polyethylene glycol 400, The British Journal of Radiology, November 1992
Radioprotection by calcium antagonists
sodium citrate and citric acid (courtesy Prof. S. Kazda). Since standard solvent and ethanol alone, in the quantity contained in the solvent, afforded equivalent radioprotection (Fig. 2), it can be assumed that the components of the standard solvent other than ethanol did not contribute to the protective activity afforded by the standard solvent. It was ascertained in separate experiments that polyethylene glycol 400 alone had no radioprotective activity. Nitrendipine (Bayer) was administered as a suspension in distilled water (0.1 ml/10 g body weight) by the intraperitoneal route. Isradipine (Sandoz) was dissolved in olive oil and was also injected intraperitoneally (0.1 ml/10 g body weight). Olive oil alone did not protect. Verapamil (Knoll) was administered intraperitoneally in distilled water (0.1 ml/10 g body weight) and flunarizine (Janssen) was given orally as a suspension in distilled water (0.1 ml/10 g body weight). Statistical evaluation The statistical significance of the differences in survival rates, as compared to controls, was determined according to the two-tailed chi-square test. Differences in survival time were evaluated according to a two-tailed Mest. Results The survival at 30 days of female C3H mice irradiated over the whole body with y-rays at approximately LD100 was increased to 93% by HOmg/kg of subcutaneous diltiazem, a dose corresponding to 50% of the LD50 of the drug (Fig. 1). Survival with 55mg/kg and 27.5mg/kg of diltiazem was 58% and 17% respectively (Fig. 1). Significant survival (42%) was also obtained when diltiazem was administered lOmin after the completion of irradiation (Fig. 1). The protection produced by diltiazem occurred irrespective of the dose rate at which the radiation was given. A similar dose-dependent improvement in survival in lethally irradiated female C3H mice was achieved following the intraperitoneal administration of nifedipine and nimodipine. These agents were administered in combination with their standard solvents, which contained ethanol (18% for nifedipine and 23.7% for nimodipine). Ethanol has long been known to exert radioprotective activity (Paterson & Matthews, 1950). Therefore to avoid confounding the additive or synergistic effects of ethanol with those of nifedipine and nimodipine, it was necessary to distinguish between the relative protective contributions of solvent and calcium antagonist. Figures 2a and 2b present the results of experiments in which the efficacy of nifedipine and nimodipine (both at a dose corresponding to 25% of their LD50) are compared with those of their respective standard solvents, and with that of ethanol at an appropriate standard solvent concentration. The survival in lethally irradiated female C3H mice was 100% with 1.5mg/kg nifedipine, 61% with the standard solvent and 50% with ethanol (Fig. 2a). With 2.0 mg/kg nimodipine, 82% of the mice Vol. 65, No. 779
survived, but only 42% with the standard solvent and 50% with ethanol (Fig. 2b). The radioprotective effects afforded by standard solvents and ethanol per se were not significantly different. The differences in survival rate (chi-square test) between either nifedipine or nimodipine (each in standard solvents) and their respective solvents alone, were statistically significant (p < 0.001 and p < 0.01, respectively). Accordingly, after subtractive correction for the solvent effect, the residual radioprotection (39% with nifedipine and 40% with nimodipine) can be ascribed to the calcium antagonists. The intrinsic radioprotective potential of these calcium antagonists was confirmed in irradiation experiments on male C3H mice, which are more radiosensitive than female mice. For mice irradiated with 8.1 Gy (Fig. 3a), survival after treatment with 3 mg/kg nifedipine or 4 mg/kg nimodipine (i.e. at doses corresponding to 50% of their LD50) was 67% (p < 0.005) and 75% (p < 0.001), respectively, compared to survival on treatment with the corresponding doses of ethanol alone of 25% and 8%, respectively. For mice irradiated with the supralethal dose of 9.0 Gy (Fig. 3b), survival was 58% (p < 0.0025) with 3 mg/kg nifedipine and 55% (p < 0.0025) with 4 mg/kg nimodipine as compared with the corresponding doses of ethanol which afforded no survival advantage at 30 days, only a prolongation of the mean survival time from 6.6+ 1.6 days (controls) to 11.1+2.1 and 11.0±2.2 days (p < 0.01; Mest), respectively. The results obtained with other calcium antagonists are presented in Table I. Survival of 58% (p < 0.001) was achieved with isradipine applied at 27.5 mg/kg (n — 24) to female C3H mice irradiated with 8.5 Gy at a dose rate of 0.9 Gy/min. Nitrendipine (100 or 50 mg/kg) administered intraperitoneally as a suspension in distilled water to female C3H mice irradiated with 10.5 Gy (dose rate of 0.15 Gy/min) resulted in survival of 29% or 25%, respectively. No animal survival was produced by pretreatment with verapamil or flunarizine. Discussion These experiments demonstrate that some calcium antagonists—particularly diltiazem, but also nifedipine and nimodipine—are effective radioprotectors. One explanation for the observed effect could be related to the radiation-induced lipid peroxidation of cell membrane structures by oxygen-derived free radicals with ionic leakage through cellular membranes and excessive calcium influx with ensuing cellular dysfunction and death from Ca 2+ overload. In reperfusion injury, another situation involving peroxidative membrane injury by free radicals (Ip & Levin, 1988; Janero et al, 1988; Bolli et al, 1989; Opie, 1989; Nayler et al, 1990; Watts et al, 1990), Ca 2+ channel blockers have also been shown to be protective. On the other hand, antioxidative effects by calcium antagonists such as nifedipine (Shridi & Robak, 1988; Ondrias et al, 1989) and diltiazem (Koller & Bergmann, 1989) have been reported and their radioprotective activity could also be linked more generally to the 1027
G. L. Floersheim Table I. Survival (30 days) of lethally irradiated female C3H mice pretreated with various calcium antagonists Treatment Controls (0.1 ml aq. dest./lO g) Controls (0.1 ml olive oil/10 g Isradipine Isradipine Nitrendipine Nitrendipine Verapamil Flunarizine
Dose (mg/kg)
Route of application
Time (min)
Survival0
%
27.5 27.5 100 50 16 25
i.p. i.p. i.p. i.p. i.p. i.p. i.p. p.o.
-30 -30 -30 -120 -30 -30 -30 -30
0/30 0/6 14/24 5/18 7/24 3/12 0/12 0/12
0 0 58 28 29 25 0 0
< 0.001 < 0.01 < 0.005 < 0.005 n.s. n.s.
"No. of surviving mice/total no. of mice. *Chi-square test, compared to the relative controls, n.s. = Not significant.
scavenging of free radicals (Janero et al, 1988). Thiol radioprotectors are believed to act principally by neutralizing radiation-induced free radicals via the formation of disulphides. Therefore, the particular efficacy of diltiazem may be explained by the fact that this molecule is capable of functioning not only as a calcium antagonist, but also as a thiol. No class dependency can be observed, since both a benzothiazepine (diltiazem) and dihydropyridine (nifedipine and nimodipine) calcium antagonists were active. To the latter class belong also the moderately effective compounds nitrendipine and isradipine (Table I). Variations in efficacy between these calcium antagonists may be due to differences in bioavailability or tissue specificity. The relatively high dose of diltiazem required for radioprotection in mice may be explained by its low bioavailability and high metabolic rate in the mouse. The calcium channel blockers flunarizine and verapamil which differ in their chemical structure from benzothiazepines and dihydropyridines exhibited no radioprotective activity (Table I). Diltiazem also displayed some activity when administered after the completion of irradiation (Fig. 1). This important property is not shared with thiols such as WR 2721, cysteamine or cysteine. Whether calcium antagonists protect from ionizing radiation has not been studied previously. Some attenuation of the radiation response has been reported by treatment with nifedipine in patients developing radiation oesophagitis after thoracic radiation (Finkelstein, 1986). Nifedipine was applied when pain on swallowing was severe, after radiation, and was thought to reduce oesophageal spasm. In other work, variation in blood flow caused by calcium channel blockers was proposed to explain a modified sensitivity of mouse tumours to X rays (Wood & Hirst, 1989). With non-ionizing radiation, in vitro studies revealed that cell changes occurring after exposure to magnetic fields were influenced by calcium channel blockers (Papatheofanis, 1990). Since calcium antagonists lower blood pressure, antihypertensive agents from other classes were tested (unpublished). Neither the thiol captopril, an angio1028
tensin-converting enzyme inhibitor, nor the /J-adrenergic blocking agent propranolol or various other vasoactive drugs used in the management of hypertension, coronary disease and cardiac arrhythmias (including clonidine, diazoxide, prazosine, hydrochlorothiazide, amiodarone, debrisoquine, sodium nitroprusside, molsidamine and ketanserine) exhibited significant radioprotective activity, suggesting that hypotension accompanied by tissue hypoxia is not the cause of the radioprotection produced by calcium antagonists. Calcium antagonists are well tolerated and thus display a significant advantage over other agents providing effective radioprotection. In addition to their use in nuclear emergencies, more gentle radioprotectors may be useful in cancer radiotherapy and in situations involving internal radionuclide contamination. If it could be established that calcium antagonists inhibit radiation carcinogenesis, they might be useful in extensive medical radiological procedures, exposure to radon or to cosmic radiation at high altitudes. Since radiation injury typically exemplifies tissue damage due to reactive oxygen species, radioprotective calcium antagonists may also be viewed in the context of an evergrowing body of degenerative diseases in which free radicals are thought to be implicated in the pathogenesis (Halliwell & Gutteridge, 1985; Cross et al, 1987; Cotgreave et al, 1988). Acknowledgments This work was supported by Grant No. 3981-0.87 of the Swiss National Foundation and the Mildner Stueckrath Foundation for Cancer Research. I thank F. R. Biihler for discussions and Armin Bieri, Ronald Konig and Chantal Racine for technical assistance. References BOLLI, R., TRIANA, F. & JEROUDI, M. O., 1989. Postischemic
mechanical and vascular dysfunction (myocardial 'stunning.' and micro vascular 'stunning') and the effects of calciumchannel blockers on ischemia/reperfusion injury. Clinical Cardiology, 12, 16-25. CAIRNIE, A. B., 1983. Adverse effects of the radioprotector WR 2721. Radiation Research, 94, 221-226. COTGREAVE, I. A., MOLDEUS, P. & ORRENIUS, S., 1988. Host
The British Journal of Radiology, November 1992
Radioprotection by calcium antagonists biochemical defense mechanism against prooxidants. Annual Review of Pharmacology and Toxicology, 28, 189-212. CROSS, E. C , HALLIWELL, B., BORISH, E. T., PRYOR, W. A., AMES, B. N., SAUL, R. L., MCCORD, J. M. & HARMANN, D.,
1987. Oxygen radicals and human disease. Annals of Internal Medicine, 107, 526-545. FINKELSTEIN, E., 1986. Nifedipine for radiation oesophagitis. Lancet, i, 1205-1206. HALLIWELL, B. & GUTTERIDGE, J. M. C , 1985. Free Radicals in
Biology and Medicine (Clarendon, Oxford). IP, J. H. & LEVIN, R. I., 1988. Myocardial preservation during ischemia and reperfusion. American Heart Journal, 115, 1094-1104. JANERO, D. R., BURGHARDT, B. & LOPEZ, R., 1988. Protection
of cardiac membrane phospholipid against oxidative injury by calcium antagonists. Biochemical Pharmacology, 37, 4197-4203. KOLLER, P. T. & BERGMANN, S. R., 1989. Reduction of lipid
peroxidation in reperfused isolated rabbit diltiazem. Circulation Research, 65, 838-846.
hearts by
OPIE, L. H., 1989. Reperfusion injury and its pharmacologic modification. Circulation, 80, 1049-1062. PAPATHEOFANIS, F. J., 1990. Use of calcium channel antagonists as magnetoprotective agents. Radiation Research, 122, 24-28. PATERSON, E. & MATTHEWS, J. J., 1950. Protective action of
ethyl alcohol on irradiated mice. Nature, 168, 1126-1127. SHRIDI, F. & ROBAK, J., 1988. The influence of calcium channel blockers on superoxide anions. Pharmacological Research Communications, 20, 13-21. SWEENEY, T. R., 1979. A Survey of Compounds from the Anti-radiation Drug Development Program of the U.S. Army Medical Research and Development Command. (Walter Reed Army Institute of Research, Washington, DC). TURRISI, A. T., KLIGERMAN, M. M., GLOVER, D. J., GLICK, J. H., NORFLEET, L. & GRAMKOWSKI, M., 1983. Experience
with phase I trials of WR 2721 preceding radiation therapy. In Radioprotectors and Anticarcinogens. Ed. by Nygaard, F. O. & Simic, M. G. (Academic Press, London), pp. 681-694.
1990.
WATTS, J. A., NORRIS, T. A., LONDON, R. E., STEENBERGEN, C.
Nifedipine and experimental cardioprotection. Cardiovascular Drugs and Therapy, 4, 879-886.
& MURPHY, E., 1990. Effects of diltiazem on lactate, ATP, and cytosolic free calcium levels in ischemic hearts. Journal of Cardiovascular Pharmacology, 15, 44-49.
NAYLER,
W. G.,
LIU, J. & PANAGIOTOPOULOS,
S.,
ONDRIAS, K., MISIK, V., GERGEL, D. & STASKO, A., 1989. Lipid
peroxidation of phosphatidilcholine liposomes depressed by the calcium channel blockers nifedipine and verapamil and by the antiarrhythmic-antihypoxic drug stobadine. Biochimica Biophysica Ada, 1003, 238-245.
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WOOD, P. J. & HIRST, D. G., 1989. Calcium antagonists as
radiation modifiers: site specificity in relation to tumor response. International Journal of Radiation Oncology, Biology Physics, 16, 1141-1144.
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Calcium antagonists protect mice against lethal doses of ionizing radiation By G. L. Floersheim Department of Research, University Hospitals, Hebelstrasse 20, 4031 Basel, Switzerland (Received 28 October 1991 and in final form 8 June 1992, accepted 29 June 1992) Keywords: Radioprotection, Calcium antagonists, Diltiazem, Nifedipine, Nimodipine
Abstract. Currently available radioprotectors are poorly tolerated in man and the general use of aminothiol radioprotectors is compromised by their side-effects. In a search for less toxic radioprotective agents, diltiazem, a calcium antagonist with a benzothiazepine structure, was found to protect mice against a lethal (LD100) y radiation dose allowing survival of up to 93%. Dihydropyridine calcium antagonists such as nifedipine, nimodipine, isradipine and nitrendipine also provided radioprotection. Calcium antagonists might attenuate radiation-induced injury by inhibiting cellular calcium overload, subsequent to cell: membrane damage caused by radiation-generated free radicals. In view of their good tolerance, calcium antagonists may be applied safely in situations of radiation exposure, including radiotherapy and internal radionuclide contamination. These calcium antagonists may also be viewed in other contexts where free radicals are implicated in pathological processes.
The search for chemical agents which protect against the tissue damage caused by exposure to ionizing radiation has led to the identification of thiol (sulfhydryl) compounds with marked activity in experimental models (Sweeney, 1979). However, the use of the most promising agent, WR 2721 (S-2-(3-aminopropylamino)ethylphosphorothioic acid), has been limited by its poor clinical tolerance (Cairnie, 1983; Turrisi et al, 1983). In this study a different approach has been initiated by testing thiol substances not designed for radioprotective activity but known from their use in other contexts to have a favourable therapeutic index. This included diltiazem, a benzothiazepine whose use as a calcium antagonist in cardiovascular pharmacology is based on extensive clinical experience. Since diltiazem protected against radiation-induced damage, the effects of other calcium antagonists were also investigated. Materials and methods
Mice and irradiation The experiments were carried out using C3H mice of both sexes (Gl. Bomholtgard, Ry, Denmark) weighing 22-24 g and fed Nafag pellets and water ad libitum. Groups of mice were irradiated in a perforated plexiglass chamber from a 60Co source (Gammatron). The target distance was 80 cm. Dosimetry was performed with a strontium-calibrated ionization chamber. For the experiments with diltiazem, the results of which are illustrated in Fig. 1, female mice {n — 173) were irradiated with 10.5 Gy (dose rate ~ 0.15 Gy/min). A smaller scale experiment (n = 18) was also peformed with a dose of 8.5 Gy from a source delivering 0.9 Gy/min. The doses delivered by the two radiation sources corresponded to the respective LD100 values and resulted in the same mean survival times. Accordingly, they were assumed to be biologically isoeffective and Vol. 65, No. 779
100
Figure 1. Survival of female C3H mice irradiated with 10.5 Gy (dose rate ~ 0.15 Gy/min). A small number of the mice (6 treated with HOmg/kg diltiazem at — 15min, 6 treated with HOmg/kg diltiazem at +10 min and 6 controls) were irradiated with 8.5 Gy at a dose rate of 0.9 Gy/min. The mice were treated subcutaneously with diltiazem before or after irradiation. The doses of diltiazem were as follows: HOmg/kg (n = 29) at - 15 min (O); 55 mg/kg (n = 24) at - 1 5 min ( • ) ; 27.5 mg/kg (n = 12) at - 1 5 min (A); 110 mg/kg (« = 12) at + 10 min (V)- Controls (n = 114) were injected with 0.1 ml distilled water per 10 g body weight ( # ) .
results obtained with both radiation dose rates were pooled. The female mice used for the experiments whose results are shown in Fig. 2 received 8.5 Gy at a rate of 0.9 Gy/min. In the experiments with male mice, the results of which are illustrated in Fig. 3, the irradiation doses were 8.1 and 9.0 Gy at a rate of 0.9 Gy/min. The efficacy of a compound was measured by its ability to protect mice against a lethal dose of whole1025
G. L. Floersheim
(b) Figure 2. Groups of 12 female C3H mice irradiated with 8.5 Gy (dose rate 0.9 Gy/min). Their radiation LD 50 was ~ 7.75 Gy. The mice were injected intraperitoneally 30 min before irradiation. For a better detection of the relative contributions, the agents were applied at only half their optimal dose, (a) The mice received 1.5 mg/kg body weight nifedipine in 0.15 ml/10 g standard solvent ( • ) , 0.15 ml/10 g standard solvent containing 18% ethanol (A), or 0.15 ml/10 g of an 18% ethanol solution in distilled water, corresponding to 2295 mg/kg ethanol (V)Controls received 0.15 ml/10 g distilled water ( # ) . (b) The mice were injected with 2.0 mg/kg nimodipine in 0.1 ml/10 g standard solvent ( • ) ; 0.1 ml/10 g standard solvent containing 23.7% ethanol (A), or 0.1 ml/10 g of a 23.7% ethanol solution in distilled water corresponding to 2015 mg/kg ethanol (V)Controls recevied 0.1 ml/10 g distilled water ( # ) .
body 7 radiation, protection being identified by animal survival at 30 days. The agents were first tested at 50% of the LD50 and, in case of efficacy, at progressively halved doses. The figures for survival after the respective treatments are presented in the Figures. Since, in general, the mice succumbed at approximately day 10 after irradiation, their death can be attributed to bone marrow damage. However, in the experiments in which a supralethal radiation dose was applied, the results of which are depicted in Figure 3b, gut-related deaths may have occurred. 1026
Figure 3. Survival in groups of 12 male C3H mice irradiated with 8.1 Gy (a) or 9.0 Gy (b) at the dose rate 0.9 Gy/min. The radiation LD 50 in these mice was ~ 6.35 Gy (dose rate 0.9 Gy/min). The mice were injected intraperitoneally 30 min before irradiation with either nifedipine or nimodipine in their solvents or with ethanol at the dose corresponding to that present in the solvent. Treatments were as follows: Controls: 0.2 ml distilled water/10 g body weight ( # ) ; nifedipine, 3 mg/kg in 0.3 ml solvent/10 g ( • ) ; 0.3 ml/10 g of an 18% ethanol solution in distilled water corresponding to 4590 mg/kg ethanol (A); nimodipine, 4 mg/kg in 0.2 ml solvent/10 g ( • ) ; 0.2 ml/10 g of an 23.7% ethanol solution (4050 mg ethanol/kg) in distilled water (A)-
Drugs Diltiazem was provided in ampoules containing 25 mg diltiazem hydrochloride and 150 mg mannitol (Warner-Lambert). The content of the ampoules was dissolved in distilled water and 0.1 ml/10 g body weight was administered by the subcutaneous route to the mice. In control experiments, it was ascertained that mannitol alone did not exert any radioprotection. Nifedipine and nimodipine were available as commercial solutions (Bayer) and applied by the intraperitoneal route. The solvents for nifedipine and nimodipine contained 18% and 23.7% ethanol, respectively. In addition, the solvent included polyethylene glycol 400, The British Journal of Radiology, November 1992
Radioprotection by calcium antagonists
sodium citrate and citric acid (courtesy Prof. S. Kazda). Since standard solvent and ethanol alone, in the quantity contained in the solvent, afforded equivalent radioprotection (Fig. 2), it can be assumed that the components of the standard solvent other than ethanol did not contribute to the protective activity afforded by the standard solvent. It was ascertained in separate experiments that polyethylene glycol 400 alone had no radioprotective activity. Nitrendipine (Bayer) was administered as a suspension in distilled water (0.1 ml/10 g body weight) by the intraperitoneal route. Isradipine (Sandoz) was dissolved in olive oil and was also injected intraperitoneally (0.1 ml/10 g body weight). Olive oil alone did not protect. Verapamil (Knoll) was administered intraperitoneally in distilled water (0.1 ml/10 g body weight) and flunarizine (Janssen) was given orally as a suspension in distilled water (0.1 ml/10 g body weight). Statistical evaluation The statistical significance of the differences in survival rates, as compared to controls, was determined according to the two-tailed chi-square test. Differences in survival time were evaluated according to a two-tailed Mest. Results The survival at 30 days of female C3H mice irradiated over the whole body with y-rays at approximately LD100 was increased to 93% by HOmg/kg of subcutaneous diltiazem, a dose corresponding to 50% of the LD50 of the drug (Fig. 1). Survival with 55mg/kg and 27.5mg/kg of diltiazem was 58% and 17% respectively (Fig. 1). Significant survival (42%) was also obtained when diltiazem was administered lOmin after the completion of irradiation (Fig. 1). The protection produced by diltiazem occurred irrespective of the dose rate at which the radiation was given. A similar dose-dependent improvement in survival in lethally irradiated female C3H mice was achieved following the intraperitoneal administration of nifedipine and nimodipine. These agents were administered in combination with their standard solvents, which contained ethanol (18% for nifedipine and 23.7% for nimodipine). Ethanol has long been known to exert radioprotective activity (Paterson & Matthews, 1950). Therefore to avoid confounding the additive or synergistic effects of ethanol with those of nifedipine and nimodipine, it was necessary to distinguish between the relative protective contributions of solvent and calcium antagonist. Figures 2a and 2b present the results of experiments in which the efficacy of nifedipine and nimodipine (both at a dose corresponding to 25% of their LD50) are compared with those of their respective standard solvents, and with that of ethanol at an appropriate standard solvent concentration. The survival in lethally irradiated female C3H mice was 100% with 1.5mg/kg nifedipine, 61% with the standard solvent and 50% with ethanol (Fig. 2a). With 2.0 mg/kg nimodipine, 82% of the mice Vol. 65, No. 779
survived, but only 42% with the standard solvent and 50% with ethanol (Fig. 2b). The radioprotective effects afforded by standard solvents and ethanol per se were not significantly different. The differences in survival rate (chi-square test) between either nifedipine or nimodipine (each in standard solvents) and their respective solvents alone, were statistically significant (p < 0.001 and p < 0.01, respectively). Accordingly, after subtractive correction for the solvent effect, the residual radioprotection (39% with nifedipine and 40% with nimodipine) can be ascribed to the calcium antagonists. The intrinsic radioprotective potential of these calcium antagonists was confirmed in irradiation experiments on male C3H mice, which are more radiosensitive than female mice. For mice irradiated with 8.1 Gy (Fig. 3a), survival after treatment with 3 mg/kg nifedipine or 4 mg/kg nimodipine (i.e. at doses corresponding to 50% of their LD50) was 67% (p < 0.005) and 75% (p < 0.001), respectively, compared to survival on treatment with the corresponding doses of ethanol alone of 25% and 8%, respectively. For mice irradiated with the supralethal dose of 9.0 Gy (Fig. 3b), survival was 58% (p < 0.0025) with 3 mg/kg nifedipine and 55% (p < 0.0025) with 4 mg/kg nimodipine as compared with the corresponding doses of ethanol which afforded no survival advantage at 30 days, only a prolongation of the mean survival time from 6.6+ 1.6 days (controls) to 11.1+2.1 and 11.0±2.2 days (p < 0.01; Mest), respectively. The results obtained with other calcium antagonists are presented in Table I. Survival of 58% (p < 0.001) was achieved with isradipine applied at 27.5 mg/kg (n — 24) to female C3H mice irradiated with 8.5 Gy at a dose rate of 0.9 Gy/min. Nitrendipine (100 or 50 mg/kg) administered intraperitoneally as a suspension in distilled water to female C3H mice irradiated with 10.5 Gy (dose rate of 0.15 Gy/min) resulted in survival of 29% or 25%, respectively. No animal survival was produced by pretreatment with verapamil or flunarizine. Discussion These experiments demonstrate that some calcium antagonists—particularly diltiazem, but also nifedipine and nimodipine—are effective radioprotectors. One explanation for the observed effect could be related to the radiation-induced lipid peroxidation of cell membrane structures by oxygen-derived free radicals with ionic leakage through cellular membranes and excessive calcium influx with ensuing cellular dysfunction and death from Ca 2+ overload. In reperfusion injury, another situation involving peroxidative membrane injury by free radicals (Ip & Levin, 1988; Janero et al, 1988; Bolli et al, 1989; Opie, 1989; Nayler et al, 1990; Watts et al, 1990), Ca 2+ channel blockers have also been shown to be protective. On the other hand, antioxidative effects by calcium antagonists such as nifedipine (Shridi & Robak, 1988; Ondrias et al, 1989) and diltiazem (Koller & Bergmann, 1989) have been reported and their radioprotective activity could also be linked more generally to the 1027
G. L. Floersheim Table I. Survival (30 days) of lethally irradiated female C3H mice pretreated with various calcium antagonists Treatment Controls (0.1 ml aq. dest./lO g) Controls (0.1 ml olive oil/10 g Isradipine Isradipine Nitrendipine Nitrendipine Verapamil Flunarizine
Dose (mg/kg)
Route of application
Time (min)
Survival0
%
27.5 27.5 100 50 16 25
i.p. i.p. i.p. i.p. i.p. i.p. i.p. p.o.
-30 -30 -30 -120 -30 -30 -30 -30
0/30 0/6 14/24 5/18 7/24 3/12 0/12 0/12
0 0 58 28 29 25 0 0
< 0.001 < 0.01 < 0.005 < 0.005 n.s. n.s.
"No. of surviving mice/total no. of mice. *Chi-square test, compared to the relative controls, n.s. = Not significant.
scavenging of free radicals (Janero et al, 1988). Thiol radioprotectors are believed to act principally by neutralizing radiation-induced free radicals via the formation of disulphides. Therefore, the particular efficacy of diltiazem may be explained by the fact that this molecule is capable of functioning not only as a calcium antagonist, but also as a thiol. No class dependency can be observed, since both a benzothiazepine (diltiazem) and dihydropyridine (nifedipine and nimodipine) calcium antagonists were active. To the latter class belong also the moderately effective compounds nitrendipine and isradipine (Table I). Variations in efficacy between these calcium antagonists may be due to differences in bioavailability or tissue specificity. The relatively high dose of diltiazem required for radioprotection in mice may be explained by its low bioavailability and high metabolic rate in the mouse. The calcium channel blockers flunarizine and verapamil which differ in their chemical structure from benzothiazepines and dihydropyridines exhibited no radioprotective activity (Table I). Diltiazem also displayed some activity when administered after the completion of irradiation (Fig. 1). This important property is not shared with thiols such as WR 2721, cysteamine or cysteine. Whether calcium antagonists protect from ionizing radiation has not been studied previously. Some attenuation of the radiation response has been reported by treatment with nifedipine in patients developing radiation oesophagitis after thoracic radiation (Finkelstein, 1986). Nifedipine was applied when pain on swallowing was severe, after radiation, and was thought to reduce oesophageal spasm. In other work, variation in blood flow caused by calcium channel blockers was proposed to explain a modified sensitivity of mouse tumours to X rays (Wood & Hirst, 1989). With non-ionizing radiation, in vitro studies revealed that cell changes occurring after exposure to magnetic fields were influenced by calcium channel blockers (Papatheofanis, 1990). Since calcium antagonists lower blood pressure, antihypertensive agents from other classes were tested (unpublished). Neither the thiol captopril, an angio1028
tensin-converting enzyme inhibitor, nor the /J-adrenergic blocking agent propranolol or various other vasoactive drugs used in the management of hypertension, coronary disease and cardiac arrhythmias (including clonidine, diazoxide, prazosine, hydrochlorothiazide, amiodarone, debrisoquine, sodium nitroprusside, molsidamine and ketanserine) exhibited significant radioprotective activity, suggesting that hypotension accompanied by tissue hypoxia is not the cause of the radioprotection produced by calcium antagonists. Calcium antagonists are well tolerated and thus display a significant advantage over other agents providing effective radioprotection. In addition to their use in nuclear emergencies, more gentle radioprotectors may be useful in cancer radiotherapy and in situations involving internal radionuclide contamination. If it could be established that calcium antagonists inhibit radiation carcinogenesis, they might be useful in extensive medical radiological procedures, exposure to radon or to cosmic radiation at high altitudes. Since radiation injury typically exemplifies tissue damage due to reactive oxygen species, radioprotective calcium antagonists may also be viewed in the context of an evergrowing body of degenerative diseases in which free radicals are thought to be implicated in the pathogenesis (Halliwell & Gutteridge, 1985; Cross et al, 1987; Cotgreave et al, 1988). Acknowledgments This work was supported by Grant No. 3981-0.87 of the Swiss National Foundation and the Mildner Stueckrath Foundation for Cancer Research. I thank F. R. Biihler for discussions and Armin Bieri, Ronald Konig and Chantal Racine for technical assistance. References BOLLI, R., TRIANA, F. & JEROUDI, M. O., 1989. Postischemic
mechanical and vascular dysfunction (myocardial 'stunning.' and micro vascular 'stunning') and the effects of calciumchannel blockers on ischemia/reperfusion injury. Clinical Cardiology, 12, 16-25. CAIRNIE, A. B., 1983. Adverse effects of the radioprotector WR 2721. Radiation Research, 94, 221-226. COTGREAVE, I. A., MOLDEUS, P. & ORRENIUS, S., 1988. Host
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1987. Oxygen radicals and human disease. Annals of Internal Medicine, 107, 526-545. FINKELSTEIN, E., 1986. Nifedipine for radiation oesophagitis. Lancet, i, 1205-1206. HALLIWELL, B. & GUTTERIDGE, J. M. C , 1985. Free Radicals in
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of cardiac membrane phospholipid against oxidative injury by calcium antagonists. Biochemical Pharmacology, 37, 4197-4203. KOLLER, P. T. & BERGMANN, S. R., 1989. Reduction of lipid
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