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Effects of ICRF 159 on adriamycin-induced cardiomyopathy in rats
77
Cancer Letters, 19 (1983) 77-83 Elsevier Scientific Publishers Ireland Ltd.
EFFECTS OF ICRF 159 ON ADRIAMYCIN-INDUCED CARDIOMYOPATHY IN RATS
G. DECORTIa, F. BARTOLI KLUGMANNa, F. MALLARDIb, B. BENUSSIC, V. GRILLa and L. BALDINIa aIn.stitute of Pharmacology, bZnstitute of Anatomy University of Trieste, 34100, Trieste (Italy)
S. KLUGMANNC,
and ‘Department
of Cardiology,
(Received 26 July 1982) (Revised version received 22 December 1982) (Accepted 6 January 1983)
SUMMARY
The effect of ICRF 159 on adriamycin (ADR) cardiotoxicity and on total myocardial calcium content was examined in rats. ICRF 159 did not increase the survival time of ADR-treated animals; however the histological findings showed a significant prevention of ADR-induced cardiomyopathy (CMP) by ICRF. The total myocardial calcium content of animals treated with ADR was significantly higher, while no significant difference was seen in animals pretreated with ICRF 159 as compared with controls. As these findings suggested a role of calcium in ADR CMP and in the pharmacological action of ICRF in this disease, we also tested a closely related chelating agent, EDTA. This molecule decreased myocardial calcium levels in ADR-treated ani’mals almost to normal values; however the histological cardiac alterations were not prevented.
INTRODUCTION ADR is an anthracycline antibiotic useful in a large number of neoplasms [ 13 ; its clinical use has however been limited by ,a dose-related CMP [7]. Many attempts to modify this toxic effect have involved the development of less cardiotoxic analogs and the administration of agents which would protect the myocardium from ADR action without. interfering with its effectiveness as an antitumor agent. ICRF 159 and its d-isomer ICRF 187, initially tested in combination with ADR in the attempt to improve the antineoplastic activity [lo], have been proven to be effective also on the cardiotoxic action of ADR. Various experiments have been carrried out in a number of experimental models to study this interesting effect, with different results [ 4-6, lo]. As some 0304-3835/83/$03.00 o 1983 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland
authors hypothesize an alteration in calcium metabolism as one of the pathogenetic causes of the disease [ 81, it has been suggested that ICRF could exert its effect by acting as a chelating agent, and hence reducing the intracellular concentration of divalent cations. Therefore the aim of our study was to evaluate the effect of ICRF 159 on ADR-induced CMP in the rat, which is, with the rabbit, the most useful and most extensively used animal model of the ADR CMP [3], and to clarify the possible role of calcium in the pathogenesis of the CMP and in the pharmacological action of ICRF 159 on the myocardium. MATERIALS
AND METHODS
For the first experiment, 60 male Wistar rats, average weight 200 g, were used and were divided into 4 groups treated as follows: group 1 (20 animals) ADR 10 mg/kg i.v.; group 2 (20 animals) ADR 10 mg/kg iv. + ICRF 159 100 mg/kg i.p. 24 h before ADR; group 3 (10 animals) ICRF 159 100 mg/kg i.p.; group 4 (10 animals) controls. For a second experiment, designed to evaluate the activity of EDTA, 30 male Wistar rats, average weight 200 g, were used and divided in 3 groups of 10 animals each: group 1 ADR 10 mg/kg i.v.; group 2 ADR 10 mg/kg i.v. + EDTA 124 mg/kg i.p. 24 h before ADR; group 3 EDTA 124 mg/kg i.p. The dosages of ICRF 159 and EDTA were equimolar. ICRF and EDTA were dissolved in 1% carboxy-methyl cellulose (CMC) in 0.9% saline. All animals were anesthetized with uretan 1.2 g/kg i.p. and ECGs were recorded with a Battaglia Rangoni poligraph 2 days before treatment and 7 and 30 days, thereafter. Needle electrodes were inserted under the skin of the limbs; the paper speed was 100 mm/s. All the limb derivations were recorded; ECGs were coded for blind evaluation. A necropsy was performed in all animals shortly after death and in the survivors after completion of the experiment (10 weeks). Hearts were removed, fixed in buffered picric acid-formaldehyde [ll] and processed with conventional histological techniques for optical microscopy. Sections were cut at 7 pm and stained with hematoxylin and eosin. Five sections per organ were examined by a double blind method. In a third experiment designed to study the calcium concentration of the myocardium, 5 additional animals per group (10 for ADR alone and 8 for controls) were killed 7 days after treatment and the ventricular myocardium was collected for total calcium analysis by atomic absorption spectrophotometry. RESULTS
Survival study
The cumulative mortality data for all groups of animals of the first experiment are presented in Fig. 1. Most of the animals treated with ADR started
79
I,,),,,,,,, 123656
7
8
910
weeks Fig. 1. Cumulative mortality data for animals receiving ADR 10 mg/kg i.v. (o), ADR 10 mg/kg i.v. + ICRF 159 100 mg/kg i.p. (A), ICRF 159 100 mg/kg i.p. (0) and controls (A).
to die after 45 days, while a severe mortality was observed in the first 3 weeks in the animals pretreated with ICRF 159; no mortality was seen in control animals,and in those treated with ICRF alone. In the second experiment the mortality rate of ADR-treated animals was identical to the first one. The mortality rate of ADR plus EDTA treated animals was not significantly different from ADR-treated animals; no mortality was seen in animals treated with EDTA alone (data not shown). ECG findings
An evaluation of ECGs showed only aspecific alterations in animals treated with ADR and ADR plus EDTA; they included a widening of PQ and QT intervals. These abnormalities were almost completely absent in animals pretreated with ICRF 159 and in those treated with ICRF or EDTA alone. These alterations were too aspecific and hence could not be used as a reliable method in studying the evolution of the CMP in this model system. Histopathological
findings
The hearts of the animals treated with ADR alone showed severe lesions consistent with the CMP, and in particular we observed marked cellular vacuolization, myocytolysis, and lymphomonocitic infiltration (Fig. 2); furthermore in 50% of the animals, myocardial hemorrhages were present
Fig. 3. Myocardial haemorrhages 10 mg/kg i.v. (H and E, 360X ).
in the ventricular tissue from a rat receiving ADR
Fig. 4. Light microscopic appearance of ventricular myocardium ADR 10 mg/kg i.v. + ICRF 159 100 mg/kg i.p. (H and E 360X ).
from a rat receiving
(Fig. 3). The same severe lesions were present in EDTA pretreated animals. All these lesions were almost completely absent in the hearts of animals pretreated with ICRF 159 (Fig. 4). Myocardial
calcium
content
(Table
1)
The treatment with ADR caused a significant increase in ventricular calcium content. Combined administration of ADR and ICRF 159 or of ADR and EDTA decreased the elevated myocardial calcium levels by ADR. The total calcium concentration in the hearts of rats treated with ICRF 159 alone was significantly below the normal values. A slight decrease in the total calcium concentration in the hearts of rats treated with EDTA alone was also present but it was not significantly different from controls. DISCUSSION
Previous contrasting findings [4-6, lo] about the action of ICRF 159 and its isomer ICRF 187 on ADR-induced CMP, led us to determine whether pretreatment with ICRF 159 could reduce the cardiac side effe.cts in the rat. In agreement with the results obtained with mice by Giuliani et al. [4] the combined treatment with ICRF 159 and ADR increased the acute
82 TABLE 1 CALCIUM CONTENT OF VENTRICULAR Controls N=8
ADR (10 mgbg) N= 10
42.7 f 3.6
57.3 f 6.Bb
.
MYOCARDIUM
ADR (10 mg/kg) + ICRF 159 (100 mg/kg) N=5
ADR (IO mg/kg) + EDTA (124 mg/kg) N=5
ICRF 159 ‘cl00 mg/kg) N=5
EDTA (124 mg/kg) N= 5
48.3 + 5.3
46.3 + 4.4
33.4 * 6.9’
38.8 + 5.3
Values are mean f S.D. expressed as micrograms of calcium per gram of tissue wet weight; test of significance (Student’s t-test for unpaired data)’ indicate experimental group versus control. %ignificantly different from controls at P < 0.05. bSignificantly different from controls at P < 0.01.
antibiotic toxicity, while, at the end of the experimental period, the number of deaths was similar in both groups; these data suggest a synergistic acute action of the 2 drugs on organs which are usual targets of ADR acute toxicity. Nevertheless, our results on myocardial calcium content are of interest; the elevated calcium levels in the hearts of animals treated with ADR found in other studies [ 8,9] and in our own have been reported to be the possible pathogenetic cause of the CMP; where rats were pretreated with ICRF 159, the myocardial calcium concentration was reduced to almost normal values. Furthermore, ICRF 159 alone significantly reduced ventricular calcium concentrations as compared with untreated controls; these data could be explained by the chelating action of ICRF, which is a non-polar derivative of EDTA [2]. Pretreatment with EDTA also reduced to almost normal values the myocardial calcium content. As the pretreatment with ICRF 159 significantly ameliorated the histopathologic lesions of the CMP while the pretreatment with EDTA did not have any protective action, we suggest that the myocardial calcium metabolism may not have a primary role in the pathogenesis of ADR CMP and that the effect of ICRF 159 on the myocardium is probably due to other, still unclear pharmacologic activity. However, as the reduction in ADR-induced myocardial injury by ICRF 159 is not accompanied by other signs of protection and, in fact, there is even an increase in the incidence of acute deaths, it seems appropriate to further investigate this combination therapy. ACKNOWLEDGEMENT
This work was supported by a special grant from the University of Trieste. We thank Dr. Remoli and Dr. Orpelli for their help with tissue calcium determination.
83 REFERENCES 1 Blum, R.H. and Carter, SK. (1974) Adriamycin - a new anticancer drug with significant clinical activity. Ann. Intern. Med., 80, 249-259. 2 Creighton, A.M., Hellmann, K. and Whitecross, S. (1969) Antitumor activity in a series of bis-diketopiperazines. Nature, 222, 384-385. 3 Dorochow, J.H., Locker, G.Y. and Myers, C.E. (1979) Experimental animal models of adriamycin cardiotoxicity. Cancer Treat. Rep., 63, 855-860. 4 Giuliani, F., Casazza, A.M., Di Marco, A. and Savi, G. (1981) Studies in mice with ICRF 159 combined with daunorubicin and doxorubicin. Cancer Treat. Rep., 65, 267-276. 5 Herman, E.H. and Ferrans, V.J. (1981) Reduction of chronic doxorubicin cardiotoxicity in dogs by pretreatment with (*)-1,2-bis(3,5-dioxopiperazinyl-l-yl)propane (ICRF-187). Cancer Res., 41, 3436-3440. 6 Herman, E.H., Mhatre, R.M., Lee, I.P. and Waravdekar, V.S. (1972) Prevention of the cardiotoxic effects-of adriamycin and daunomycin in the isolated dog heart (36432). Proc. Exp. Biol. Med., 140, 234-239. 7 Minow, R.A., Benjamine, R.S. and Gottlieb, J.A. (1975) Adriamycin (NSC-123127) cardiomyopathy: an overview with determination of risk factors. Cancer Chemother. Rep., Part 3, 6, 195-201. 8 Olson, H.M. and Capen, C.C. (1977) Subacute cardiotoxicity of adriamycin in the rat. Lab. Invest., 37, 386-394. 9 Olson, H.M., Young, D.M., Prieur, D., Le Roy, A. and Reagan, R. (1974) Electrolyte and morphologic alterations of myocardium in adriamycin-treated rabbits. Am. J. Pathol., 77,439-454. 10 Woodman, R.J., Cysyk, R.L., Kline, I., Gang. M. and Venditti, J.M. (1975) Enhancement of the effectiveness of daunorubicin (NSC-82151) or adriamycin (NSC-123127) against early mouse L1210 leukemia with ICRF 159 (NSC-129943). Cancer Chemother. Rep., 59, 689-695. 11 Zamboni, L. and De Martino, C. (1967) Buffered picric acid-formaldehyde: a new rapid fixative for electron microscopy. J. Cell. Biol., 35, 148 A.
Cancer Letters, 19 (1983) 77-83 Elsevier Scientific Publishers Ireland Ltd.
EFFECTS OF ICRF 159 ON ADRIAMYCIN-INDUCED CARDIOMYOPATHY IN RATS
G. DECORTIa, F. BARTOLI KLUGMANNa, F. MALLARDIb, B. BENUSSIC, V. GRILLa and L. BALDINIa aIn.stitute of Pharmacology, bZnstitute of Anatomy University of Trieste, 34100, Trieste (Italy)
S. KLUGMANNC,
and ‘Department
of Cardiology,
(Received 26 July 1982) (Revised version received 22 December 1982) (Accepted 6 January 1983)
SUMMARY
The effect of ICRF 159 on adriamycin (ADR) cardiotoxicity and on total myocardial calcium content was examined in rats. ICRF 159 did not increase the survival time of ADR-treated animals; however the histological findings showed a significant prevention of ADR-induced cardiomyopathy (CMP) by ICRF. The total myocardial calcium content of animals treated with ADR was significantly higher, while no significant difference was seen in animals pretreated with ICRF 159 as compared with controls. As these findings suggested a role of calcium in ADR CMP and in the pharmacological action of ICRF in this disease, we also tested a closely related chelating agent, EDTA. This molecule decreased myocardial calcium levels in ADR-treated ani’mals almost to normal values; however the histological cardiac alterations were not prevented.
INTRODUCTION ADR is an anthracycline antibiotic useful in a large number of neoplasms [ 13 ; its clinical use has however been limited by ,a dose-related CMP [7]. Many attempts to modify this toxic effect have involved the development of less cardiotoxic analogs and the administration of agents which would protect the myocardium from ADR action without. interfering with its effectiveness as an antitumor agent. ICRF 159 and its d-isomer ICRF 187, initially tested in combination with ADR in the attempt to improve the antineoplastic activity [lo], have been proven to be effective also on the cardiotoxic action of ADR. Various experiments have been carrried out in a number of experimental models to study this interesting effect, with different results [ 4-6, lo]. As some 0304-3835/83/$03.00 o 1983 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland
authors hypothesize an alteration in calcium metabolism as one of the pathogenetic causes of the disease [ 81, it has been suggested that ICRF could exert its effect by acting as a chelating agent, and hence reducing the intracellular concentration of divalent cations. Therefore the aim of our study was to evaluate the effect of ICRF 159 on ADR-induced CMP in the rat, which is, with the rabbit, the most useful and most extensively used animal model of the ADR CMP [3], and to clarify the possible role of calcium in the pathogenesis of the CMP and in the pharmacological action of ICRF 159 on the myocardium. MATERIALS
AND METHODS
For the first experiment, 60 male Wistar rats, average weight 200 g, were used and were divided into 4 groups treated as follows: group 1 (20 animals) ADR 10 mg/kg i.v.; group 2 (20 animals) ADR 10 mg/kg iv. + ICRF 159 100 mg/kg i.p. 24 h before ADR; group 3 (10 animals) ICRF 159 100 mg/kg i.p.; group 4 (10 animals) controls. For a second experiment, designed to evaluate the activity of EDTA, 30 male Wistar rats, average weight 200 g, were used and divided in 3 groups of 10 animals each: group 1 ADR 10 mg/kg i.v.; group 2 ADR 10 mg/kg i.v. + EDTA 124 mg/kg i.p. 24 h before ADR; group 3 EDTA 124 mg/kg i.p. The dosages of ICRF 159 and EDTA were equimolar. ICRF and EDTA were dissolved in 1% carboxy-methyl cellulose (CMC) in 0.9% saline. All animals were anesthetized with uretan 1.2 g/kg i.p. and ECGs were recorded with a Battaglia Rangoni poligraph 2 days before treatment and 7 and 30 days, thereafter. Needle electrodes were inserted under the skin of the limbs; the paper speed was 100 mm/s. All the limb derivations were recorded; ECGs were coded for blind evaluation. A necropsy was performed in all animals shortly after death and in the survivors after completion of the experiment (10 weeks). Hearts were removed, fixed in buffered picric acid-formaldehyde [ll] and processed with conventional histological techniques for optical microscopy. Sections were cut at 7 pm and stained with hematoxylin and eosin. Five sections per organ were examined by a double blind method. In a third experiment designed to study the calcium concentration of the myocardium, 5 additional animals per group (10 for ADR alone and 8 for controls) were killed 7 days after treatment and the ventricular myocardium was collected for total calcium analysis by atomic absorption spectrophotometry. RESULTS
Survival study
The cumulative mortality data for all groups of animals of the first experiment are presented in Fig. 1. Most of the animals treated with ADR started
79
I,,),,,,,,, 123656
7
8
910
weeks Fig. 1. Cumulative mortality data for animals receiving ADR 10 mg/kg i.v. (o), ADR 10 mg/kg i.v. + ICRF 159 100 mg/kg i.p. (A), ICRF 159 100 mg/kg i.p. (0) and controls (A).
to die after 45 days, while a severe mortality was observed in the first 3 weeks in the animals pretreated with ICRF 159; no mortality was seen in control animals,and in those treated with ICRF alone. In the second experiment the mortality rate of ADR-treated animals was identical to the first one. The mortality rate of ADR plus EDTA treated animals was not significantly different from ADR-treated animals; no mortality was seen in animals treated with EDTA alone (data not shown). ECG findings
An evaluation of ECGs showed only aspecific alterations in animals treated with ADR and ADR plus EDTA; they included a widening of PQ and QT intervals. These abnormalities were almost completely absent in animals pretreated with ICRF 159 and in those treated with ICRF or EDTA alone. These alterations were too aspecific and hence could not be used as a reliable method in studying the evolution of the CMP in this model system. Histopathological
findings
The hearts of the animals treated with ADR alone showed severe lesions consistent with the CMP, and in particular we observed marked cellular vacuolization, myocytolysis, and lymphomonocitic infiltration (Fig. 2); furthermore in 50% of the animals, myocardial hemorrhages were present
Fig. 3. Myocardial haemorrhages 10 mg/kg i.v. (H and E, 360X ).
in the ventricular tissue from a rat receiving ADR
Fig. 4. Light microscopic appearance of ventricular myocardium ADR 10 mg/kg i.v. + ICRF 159 100 mg/kg i.p. (H and E 360X ).
from a rat receiving
(Fig. 3). The same severe lesions were present in EDTA pretreated animals. All these lesions were almost completely absent in the hearts of animals pretreated with ICRF 159 (Fig. 4). Myocardial
calcium
content
(Table
1)
The treatment with ADR caused a significant increase in ventricular calcium content. Combined administration of ADR and ICRF 159 or of ADR and EDTA decreased the elevated myocardial calcium levels by ADR. The total calcium concentration in the hearts of rats treated with ICRF 159 alone was significantly below the normal values. A slight decrease in the total calcium concentration in the hearts of rats treated with EDTA alone was also present but it was not significantly different from controls. DISCUSSION
Previous contrasting findings [4-6, lo] about the action of ICRF 159 and its isomer ICRF 187 on ADR-induced CMP, led us to determine whether pretreatment with ICRF 159 could reduce the cardiac side effe.cts in the rat. In agreement with the results obtained with mice by Giuliani et al. [4] the combined treatment with ICRF 159 and ADR increased the acute
82 TABLE 1 CALCIUM CONTENT OF VENTRICULAR Controls N=8
ADR (10 mgbg) N= 10
42.7 f 3.6
57.3 f 6.Bb
.
MYOCARDIUM
ADR (10 mg/kg) + ICRF 159 (100 mg/kg) N=5
ADR (IO mg/kg) + EDTA (124 mg/kg) N=5
ICRF 159 ‘cl00 mg/kg) N=5
EDTA (124 mg/kg) N= 5
48.3 + 5.3
46.3 + 4.4
33.4 * 6.9’
38.8 + 5.3
Values are mean f S.D. expressed as micrograms of calcium per gram of tissue wet weight; test of significance (Student’s t-test for unpaired data)’ indicate experimental group versus control. %ignificantly different from controls at P < 0.05. bSignificantly different from controls at P < 0.01.
antibiotic toxicity, while, at the end of the experimental period, the number of deaths was similar in both groups; these data suggest a synergistic acute action of the 2 drugs on organs which are usual targets of ADR acute toxicity. Nevertheless, our results on myocardial calcium content are of interest; the elevated calcium levels in the hearts of animals treated with ADR found in other studies [ 8,9] and in our own have been reported to be the possible pathogenetic cause of the CMP; where rats were pretreated with ICRF 159, the myocardial calcium concentration was reduced to almost normal values. Furthermore, ICRF 159 alone significantly reduced ventricular calcium concentrations as compared with untreated controls; these data could be explained by the chelating action of ICRF, which is a non-polar derivative of EDTA [2]. Pretreatment with EDTA also reduced to almost normal values the myocardial calcium content. As the pretreatment with ICRF 159 significantly ameliorated the histopathologic lesions of the CMP while the pretreatment with EDTA did not have any protective action, we suggest that the myocardial calcium metabolism may not have a primary role in the pathogenesis of ADR CMP and that the effect of ICRF 159 on the myocardium is probably due to other, still unclear pharmacologic activity. However, as the reduction in ADR-induced myocardial injury by ICRF 159 is not accompanied by other signs of protection and, in fact, there is even an increase in the incidence of acute deaths, it seems appropriate to further investigate this combination therapy. ACKNOWLEDGEMENT
This work was supported by a special grant from the University of Trieste. We thank Dr. Remoli and Dr. Orpelli for their help with tissue calcium determination.
83 REFERENCES 1 Blum, R.H. and Carter, SK. (1974) Adriamycin - a new anticancer drug with significant clinical activity. Ann. Intern. Med., 80, 249-259. 2 Creighton, A.M., Hellmann, K. and Whitecross, S. (1969) Antitumor activity in a series of bis-diketopiperazines. Nature, 222, 384-385. 3 Dorochow, J.H., Locker, G.Y. and Myers, C.E. (1979) Experimental animal models of adriamycin cardiotoxicity. Cancer Treat. Rep., 63, 855-860. 4 Giuliani, F., Casazza, A.M., Di Marco, A. and Savi, G. (1981) Studies in mice with ICRF 159 combined with daunorubicin and doxorubicin. Cancer Treat. Rep., 65, 267-276. 5 Herman, E.H. and Ferrans, V.J. (1981) Reduction of chronic doxorubicin cardiotoxicity in dogs by pretreatment with (*)-1,2-bis(3,5-dioxopiperazinyl-l-yl)propane (ICRF-187). Cancer Res., 41, 3436-3440. 6 Herman, E.H., Mhatre, R.M., Lee, I.P. and Waravdekar, V.S. (1972) Prevention of the cardiotoxic effects-of adriamycin and daunomycin in the isolated dog heart (36432). Proc. Exp. Biol. Med., 140, 234-239. 7 Minow, R.A., Benjamine, R.S. and Gottlieb, J.A. (1975) Adriamycin (NSC-123127) cardiomyopathy: an overview with determination of risk factors. Cancer Chemother. Rep., Part 3, 6, 195-201. 8 Olson, H.M. and Capen, C.C. (1977) Subacute cardiotoxicity of adriamycin in the rat. Lab. Invest., 37, 386-394. 9 Olson, H.M., Young, D.M., Prieur, D., Le Roy, A. and Reagan, R. (1974) Electrolyte and morphologic alterations of myocardium in adriamycin-treated rabbits. Am. J. Pathol., 77,439-454. 10 Woodman, R.J., Cysyk, R.L., Kline, I., Gang. M. and Venditti, J.M. (1975) Enhancement of the effectiveness of daunorubicin (NSC-82151) or adriamycin (NSC-123127) against early mouse L1210 leukemia with ICRF 159 (NSC-129943). Cancer Chemother. Rep., 59, 689-695. 11 Zamboni, L. and De Martino, C. (1967) Buffered picric acid-formaldehyde: a new rapid fixative for electron microscopy. J. Cell. Biol., 35, 148 A.