WO1993002670A1

WO1993002670A1 - Radioprotection by calcium antagonists

Info

Publication number: WO1993002670A1
Application number: PCT/EP1991/001410
Authority: WO
Inventors: Georges Floersheim
Original assignee: Bayer Aktiengesellschaft
Priority date: 1991-07-27
Filing date: 1991-07-27
Publication date: 1993-02-18
Also published as: JPH06501917A; CA2092408A1; EP0550446A1

Abstract

The present invention relates to the use of calcium antagonists, combinations of two or more different calcium antagonists, or combinations of calcium antagonists and zinc salts for protecting warm blooded animals against deleterious effects of radiation, to the use for the manufacture of radioprotective pharmaceutical preparations, and to novel synergistic radioprotective pharmaceutical preparations. A representative example of a calcium antagonist is diltiazem.

Description

RADIOPROTECTION BY CALCIUM ANTAGONISTS

FIELD OF THE INVENTION

The present invention relates to the use of calcium antagonists, combinations of two or more different calcium antagonists, or combinations of calcium antagonists and zinc salts for protecting warm blooded animals against deleterious effects of radiation, to the use for the manufacture of radioprotective pharmaceutical preparations, and to novel synergistic radioprotective pharmaceutical preparations.

BACKGROUND OF THE INVENTION

Calcium antagonists are actual calcium channel bloc erε and as such wellknown in the pharmaceutical art as therapeutic agents for treating cardiovascular diseases. So far no report became known of their pro¬ tective effect against radiation.

The search for chemical compounds which protect against the tissue damage caused by exposure to ionizing radiation has led to the iden¬ tification of thiol (sulfhydryl) compounds with marked experimental activity (T.R. Sweeney, "A Survey of Compounds from the Antiradiation Drug Development Programm of the U.S. Army Medical Research and Development Command", Walter Read Institute of Research, Washington, DC 1979). However, the use of the most promising compound, namely WR 2721 (S-2-(3-aminopropylamino) ethylphosphorothioic acid), has been limited by poor clinical tolerance [A.B. Cairnie, Radiation Research 94, 221 (1983); and A.T. Turrisi, M.M. Kligerman, D.J. Glover, J.H. Glick, L. Norfleet and M. Gramkowski, in "Radioprotectorε and Anticarcinogens", F.O. Nygaard and M.G. Simic, Eds. Academic Press, New York, 1983, pp. 681-694; and A.L. Blumberg, D.F. Nelson, M. Gramkowski, D. Glover, J.H. Glick, J.M. Yuhas and M.M. Kligerman, Int. J. Radiation Oncology Biol. Phys. 8, 561 (1982)].

High toxicity of the thiol compounds, especially if applied in the doses required for achieving radioprotection, and the very short time they exert a radioprotective effect in human bodies, led to the development of products containg such thiol compounds in combination with salts of zinc as described in European Patent Application No. 245669. Such products showed as synergistic effect radioprotection by using small doses of thiols in combination with the indicated metal salts. However, the effect and the tolerance of the products are still low.

Accordingly there is a need for pharmaceutical preparations with ra¬ dioprotective effectβ, less side effects and a higher tolerance.

OBJECT OF THE INVENTION

It is an object of the present invention to overcome, or at least alleviate, one or more of the difficulties of the preparations of the prior art.

Surprisingly it was found that calcium antagonists, combinations of different calcium antagonists, or combinations of calcium antagonist and zinc salts are useful radioprotectors with a high tolerance.

Moreover, it was found that some combinations of calcium antagonists with radioprotectors from other classes or with other calcium antagonists brought forth synergiβm.

Furthermore, it was surprisingly found that radiation-induced tumor growth delay was not prevented by using radioprotecting calcium antagonists.

DETAILED DESCRIPTION OF THE INVENTION

In an aspect the invention concerns a method for protecting a warmblooded animal against deleterious effects of radiation comprising administering to said animal a radioprotectively effective amount of a calcium antagonist.

Warmblooded animals are in particular valuable mammals, e.g. farm, house or zoo animals, such as horses, cows, dogs, lions, tigers, elefants, and the like, respectively, which are subjects to veterinary medicine. The term warmblooded animal as used in this description also comprises humans as the preferred species.

Deleterious effects of radiation are in particular caused by ionizing radiation, which is radiation of high energy, such as of X-rays and radioactive decay.

The term calcium antagonist comprises all chemical compounds which block calcium channels in animal tissues. A huge number of calcium antagonists are known in the art. Of particular relevance are those of the piperazine or phenylalkylamine type, and especially those of the benzothiazepine and dihydropyridine type. Preferred are those calcium antagonists having been shown to be therapeutically useful for treatment of cardiovascular diseases in humans and of which pharmaceutical preparations are already available. Such calcium antagonists are for example diltiazem, e.g. Dilzem^R' nifedipine, e.g. Adalat^R, nimodipine, e.g. Nimotop^R, nitrendipine, e.g. Baypress , isradipine, e.g. Lomir^R, flunarizine, e.g. Sibelium^R, verapamil, e.g. Isoptin^R, and further nicardipine, niludipine, nigludipine, nisoldipine, felodipine, amlodipine, lacidipine, anipamil, ryosidine, fendiline, gallopamil, and tiapamil. More pertaining calcium antagonists are listed in Winifred G. Nayler, CALCIUM ANTAGONISTS, Academic Press, London,1988. A preferred calcium antagonist is diltiazem.

Instead of just one calcium antagonist a combination of two or more, e.g three, of such compounds, which is usually synergistic, may be administered, whereby a better radioprotective effect or less side effects are achieved. Synergistic combinations may contain both compounds in less than a useful radioprotective dose. For example admixtures of diltiazem and nifedipine, e.g. as shown in Table 6, are synergistic. Such synergistic combinations are also a subject of the present invention.

A further subject of the invention are synergistic combinations of a calcium antagonist and a radioprotective metal salt, such as a zinc salt, e.g. zinc aspartate, zinc histidine, zinc orotate or zinc acetate, for example a combination of diltiazem and zinc aspartate, e.g. as shown in Table 5.

The radioprotecive calcium antagonists or the synergistic combinations described herein before are administered before, during or after the irradiation. If administered after irradiation a surprising curative effect is observed. In cases where a deleterious effect of irradiation is expected to occur, the present radioprotectors are administered before, preferably shortly before, e.g. about 10 to 30 minutes before the irradiation takes place. In cases where the body is already contaminated with radioactive material, e. g. after a nuclear catastrophe, such as in nuclear warfare or accidents of nuclear reactors, e.g. Tschernobyl, the present radioprotectors should be administered during a long period, e.g. the entire irradiation time, i.e. as long as the radioactive material is able to create deleterious effects in the contaminated body, and advantageously also thereafter in order to take advantage of the curative effects. A prolonged administration may also be useful during a long space flight.

Surprisingly the radioprotective calcium antagonists do not protect tumors against ionizing radiation. This fact is important in the therapeutic irradiation of cancer patients, where a selective protection of normal tissues and a deleterious effect on tumor tissues is desired.

The present radioprotectors are administered orally or parenterally, e.g. subcutaneously, intraperitonealy, intramuscularly or intrave¬ nously, in amounts having the desired radioprotective, including radiocurative effect. The dosage depends on the radioprotective activity of the pharmaceutical preparation, the route of administration, the rate of its metabolism, the intensity of the irradiation, the species to be treated, the severity of the disease which is present or expected, and the weight and general condition of the patient, and has finally to be decided by the responsible physician. In general the dose is about the same as aplied for achieving the cardiovascular effect of the respective calcium antagonist, which dose is wellknown in the art, and is between about 0.2 and about 5 mg/kg. However, if need is there, also higher doses, e.g. up to about 20 mg/kg, may be administered. For example in man average daily doses of about 20 to about 6000 mg are administered orally or by parenteral infusion, however, in severe cases higher doses may have to be used.

Various tablets and ampoules for infusion are commercially available for cardiovasculare indications, e.g. of diltiazem (tablets with 60, 90, 120 and 180 mg, and ampoules with 10 or 25 mg in 2 or 5ml solvent, respectively), nifedipine (tablets with 5, 10 or 20 mg, and ampoules for infusion with 5 mg in 50 ml solvent), nimodipine (tablets with 30 mg, or ampoules for infusion with 10 mg in 50 ml solvent), nitrendipine (tablets with 10 and 20 mg), or isradipine (tablets 2,5 mg) .

The invention concerns also the use of a calcium antagonist, a combination of two or more calcium antagonists, or a combination of a calcium antagonist and a zinc salt for the manufacture of a pharmaceutical preparation for protecting a warmblooded animal against deleterious effects of radiation.

The calcium antagonists or the combinations of the present invention are administered orally or parenterally to achieve the radioprotective effect, in any of the usual pharmaceutical forms. These include solid and liquid unit oral dosage forms such as tablets, capsules, powders, suspensions, solutions, syrups and the like, including sustained release preparations, and fluid injectable forms, such as sterile solutions and suspensions. The term dosage form as used in this specification and the claims refer to physically discrete units to be administered in single or multiple dosage to animals, each unit containing a predetermined quantity of active material in association with the required diluent, carrier or vehicle. The quantity of active material is that calculated to produce the desired therapeutic effect upon administration of one or more of such units.

Powders are prepared by comminuting the compound to a suitably fine εize and mixing with a similarly comminuted diluent pharmaceutical carrier, such as an edible carbohydrate material as for example, starch. Sweetening ,flavoring, preservative, dispersing and coloring agents can also be added.

Capsules are made by preparing a powder as described above and filling formed gelatin sheaths. A lubricant, such as talc, magnesium stearate and calcium stearate can be added to the powder mixture as an adjuvant before the filling operation. A glidant such as colloidal silica may be added to improve flow properties. A disintegrating or solubilizing agent may be added to improve the availability of the medicament when the capsule is ingested.

Tablets are made by preparing a powder mixture, granulating or slugging, adding a lubricant and disintegrant and pressing into the desired form. A powder mixture is prepared by mixing the compound, suitably comminuted, with a diluent or base such as starch, sucrose, kaolin, dicalcium phosphate and the like. The powder mixture can be granulated by wetting with a binder such as syrup, starch paste, acacia mucilage or solutions of cellulosic or polymeric materials and forcing through a screen. As an alternative to granulating, the powder mixture can be run through the tablet machine and the resulting imperfectly formed slugs broken into granules. The granules can be lubricated to prevent sticking to the tablet forming dies by means of the addition of stearic acid, a stearate salt, talc or mineral oil. The lubricated mixture is then pressed into tablets. The medicaments can also be combined with free flowing inert carriers and compressed into tablets directly without going through the granulating or slugging steps. A protective coating consisting of a sealing coat of shellac, a coating of sugar or polymeric material and polish coating of wax can be provided. Dyestuffs can be added to these coatings to distinguish different unit dosages.

Oral fluids such as syrups and elixirs can be prepared in unit dosage form so that a given quantitiy, e.g. a teaspoonful, contains a predetermined amount of the compound. Syrups can be prepared by dissolving the active compound in a suitably flavored aqueous sucrose solution, while elixirs are prepared through the use of a non-toxic alcoholic, e.g. ethanolic, vehicle. Suspensions and emulsions can be formulated by dispersing the medicament in a non-toxic vehicle. For parenteral administration, fluid unit dosage forms can be prepare by suspending or dissolving a measured amount of the active material i a non-toxic liquid vehicle suitable for injection such as an aqueous, alcoholic, e.g. ethanolic, or oleaginous medium. Such fluid dosag unit forms may contain solubilizers, such as a polyethyleneglycol stabilizers, and buffers, such as a citric acid/sodium citrate buffer, to provide the desired osmotic pressure. Alternatively a measure amount of the active material is placed in a vial and the vial and it content are sterilized and sealed. An accompanying vial or vehicle ca be provided for mixing prior to administration.

Important embodiments of the present invention are the pharmaceuticall acceptable salts of the basic calcium antagonists of the presen invention. Such salts include those derived from both organic an inorganic acids such as, without limitation, hydrochloric, hydrobromic, sulfuric, phosphoric, methansulfonic, acetic, lactic, succinic, malic, maleic, aconitic, phthalic , tartaric, embonic, enanthic and the lik acids.

An important aspect in the preparation of fluid dosage forms is the use of solvents which by themselves have a certain radioprotective effect, e.g. the use of an alcohol, in particular ethanol, which may be present in amounts of about 5 to about 30%, e.g. in amounts of about 15 to 20%.

If combinations of two or more calcium antagonist or combinations of a calcium antagonist and a radioprotective salt are envisaged such combinations may be used separately and simultaneously or consecutively, or otherwise formulated together in one pharmaceutical preparation according to the methods described above.

SHORT DESCRIPTION OF THE FIGURES

FIGURE 1: Shown are the survival rates (%) of female C3H mice up to 30 days after s.c. administration of O.lml/10 g of distilled water (l;β), 110 (2;o), 55 (3;D) and 27.5 mg/kg (4;Δ) of diltiazem 15 min before, and of 110 mg/kg (5;V) of diltiazem 10 min after lethal irradiation with a ⁶⁰cobalt source.

FIGURE 2A: Shown are the survival rates (%) of female C3H mice up to 30 days after i.p. administration of 1.5 mg/kg of nifedipine (11;H) and 0.15 ml/kg of the solvent of nifedipine (12; )30 min before irradiation with 8-5.Gy (0.9 Gy/min) from a ^°cobalt source, and of the control group treated with 0.15 ml/10 g of distilled water (c;#).

FIGURE 2B: Shown are the survival rates (%) of female C3H mice up to 30 days after i.p. administration of 2 mg/kg of nimodipine (13;Q) and O.lml/lOg of the solvent of nimodipine (14;Δ) 30 min before lethal irradiation with 8.5 Gy (0.9 Gy/min) from a ⁶⁰cobalt source, and of the control group treated with 0.1 ml/10 g of distilled water (c;#).

FIGURE 3Aϊ Shown are the survival rates (%) of male C3H mice up to 30 days after i.p. administration of 4 mg/kg of nimodipine (15;D), 0.2 ml/lOg of ethanol (23.7%) (16;Δ), 3 mg/kg of nifedipine (17;B) and 0.3 ml/10 g of ethanol (18%) (18;_fc) 30 min before irradiation with 8.1 Gy (0.9 Gy/min) from a ^°cobalt source, and of the control group treated with 0.2 ml/10 g of distilled water (c;#) .

FIGURE 3B: Shown are the survival rates (%) of male C3H mice up to 30 days after i.p. administration of 4 mg/kg of nimodipine (19;D). 0.2 ml/10 g of ethanol (23.7.%) (20;A). 3 mg/kg of nifedipine (21;B) and 0.3 ml/lOg of ethanol (18%)(22;Λ) 30 min before irradiation with a supralethal dose of 9 Gy (0.9 Gy/min) from a ⁶⁰cobalt source, and of the control group treated with 0.2 ml/10 g of distilled water (c;#).

FIGURE 4: Shows the synergistic effect of a combination of diltiazem and zinc aspartate.

FIGURE 5: Shows the synergistic effect of a combination of diltiazem and nifedipine (see legend to FIGURE 4) .

FIGURE 6: Shows the average volumes (mm^) up to 30 days of an Ewing's sarcoma transplanted as xenografts into male C3H mice untreated (l;o) and pretreated intraperitoneally 30 min or subcutaneously 15 min before irradiation at day 12 with a ⁶⁰cobalt source with 110 mg/kg s.c. of diltiazem (2;B), 3 mg/kg i.p. of nifedipine (3;^) or 4 mg/kg i.p. of nimodipine (4;ψ), or 3 ml/10 g solvent of nifedipine (6; ) and of the unirradiated statistically significant (xxx) controls (5;*).

FIGURE 7: Shows the average volumes (mm³) up to 27 days of an adenocarcinoma of the colon, transplanted as xenograft into male C3H mice untreated (l;o) and pretreated intraperitoneally 30 min or subcutaneously 15 min before irradiation at day 12 with a ⁶⁰cobalt source with 100 mg/kg nitrendipine (2;4) or 3 mg/kg of nifedipine (3;A) and of the unirradiated controls (4;*), (xxx) indicating statistical significant values.

FIGURE 8: Shows the average volumes (mm³) up to 23 days of an adenocarcinoma of the colon, transplanted as xenograft into male C3H mice pretreated intraperitoneally 30 min or subcutaneously 15 min before irradiation at day 12 with 5.625 Gy of a ^°cobalt source with 110 mg/kg s.c. of diltiazem (2;B), 3 mg/kg i.p. of nifedipine (3;A) or 4 mg/kg i.p. of nimodipine (4; ), and of unirradiated (1;#) and irradiated controls (5;o), (xxx) indicating statistical significant values.

FIGURE 9: Shows the average volumes (mm³) up to 23 days of an adenocarcinoma of the colon, transplanted as xenograft into male C3H mice, pretreated intraperitoneally 30 min before irradiation at day 12 with 3.5 Gy of a ⁶⁰cobalt source with 3 mg/kg of nifedipine (2; i) or 4 mg/kg of nimodipine (3;^), and of irradiated (l;o) and unirradiated controls (4;β), (xxx) indicating statistical significant values.

The surprising advantageous properties of the calcium antagonists, the combinations of calcium antagonists or combinations of calcium antagonists with metal salts have been confirmed and verified by the following tests.

GENERAL PROCEDURE OF EXAMPLES 1-7

Calcium antagonists, combinations of calcium antagonists or combinations of calcium antagonists with zinc aspartate were tested at their highest tolerated dose against a lethal radiation ( D^QO) -ⁿ πtice (groups of at least 12 mice were used) and for their ability to confer protection when administered at sublethal doses. For this purpose the L 50 of each calcium antagonist and of zinc aspartate was first determined and then tested at the 1/2 LD50 for antiradiation activity.

The efficacy of the test compounds were measured by their ability to protect the mice against a lethal dose of X-rays, protection being defined as survival after 30 days. An attempt was made to investigate the therapeutic ratio of effective calcium antagonists by reducing each dose by one half. The results are shown in the TABLES and the FIGURES.

The test compounds were administered subcutaneously, intraperitoneally or orally, however, other routes may be applied as well.

EXAMPLE 1: RADIOPROTECTIVE EFFECT OF DILTIAZEM

Male and female C3H mice of a weight of 22 to 24 g were used for the experiments. Commercially available ampoules of diltiazem containing 25 mg of diltiazem hydrochloride (in the following only the term diltiazem is used) and 150 mg of mannitol were used. The content of the ampoules was dissolved in distilled water. Control experiments ascertained that mannitol alone did not exert radioprotection. Different doses of diltiazem were administered subcutaneously (Groups 1 to 5), orally (Group 6) or intraperitoneally (Groups 7a to 10c) before or after irradiation according to TABLE 1.

The control group of mice received subcutaneously or intraperitoneally 0.1 ml/10 g body weight of distilled water.

The groups of at least 12 mice were irradiated in a perforated plexiglass chamber with X-rays from a ⁶°cobalt source (Gammatron) at a targent distance of 80 cm. Dosimetry was performed with a strontium- calibrated ionization chamber. Irradiations were performed either with 1Q.5 Gy (1 Gy = 100 rad) at a dose rate of 0.15 Gy/min, or with 8.5 Gy with a dose rate of 0.9 Gy/min. These doses, delivered by two different 6°cobalt sources, corresponded to the respective LD^oo and yielded equal medial survival times. Accordingly, they were biologically isoeffective and the results obtained with both radiation sources could be pooled.

The results are shown in TABLE 1 and FIGURE 1.

TABLE 1: Survival rate of female (f) and male (m) C3H mice treated with diltiazem after lethal irradiation with a ^°cobalt source

1) s.c. = subcutaneously; i.p. = intraperitoneally; p.o. = orally

2) - : time before irradiation; + : time after irradiation

3) survival rate of tested mice 30 days after administration of test compound and irradiation

The survival rates of Groups 1 to 5 over the time period of 30 days are shown in FIGURE 1.

The calcium antagonist diltiazem clearly increased the survival rate of the treated mice after irradiation with lethal doses of X-rays. Diltiazem at the dosage of 110 mg/kg (1/2 LD₅₀), 55 mg/kg and 27.5 mg/kg provided survival of 93%, 58% and 17%, respectively. A significant survival rate of 42% was also observed by administration of a curative dose of 110 mg/kg 10 min after the completion of irradiation (Group 5). No statistal significant differences were seen between male and female mice, between subcutaneous and intraperitoneal administration of diltiazem, and if the treatmentt was performed 10, 15 or 30 min before irradiation.

The radioprotective effects of diltiazem is also confirmed in similar tests with other inbred mouse strains including Balb/c and C57 Bl/6, and the outbred albino strain NMRI.

EXAMPLE 2: RADIOPROTECTIVE EFFECTS OF NIFEDIPINE, NIMODIPINE AND SOLVENTS IN FEMALE MICE

EXAMPLE 1 was followed except as described hereinafter. Commercially available pharmaceutical preparations of nifedipine and nimodipine were used in their respective solvents [see index 5) and 7) of TABLE 2] .

Groups of female mice were irradiated with 8.5 Gy (dose rate 0.9 Gy/min). Their radiation LD50 was approximately 7.75 GY. The compounds were intraperitoneally injected 30 min before irradiation. For a better detection of their relative contributions, the calcium antagonists and their solvents were applied only at half of their optimal dose.

The results are compiled in TABLE 2 and FIGURES 2A and 2B.

TABLE 2: Survival rate of female (f) C3H mice treated with nifedipine or nimodipine in their respective solvents or with the solvents alone after lethal irradiation with a ⁶⁰cobalt source

Group dose (mg/kg) adm.¹⁾ time^⁾ sex surv.rate3)

1), 2) and 3) as in EXAMPLE 1 4) in 0.15 ml/10 g of solvent 5) 5) 50 ml solvent of nifedipine contain: 7.5 g of ethanol 96%, 7.5 g of polyethyleneglycol 400, and 35 g of distilled water

6) in 0.1 ml/10 g of solvent 7)

7) 50 ml solvent of nimodipine contain: 10 g ethanol 96%, 8.5 g of polyethyleneglycol 400, 0.1 g of tertiary sodium citrate, 0.015 g of citric acid, and 31,265 g of distilled water

Similar dose dependent survival was observed with nifedipine and nimodipine in lethally irradiated mice. However, for parenteral administration, these calcium antagonist were administered in their solvents defined under 5) and 7) of TABLE 2. In order to avoid confounding the additive or synergistic effects of the solvents with those of nifedipine and nimodipine, it was necessary to differentiate between the relative contributions of the solvents and the calcium antagonists. As shown in TABLE 2 the survival of lethally irradiated mice was 100% with 1.5 mg/kg of nifedipine and 61% with its solvent alone, and 82% with nimodipine and only 42% with its solvent alone. This indicates that the solvents alone offer also some radioprotection. The differences of the survival rates between the calcium antagonists and their respective solvents are statistically significant. Accordingly, after subtraction of the solvent effects, the residual radioprotection of 39% with nifedipine and 40% with nimodipine must be ascribed to the calcium antagonists.

FIGURE 2A shows the survival rates up to 30 days of Groups 11 (■) and 12 (Y) and of the control group (c).

FIGURE 2B shows the survival rates up to 30 days of Groups 13 (D) and 14 (Δ)and of the control group (c).

It is assumed that the protective activity of the solvents is due to the wellknown radioprotective effect of the ethanol component, if administered in very high quantities. This assumption was corroborated by the fact that the solvents of the calcium antagonists as compared to the respective amounts of ethanol in the solvents afforded radioprotection to a similar and statistically not sigificantly differing extent. EXAMPLE 3: RADIOPROTECTIVE EFFECTS OF NIFEDIPINE, NIMODIPINE AND ETHANOL IN MALE MICE.

With exeption of the following, the procedure of EXAMPLE 1 was repeated. The intrinsic radioprotection by the calcium antagonists was also clearly displayed in a further experiment designed to compare nifedipine and nimodipine with ethanol in more radiosensitive male C3H mice irradiated with 8.1 Gy (Groups 15-18; FIGURE 3A) or the supralethal dose of 9.0 Gy (Groups 19-22; FIGURE 3B)

The procedure is analogous to that of EXAMPLE 1. The radiation LD_5Q in these mice was 6.35 Gy (dose rate 0.9 Gy/min). The test compounds were intraperitoneally injected 30 min. before the start of the irradiation. The mice received either nifedipine or nimodipine with their solvents, or ethanol at the dose corresponding to the quantity present in the solvents. The results are compiled in TABLE 3 and FIGURES 3A and 3B.

TABLE 3: Survival rate of male (m) C3H-mice treated with nifedipine or nimodipine in their solvents or with the ethanol alone after lethal or supralethal irradiation with a ⁶⁰cobalt source

Groupe dose (mg/kg)^⁾ adm.^⁾ time^) sex surv.rate³) ^Gv

1), 2), 3) an 4) see EXAMPLES 1 and 2

7) amount of ethanol corresponding to the amount of ethanol in the solvents of nifedipine and nimodipine. If administered i.p. with their solvents 30 min before irradiation with 8.1 Gy, nifedipine afforded a survival rate of 67 % and nimodipine of 75 %, as compared to a survival rate of 25 % and 8 %, respectively, provided by ethanol administered i.p. in doses corresponding to the quantities contained in the solvent (FIGURE 3A) . Mice similarly treated but irradiated with 9.0 Gy showed a survival of 58 % with nifedipine and 55 % with nimodipine applied in their solvents while the corresponding doses of ethanol allowed no survival but only a prolongation of the mean survival time from 6.6 + 1.6 days (controls) to 11.1 + 2.1 days (FIGURE 3B).

Treatments were as follows:

- Controls: 0.2ml dist. water/lOg body weight (c); nifedipine, 3mg/kg in 0.3ml solvent/10 g (17 and 21);

0.3ml/10g of an 18 % ethanol solution in dist water corresponding to 4590mg/kg ethanol (18 and 22);

- nimodipine, 4mg/kg in 0.2ml solvent/10 g (15 or 19);

0.2ml/10g of an 23.7 % ethanol solution (4050mg ethanol/kg) in dist. water (16 and 20).

For reference reasons, less radiosensitive female mice were treated as stated for group 17, 18, 19 and 20. Survival rates were 83%, 28%, 100% and 39%, respectively.

A further attempt has been made in order to distinguish the effects of nifedipine and nimodipine from those due to their solvents by determining the average weight of the mice used in the test. The weight loss after irradiation with 9.75 Gy in groups of female C3H mice and treatment with nimodipine and nifedipine was from day 8 to day 29 with a difference of 2 to 4 g significantly lower than in groups treated with the respective solvents.

EXAMPLE 4: RADIOPROTECTIVE EFFECTS OF ISRADIPINE AND NITRENDIPINE

With exception of the following, the procedure of EXAMPLE 1 was repeated. The results are summarized in Table 4. TABLE 4: Survival rate of female (f) C3H mice treated with isradipine (in an oily solution) or nitrendipine (suspended in water) after lethal irradiation with a ^°cobalt source

Groupe dose (mg/kg) adm. ⁾ time^⁾ sex surv.rate 3)

23 27.5 isradipine i.p. 24 27.5 isradipine i.p.

25 27.5 isradipine i.p. 26 100 nitrendipine i.p. 27 100 nitrendipine i.p. 28 50 nitrendipine i.p. 29 50 nitrendipine i.p.

1), 2) and 3) as in EXAMPLE 1

EXAMPLE 5s SYNERGISTIC RADIOPROTECTIVE EFFECTS OF A COMBINATION OF DILTIAZEM AND ZINC ASPARTATE, A COMBINATION OF DILTIAZEM AND NIFEDIPINE, AND A COMBINATION OF NITRENDIPINE AND ZINC- ASPARTATE.

With exception of the following, the procedure of EXAMPLE 1 was repeated. Isobolograms were used to determine synergistic effects by the combination of two radioprotectors which alone allowed at optimal dosage survival of not less than 50 % against the radiation LD 00.

Isoeffective doses of each compound were marked on the coordinates [see FIGURE 4 (zinc aspartate and diltiazem) and FIGURE 5 (nifedipine and diltiazem)]. A hyphenated line was drawn between the lowest tested doses of two compounds wich confered each survival of not less than 50 % (lowest effective dose, LED). Lowest effective doses were as follows: diltiazem: 110 mg/kg nifedipine: 3 mg/kg zinc aspartate: 30 mg/kg The dose combinations lying on the hyphenated line drawn between the LED of the two combined compounds represent the hypothetical amounts of both compounds required to allow not less than 50 % survival if the interactions were additive. Points at the left of this line indicate synergistic interaction between two compounds and can be used to construct the concave isobols. Both compounds were tested in a checker¬ board fashion and in general as serially twofold reduced fractions. Synergism between two compounds is said do occur from a combination where each compound is used at a dose of less than 1/2 LED or at least one compound being used at the 1/2 LED an the other below the 1/2 LED. Accordingly, all dose combinations providing survival of not less than 50 % and situated left of the line representing additive interactions indicate synergism.

The synergistic effect can also be demonstrated by the test results shown in TABLE 5 and TABLE 6.

Table 5: Survival of C3H mice irradiated with 10.5 Gy and treated with diltiazem, zinc aspartate or combinations of both.

1) p<0.005; 2) p<0.02; 3) p<0.01; n.d.= not done

1), 2) and 3) indicate synergism and the statistical significance.

In combinations of diltiazem with zinc aspartate, survival of 44 % obtained with 55 mg/kg of diltiazem alone was increased by combining it with the alone ineffective doses of 5 or 10 mg/kg of zinc aspartate to 92% and 75%, respectively. Similarly, survival of 17% with 27,5 mg/kg of diltiazem was enhanced to 50 % with 15 mg/kg of zinc aspartate and to 58% with 10 mg/kg of zinc aspartate. Table 6: Survival of C3H mice irradiated with 10.5 GY and treated with diltiazem, nifedipine and combinations of both:

1) p<0.05; n.d. = not done

1) indicates the occurrence of synergism and the statistical significance.

Survival of 44% obtained with 55 mg/kg of diltiazem could be increased to 83% by combining it with the alone ineffective dose of 0.375 mg/kg of nivedipine. Survival of 17 % seen with 27.5 mg/kg of diltiazem was enhanced to 50 % by the addition of 0.75 mg/kg of nifedipine, which was also ineffective alone.

A further test with nitrendipine and zinc aspartate enhanced the survival afforded by 15 mg/kg of zinc aspartate from 0% to 100% by adding 50 mg/kg of nitrendipine.

EXAMPLE 6: RADIOPROTECTIVE EFFECT OF DILTIAZEM BEFORE AND AFTER IRRADIATION.

The procedure according EXAMPLE 1 was repeated with the following exceptions. The survival rate of C3H mice was measured when irradiated with 10.5 Gy and treated with 110 mg/kg of diltiazem administered either by the oral route 30 min before irradiation, which confers a significant survival rate of 56%, or subcutaneously 10 min after irradiation, which still allows a survival rate of significant 42%. Thus, diltiazem shows also curative effect. EXAMPLE 7: RADIOPROTECTIVE EFFECTS OF DILTIAZEM, NIFEDIPINE, NIMODIPINE AND EFFECT ON TUMOR GROWTH DELAY

Male C3H mice weighing 25-30 g and fed with Nafag pellets and water ad libitum were used. Groups of mice were irradiated according to the procedure of EXAMPLE 1. Total body irradiation was given with sublethal doses in general at day 12 after transplanting a human xenograft.

Three different human tumors were used, namely an Ewing'ε sarcoma and two adenocarcinomas of the colon, and transplanted into immunosup- pressed mice.

The first tumor measurement was performed on the day before or the day of irradiation. Tumor growth was then followed by measuring the three main perpendicular diameters with calipers. Tumor volumes were measured on the days indicated in FIGURES 6 to 9 and expressed as the product of the three diameters in cubic millimeters. The scoring was stopped when necrosis and exulceration of large tumors made reliable measurements impossible. The mean values of the tumor values for each group were tabulated and entered into the FIGURES.

Tumor volumes not significantly larger than in the irradiated, untreated control group indicated that the used agent failed to protect the tumor from radiation-induced regression. Smaller tumors than in the irradiated controls indicated sensitization of the tumor to radiation and larger tumors up to the values in the unirradiated controls would have indicated radioprotection of the tumors.

The content of ampoules containing 25 mg of diltiazem and 150 mg of mannitol was dissolved in distilled water and 0.1 ml/10 g was administered to the mice (mannitol alone did not show any radioprotective effect). Nifedipine and nimodipine were used in their respective solvents (see EXAMPLE 2). Nitrendipine was injected as suspension in distilled water (0.1 ml/10 g) . Nifedipine solvent and 18% ethanol were applied at 0.3 ml/10 g, and nimodipine solvent and 23.7% ethanol at 0.2 ml/10 g body weight. The drugs were administered intraperitoneally 30 min before irradiation started, with the exception of diltiazem which was subcutaneously administered 15 min before the start of irradiation. In all experiments the difference between tumor volume of the unirradiated controls and the group which was only irradiated was significant from a few days after the irradiation to the end of the experiment. On the other hand, the difference between tumor volume of the irradiated control and the pretreated irradiated groups was at no instance significant, indicating that the calcium antagonist failed to protect the human tumors from radiation induced regression.

The results can be taken from FIGURES 6 to 9.

FIGURE 6: The changes of the average volumes (mm³) of an Ewing's sarcoma irradiated at day 12 with 4.875 Gy from a ^cobalt source up to 30 days are shown. No significant differences can be seen between the irradiated controls (1) and the groups pretreated with 110 mg/kg of diltiazem (2), 3 mg/kg of nifedipine (3) or 4 mg/kg of nimodipine (4). For comparison curve (5) shows the results of unirradiated controls, and curve (6) those obtained with 3 mg/kg of the solvent of nifedipine. Asterisks . indicate statistical significance of differences between unirradiated and irradiated controls.

FIGURE 7: The changes of the average volumes (mm³) of an adenocarcinoma of the colon irradiated a day 12 with 5.625 Gy are shown. Pretreatment with nitrendipine led to tumor volumes comparable with the irradiated controls. In the group pretreated with nifedipine, delay of tumor growth was even more marked than in the irradiated controls. The curves (1), (2), (3) and (4) depict the results obtained with irradiated controls, 100 mg/kg of nitrendipine, 3mg/kg of nifedipine, and unirradiated controls, respectively. Asterisks depict statistical significance of differences between unirradiated and irradiated controls, and between groups pretreated with nifedipine and irradiated controls.

FIGURE 8: The results of another experiment are shown where groups of mice carrying an adenocarcinoma of the colon were irradiated as described under FIGURE 7. The results indicate that 110 mg/kg of diltiazem (2), 3 mg/kg of nifedipine (3) or 4 mg/kg of nimodipine (4) did not reduce radiation-induced tumor growth delay. Curves (1) and (5) show the results of unirradiated and irradiated controls, respectively. Asterisks indicate statistical differences as explained above.

FIGURE 9: Similar results as described in FIGURE 8 were obtained when groups of mice were irradiated at day 12 with 3.5 Gy after pretreatment with 3 mg/kg of nifedipine (2) or 4 mg/kg of nimodipine (3). Curves (1) and (4) show irradiated and unirradiated controls, respectively.

EXAMPLE 8: TABLETS

Tablets containig each 180 mg of diltiazem are manufactured by admixing carefully 18 kg of diltiazem with 90 kg of mannitol (or lactose) and pressing the mixture into 10⁵ tablets.

In a similar manner tablets with the desired amount of another active ingredient can be manufactured.

EXAMPLE 9: DRY AMPOULES

Dry ampoules containing each 25 mg of diltiazem are prepared by mixing carefully 25 kg of diltiazem with 150 kg of granulated mannitol and filling the mixture into 10⁶ ampoules.

In a similar manner ampoules with the desired amounts of another active ingredient can be manufactured.

EXAMPLE 10: AMPOULES FOR INJECTION

Ampoules for injection each containing 100 mg of nifedipine are manufactured by disolving 10 kg of nifedipine in 10³ kg of a solution comprising 150 kg of ethanol (96%), 150 kg of polyethyleneglycol 400 and 700 kg of distilled water, sterilizing this mixture and filling it in 10⁵ sterilized ampoules each comprising 100 mg of active compound in 10 g of solution. In a similar manner ampoules with the desired amount of another active ingredient can be manufactured.

Claims

1. A method for protecting a warmblooded animal against deleterious effects of radiation comprising administering to said animal a radioprotectively effective amount of a calcium antagonist.

2. A method according to claim 1, wherein said warmblooded animal is mammal.

3. A method according to claim 1, wherein said warmblooded animal is a human.

4. A method according to claim 1, wherein said calcium antagonist is of the piperazin or phenylalkylamine type.

5. A method according to claim 1, wherein said calcium antagonist is of the benzothiazepine or dihydropyridine type.

6. A method according to claim 1, wherein said calcium antagonist is diltiazem, nifedipine, nimodipine, nitrendipine or isradipine.

7. A method according to claim 1, wherein said calcium antagonist is flunarizine, verapamil, nicardipine, niludipine, niεoldipine, felodipine, amlodipine, lacidipine, tiapamil, nigludipine, anipamil, ryosidine, fendilene or gallopamil.

8. A method according to claim 1, wherein said calcium antagonist is in combination with one or more other calcium antagonists.

9. A method according to claim 1, wherein said calcium antagonist is in combination with a radioprotective metal salt.

10. A method according to claim 1, wherein said calcium antagoniεt is orally or parenterally adminiεtered in form of a pharmaceutical preparation.

11. The uεe of a calcium antagoniεt, a combination of two or more calcium antagonists, or a combination of a calcium antagoniεt and a zinc salt for the manufacture of a pharmaceutical preparation forpro¬ tecting a warmblooded animal against deleterious effects of radiation.

12. The use of a calcium antagonist, a combination of two or more calcium antagonists, or a combination of a calcium antagonist and a zinc salt for the manufacture of a pharmaceutical preparation to be used in a method according to anyone of claim 1 to 10.

13. A synergistic pharmaceutical combination comprising two or more calcium antagonists or a calcium antagonist and a radioprotective me¬ tal salt.

14. A combination according to.claim 13, wherein said calcium anta¬ gonists are diltiazem and nifedipine or said calcium antagonist and radioprotective metal salt are diltiazem and zinc aspartate.