Note: Descriptions are shown in the official language in which they were submitted.
93/02670 ~ Q`~ PCl/E~91/01410
R~DlOPROTEcTIoN BY CAL~U~ ANTAGONI6~8
FIELD O~ THL INVENTION
The present invention rel~tes to the u~e of calcium antagonlsts,
combination~ of two or more different calcium antagoni~tfi, or
combination~ of calclum antagonl~ts nnd ~nc ~alts for protecting warm
blooded animal~ again~t deleteriou~ effect6 of radiation, to the uae
for the manufacture of radioprotective pha~maceutical preparations, and
to novel ~ynergi~tic radioprotectlve pharmaceutical preparation~.
BACXGROUND OF ~HE INVENTION
Calcium antagonist~ are actual calcium channel blocker6 and as such
wellknown in the pharmaceutical art a6 therapeutic agent~ for treating
cardiova~cular di~ea~e6. So far no report became known of their pro-
tective effect against radiation.
The search for chemical compounds which protect again~t the ti~sue
damage cau~ed by exposure to ioni7ing radiation ha~ led to the lden-
ti~ication of thiol (sulfhydryl) compound~ with marked experimental
activity (T.R. Sweeney, "A Survey of Compounds from the Antiradiation
Drug Development Programm of the U.S. Army MedLcal Re~earch and
Development Command~, Walte~ Read In6titute o~ Re~earch, Wa~hington, DC
1979). However, the u~e of the mo6t promising compound, namely WR 2721
(S-2 (3-aminopropylamino) ethylpho3phorothioic acid)I has been limited
by poor clinical tolerance lA.B. Cairnie, Radiation ~e~earch 94, 221
(1983); ~nd A.T. Turrisi, M.M. X~igerman, D.J. Glover, J.H. Glick, L.
Norfleet and M. Gramkowski, in "Radioprotectors and AnticArcinogensn,
F.O. Nygaard and M.G. Simic, Ed~. Academic Press, New York, 1983, pp.
681-694; and A.L. Blumberg, D.E. Nel~on, M. Gramkowski, D. Glover, J.H.
Glick, J.M. Yuha~ and M.M. Rligerman, lnt. J. Radiation Oncology Biol.
Phy~. 8, 561 (1982)~.
High toxic~ty of thP thiol compound~, c~pecially lf applied in the
dose3 requlred for achieving radioprotection, and the very ~hort tlme
they exert a radioprotective eff~ct in human bodl~s, led to the
~2~
development of product0 containg such thlol ~ompound~ ~n combinatlon
wlth nalts of zinc as descrlbed in European Patent Appllc~tion No.
245669. Such products ~howed a~ ~ynergi8tLc effect radioprotectLon by
u~ing ~mall doses of thiol~ $n combination with the indicated metal
salt~. However, the effect ~nd the tolerance of the products are still
low.
Accordlngly there i~ a nesd for pharmaceutlcal preparations wlth ra
dioprotective effects, le~s ~ide eff~ct~ and a higher toleranc0.
OBJECT OE' THE INVENTION
It ia an object of the present invention to overcome, or at least
alleviate, one or mDre of the difficulties of the preparations of the
prior art.
surprisingly it was found that calcium antagonists, combinations of
different calcium antagoni3ts, or combination3 of calcium antagonist
and zinc salt6 are useful radioprotector~ with a high tolerance.
~oreover, it was found that some combinations of calcium antagonists
with radioprotectors from other classe6 or with other calcium
antagoni~ts brought ~orth synerglsm.
Furthermore, it was surpriaingly found that radiation-induced tumor
growth delay wa~ not prevented by u6ing radioprotecting calcium
antagoni~ts.
DETAIL2D DESCRIPTION 0~ THE INVENTIOW
In an aspect the invention concern~ a method for protecting awarmblooded animal against deleterious effects of radiation comprising
Administering to Qaid animal a radioprotectively effective amount of a
calcium antagoni~t.
WarmbloDded animals are in particular valuabl~ mammals, e.g. fanm,house or zoo ~nimals, ~uch A6 horses, COWG, dogs, lion6, tiger6,
el~fants, nd the like, re~pectively, which are sub~ect3 to veterinary
/U2~ 8 ~ 41
medicine. The term warmblooded animal as u~ed in this description also
comprises humans as the preferred species.
Deleterious e~fects of radiation are in particular caused by ionizlng
radiation, which is radiation of high energy, such as of X-ray~ and
radloactive decay.
The term calcium antagonist comprises all chemical compounds whichblock calcium channels in animal ti3sues. A huge number of calcium
antagoni3ts 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 di~eases in humans and of which
pharmaceutical preparations are already available. Such calcium
antagonists are for example diltiazem, e.g. DilzemR~ nifedipine, e.g.
AdalatR, nimodipine, e.g. NimotopR, nitrendipine, e.g. BaypressR,
isradipine, e.g. LomirR, flunarizine, e.g. SibeliumR, verapamil, e.g.
IsoptinR, and further nicardipine, niludipine, nigludipine,
nisoldipine, felodipine, amlodipine, lacidipine, anipamil, ryosi~ine,
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 u~ually synergistic, may be
administered, whereby a better radioprotective effect or les~ side
effects are achieved. Synergistic combinations may contain both
compound3 in less than a useful radioprotective dose. For example
admixtures o~ diltiazem and nifedipine, e.g. as shown in Table 6, are
3ynergistic. Such synergistic combinations are also a 6ubject of the
pre~ent invention.
A further 3ubject of the inv~ntion are ~ynergistic combinations of a
calcium antagonist and a radioprotective metal salt, su~h a5 a zinc
~1 I~TI-rl 17-~ ~L~T
salt, e.g. zinc a~partate, ~inc histldine, zinc orotate or zinc
acetate, for example a comblnation of diltiazem and zinc aspartate,
e.g. as shown in Table 5.
The radloprotecLve calcium antagonlsts or the synerglstic combinations
described herein bsfore are administered bafore, during or after the
irradiation. If administered after irradlation a surpri~ing curative
ef~ct is observed. In ca~es where a deleterious effect of irradiation
i8 expected to occur, the present radloprotectors are administered
before, preferably ahortly before, e.g. about 10 to 30 minutes before
the irradiation take~ place. In cases where the body is already
contaminated with radioactive material, e. g. after a nuclear
catastrophe, such Q3 in nuclear warfare or accidents of nuclear
reactor~, e.g. Tschernobyl, the pre9ent radioprotectors should be
adminlstered durlng a long period, e.g. the entlre irradiation time,
i.e. as long as the radioactivs material i9 able to create deleterioua
effects in the contaminated body, and advantageously also thereafter in
order to take advantage of the curative effects. ~ prolonged
administration may also be u~eful during a long space flight.
Surprisingly the radioprotective calcium antagoniats do not protect
tumors against ionizing radiation. This fact is important in the
therapeutlc irradiation of cancer patients, where a selective
protection of normal tissues and a deleteriou5 effect on tumor tis~ues
is deslred.
The present radioprotectors are administered orally or parenterally,
e.g. subcutaneously, intraperitonealy, intramuscularly or intrave-
nously, Ln amounts having the d~sired radioprotective, including
radiocurative effect. The do age 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 disea~e
which is pre~ent or expected, and the weight and general condition of
the patient, and has finally ~o be decided by th~ responsible
physician. In general the do~e is about the ~ame as aplied for
- achieving the cardiovascular eff~ct of the respective calclum
antagonist, which dose 19 wellknown in the art, and i9 between about
SllÇ~ST~Tl)TE SHEET
93/02~7~ PCT/FP91/01410
0.2 and about 5 mg/kg. However, $f need i~ there, 1DO higher- dosPs,
.9. up to about 20 mg/kg, may be admini~tered. For example in man
nverage daily dosec of about 20 to about 6000 mg ~re ~dmlni~tered
orally or by parenteral infusion, however, in 3evere ca3e~ higher do~e~
may have to be used.
Varioua tablets and ampoul~ for infusion are commercially available
for cardiovaDculare indications, e.g. of diltiazem (tablet~ 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 ampoule6 for infusion with 10 mg in 50 ml 301vent), nitrendipine
(tablets with 10 and 20 mg), or isradipine (tablets 2,5 mg).
The invention concerns al~o the use of a calcium antagoni~t, a
comhination of two or more calcium antagonists, or a combination of a
calcium antagonist and a zinc ~alt for the manufacture of a
pharmaceutical preparation for protecting a warmblooded animal against
deleterious effect6 of radiation.
The calcium antagonlst~ or the combinations of the prefient invention
are administered orally or parenterally to achieve the radioprotective
effect, in any of the usual pharmaceutical ~orms. The~e include solid
and liquid unit oral dosage forms such as tablets, capsules, powders,
suspenslons, solution6, ~yrups and the like, including sustained
relea~e preparations, and fluid injectable form6, such as ~terile
~olutions and ~u3pensions. The term doRage fo~m a~ used in this
~pecification and the claims refer to phy6ically di~crete units to ~e
adminiatered in single or multiple do~age to animal~, each unit
containing a predetermined quantity ~f ~ctive material in a~sociation
with the required diluent, carrler or vehicle. The quantity of activ~
material is that calculated to produce the desired therapeutic effect
upon administration of on~ or more of ~uch unit~.
Powders are prepared by comminuting the compound to a ~uitably fine
size and mixing with a ~imilarly comminuted diluent pharmaceutical
carrier, ~uch as an edible c~rbohydrate material A3 for example,
~t~2~
~tarch. Sweetening ,flavoring, preservative, dispersing and coloring
agents can also be added.
Capsules are made by preparlng a powder as descrlbed above and filling
formed gelatln sheath3. A lubrLcant, ~uch as talc, magnesium stearate
and calcium ~tearate can be added to the powder mixture as an adjuvant
before the filling operatlon. A glidant ~uch as colloidal silica may be
added to improve flow propertles. A di~integrating or ~olubilizing
agent may be added to improve the availability of the medicament when
the capsula is ingested.
Tablets are made by preparlng a powder mixture, granulating orslugging, 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 atarch, ~ucro3e,
kaolin, dicalcium phosphate and the like. The powder mixture can bs
granulated by wetting with a binder such a~ syrup, starch paste, acacia
mucilage or solutions of cellulosic or polymeric materials and forcing
through a screen. Ag an alternative to granulating, the po~der mixture
can be run through the tablet machine and the resulting imperfectly
formed slugs broken into granules. The granule~ 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 compresse~ into tablet~
directly without going through the granulating or slug~ing steps. A
protective coating consisting of a sealing coat of shellac, a coating
of ~ugar or polymeric material and polish coating of wax can be
provided. Dyestuffs can be added to these coatings to distingui3h
different unit dosages.
Oral fluids such as ~yrups and elixirs can be prepared in unit do~age
form so tha-t a given quantitiy, e.g. a teaspoonful, contains a
predetermined amount of the compound. Syrups can be prepared by
di~solving the active compound in a suitably flavored aqueous sucrose
solution, while eli~ir~ are prepared through the u~e of a non-toxic
alcoholic, e.g. ethanollc, vehicls. Su~pen~ion~ and emul~ions can be
formulated by dispersing the medicament in a non-toxic vehicle.
~;UB~3TITUTI~ SHEET
93/02670 2 ~ PCr/~Pgl/01410
For parenteral admini~trntion, fluid unit dosage form~ can be prepared
by flu~pending or di~olving a mea~ured amount of the active material ln
a non-toxic liquid vehicle Duitable for injection nUch a~ an aqueous,
alcoholic, e.g. ethanolic, or oleaginous medium. Such fluid dosage
unit forms may contain ~olubilizer~, such as a polyethyleneglycol,
~tabilizer~, and buffers, such as a citric acid/sodium citrate buffer,
to provide the desired o~motic pressure. Alternatively a measured
nmount of the active material iB placed in a vial and the vial and ~t6
content are sterilized and sealed. An accompanyinq vial or vehicle can
be provided for mixing prior to administration.
Important embodiments of the present invention are the pharmaceutically
acceptable ~alt~ of the basic calcium antagoni~t~ of the present
invention. Such ~alt6 include those derived from both organic and
inorganic acids ~uch a~, wlthout limitation, hydrochlbric, hydrobromic,
sulfuric, pho~phoric, methansulfonic, acetic, lactic, ~uccinic, malic,
maleic, acontic, phthalic , tartaric, embonic, enanthic and the like
acids.
An important aspect in the preparation of fluid dosage forms i~ the
u6e of solvents which by themselves have a certain radioprotective
effect, e.g. the use of an alcohol, in particular ethanol, which may be
pre~ent in amounts of about 5 to about 30%, e.q. in amount~ of about 15
to 20%.
If combinations of two or more calcium antagonist or combinations of a
calcium antagoni~t and a radioprotective salt are envi~aged ~uch
combinations may be u~ed 6eparately and 6imultaneously or
con~ecutively, or otherwise formulated together in one pharmaceutical
preparation according to the method~ described above.
;,
SHO~T DESCRIPTION OF THE FIGURES
FIGURE 1: Shown are the survival rate~ (~) of femal~ C3H mice up to 30
days after B.C. adminiBtration of 0.1ml/10 9 of dist11ed water (l;O),
110 (2jo), 55 (3;W) and 2~.5 mg/kg (4;~) of dlltiazem 15 min before,
d
~nd of 110 mg/kg (5tV) of diltiazem lO min after lethnl lrradiatlon
with a 60cobalt source.
FIGURE 2A: Shown are the surv~val rates (~) of female C3H m~ce up to 30d~ys after i.p. admlnistration of 1.5 mg/~g of nifediplne (11;~) and
0.15 ml/~g of the solvent of nlfedipine (12;~130 mln before lrradistlon
wlth 8.5.Gy tO.9 Gy/min) from a 60cobalt ~ource, and of the control
group treated with 0.15 ml/10 g of dlstilled water (c;~).
FIGURE 2B: Shown are the ~urvival rates ~) of female C3H mice up to 30
days aPter i.p. admlni6tratlon of 2 mg/~g of nimodiplne ~13;G) and
O.lml/lOg of the ~olvent of nimodipine ~14j~) 30 min before lethal
lrradiation with a.5 Gy (0.9 Gy/min) from a 60cobalt ~ource, and of the
control group treated with 0.1 ml/10 9 of distilled water (c;-).
FICURE 3A: Shown are the ~urvival rates (~) o male C3H mlce up to 30
days after i.p. admini~tration of 4 mg/kg of nimodipine (15;0), 0.2
ml/lOg of ethanol (23.7~)(16F~), 3 mg/kg of nlfedlplne (17;~) and 0.3
ml/10 9 of ethanol (18~)(18;~) 30 min before irradiation with 8.1 Gy
(o~9 Gy/min) from a 60cobalt source, and of the control group treated
with 0.2 ml/10 9 of distilled water (c;D).
FIGURE 3B: Shown are the survi~al rates (%) of male C3H mice up to 30
days after i.p. administration of 4 mg/kg of nimodipLne (l9;~), 0.2
ml/10 g of ethanol (23.7.~)(20;~), 3 mg/kq of nifedipine (21;~) and 0.3
ml/lOg of ethanol (1a%)(22;~ ) 30 min before irradia~ion with a
~upralethal dose of 9 Gy (0.9 Gy/min) from a 60cobalt source, and of
the control group treated with 0.2 ml/10 9 of dlstilled water (c;0)
FIGURE 4: Shows the Hynergistic effect of a combination of dilSiazem
and zinc aspartate.
FIGURE 5: Shows the Hynergistic effect of a ~ombination of dilt.iazem
and nifedipine (see legend to EIGURE 4).
FIGURF 6: Shows the average volumes (mm3) up to 30 days Df an E~ing~6
~arcoma transplanted as xenograft6 into male C3H mice untreated (l;o)
and pretreated intraperitoneally 30 min or ~ubcutaneously 15 min before
)93/02670 ~ 8 PCT/EPgl/01410
irradiation at day 12 with a 60cobalt aource with 110 mg/kg a.c. of
diltiazem (2;~), 3 mg/kg i.p. of nifedipine (3;~) or 4 mg/kg i.p. of
nimodipine (4;~), or 3 ml/10 g solvent of nifedipine (6j~) and of the
unirradiated ~tatistically aignificant (xxx) control~ (5;-).
FIGU~E 7: Show~ the average volume~ (mm3) up to 27 daya of an
adanocarcinoma of the colon, tranaplanted aa xenograft into male C3H
mice untreated (l;o) and pretreated intraperitoneally 30 min or
aubcutaneouRly lS min before irradiation at day 12 wlth a 50cobalt
aource with 100 mg/kg nitrendipine (2;0t or 3 mg/kg of nifedipine (3;~)
and of the unirradiated controls (4;~), (xxx) indicating statistical
significant values.
FIGURE 8: Shows the ave~age volumes (mm3) up to 23 days of an
adenocarcinoma of the colon, transplanted as xenograft into male C3H
mice pretreated intraperitoneally 30 min or ~ubcutaneously 15 min
before irradiation at day 12 with 5.625 Gy of a 60cobalt source with
110 mg/kg 9.C. of diltiazem (2;~), 3 mg/kg i.p. of nifedipine (3;~) or
4 mg/kg i.p. of nimodipin~ (4; ~), and of unirradiated (1;~) and
irradiated controls (5;o), ~xxx) indicating ~tati6tical significant
values .
FIGURE 9: Shows the avera~e volumes (mm3) up to 23 daya of an
adenocarcinoma of the colon, tran~planted aa xenograft into male C3
mice, pretreated intraperitoneally 30 min befare irradiation at day 12
with 3.5 Gy of a 60cobalt ~ource with 3 mg/kg of nifedipine (2~) or 4
mg/~g of nimodipine (3;~), and of irradiated (l;o) and unirradiated
controls (4;o), (xxx) indicating stati~tical 0ignificant value~.
The ~urpri~ing advantageous propertiea of the calcium antagoni~ts, the
combinatLon~ of calcium antagoni~ta or combination~ of calclum
antagonist~ with metal 6alta have bsen confirmed and verified by the
follow;ng te~t~.
GENERhL PROCED~RE OF EXAMPLES 1-7
Calcium antagoni~t6, combination~ of calcium antagoni~ts or
combination~ of calcium antagoniRt~ with zinc a5pa~tate wera te~ted at
thelr higheat tolerated do~e against a lethal radiatlon (LDloo) in mlce
(groups of at least 12 mlce were uaed) and for their abil~ty to confer
protection when administered at aublethal doae5. For thia purpoae the
LD50 of ~nch calcium antagonl~t and of zlnc aapartate was first
determined and then teated at the 1/2 LDso for antiradintion activity.
The efficacy o~ the test compounds were measured by their abillty to
protect the mica again~t a lethal dose of X~rays, protection being
defined as survival after 30 days. ~n attempt was made to investigate
the therapeutic ratio of effective calcium antagonists by reducing each
do~e by one half. The reaults are ~hown in the TABLES and the FICURES.
The teat compounds were admini~tered subcutaneously, intraperitoneally
or orally, however, other routes may be applied as well.
EXAMPLE 1: RADIOPROTECTIVE EFFECT OF DILTIA~EM
Male and female C3H mice of a weight of 2~ to 24 g were u~ed for the
experiments. Commercially availahle ampoule5 of diltiazem containing 25
mg of diltiazem hydrochloride ~in the following only the term diltiazem
i9 u~ed) and 150 mg of mannitol were u~ed. The content of the ampoules
was dissolved in distilled water. Control experiments ascartained that
mannltol alone did not exert radioprotection. Different doses of
diltiazem we-e administered aubcutaneously ~Groups 1 to 5), orally
~Group 6) or intraperitoneally (Groups 7a to lOc) before or after
irradiation according to TABLE 1.
The control group of mice received ~ubc~taneously 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 60cobalt 60urce ~Gammatron) at a
targent diatance of 80 cm. Dosimetry was performed with a strontium-
cal~brated ionization chamber. Irradiat$ons were performed either with
10.5 Gy (1 Gy = 100 rad) at a dose rate of 0.15 Gy/min, or with 8.5 Gy
with a doae rate of 0.9 Gy/mln. The e dosa~, delivered hy two diÇferent
60cobalt ~ources, correaponded to the reapective LDloo and yielded
equal medial aurvival times. Accordingly, they wer~ biologically
~ 93/0~67~ a ~ pCT/EP91/0141~
1 1
lzoeffactlv~ and the r~nultu obt~i~cd wlth both radl~tion ~ource~ could
be pooled.
The sesults are ~hown 1A TABLE 1 and FIGURE 1.
TA~LE 1: Survival rate of female ~f) and m~le ~m) C3H mlce treated wlth
dLltiazem after lethal lrradlation with ~ 60cobalt source
. _
Group do~e ~mg/~g) ndm.1) tlme2) aex ~urv.rAte3)
-
1 0.1 ml/lOg di~t.water ~.c. -15 min f 2
2 10.0 diltiazem ~.c. -lS min f 93
3 55.0 diltiazem s.c. -15 min f 58
4 27.5 diltiazem 8 .c. -15 min f 1?~
110.0 diltiazem s.c. ~10 min f 42
6 110.0 diltiazem p.o. -30 min f 56
7a 0.1 ml/lOg dist.water i.p. -30 min f 0
7b o.l ml/lOg dist.water i.p. -30 min m 8~
8 110.0 diltiazem i.p. -10 min f 54t
9 110.0 diltiazem i.p. -30 min f 58
lOa 110.0 diltiazem i.p. -120 minf 19
lOb 110.0 diltiazem i.p. -10 mLn m 100
lOc 110.0 diltiazem i.p. 30 minm 80~
1) s.c. - ~ubcutaneously; l.p. = intraperitoneally; p.o. - orally
2) - : tLme before irr~diation; + : tlme after ~rradiation
3) survival rate of te~ted mice 30 days ~fter ~dmini~tration of test
compound and irradiation
The ~urvival rates of Group~ 1 to 5 over the time period of 30 day~ ~re
~hown in FIGURE 1.
The calcium antagonist dlltiazem clearly incr~a~ed the 3urvival r~te of
th~ tseated mlc~ after irradL~tlon wlth lethal do~e~ o~ X-r~ys.
Diltlazem at th~ dos~ge of llD mg/kg tl/2 LDso), 55 mq/~g and 27.5
mg/~g provld~d ~urviv~l of ~3S, 58~ And 17~, reapectlvely.
~ r~~ r~ ~ ~ ~ T
~ 12 ~2~
A ~ignif$cant ~urvlval r~te of 42~ wa~ al~o ob~erved by adminintration
of a curative do~e of 110 mg/kq 10 mln after the completion of
irradiation (Group 5). No ~tati~tal 8ign~ficant differences were aeen
between male and female mice, between ~ubcutaneou~ And intraperltoneal
administration of diltia~em, and lf the treatmentt was performed 10, 15
or 30 mln before irradiation.
The radioprotective effect~ of d~ltiaze~ ie al~o confirrned in ~imilar
test~ with other inbred mou~e 8tr~1ns lncluding ~nlb/c nnd C57 ~1/6,
and the outbrsd albino strain NMRI.
EXAMPLE 2: ~DIOPROTECTIVE EFFECTS OF NIFEDIPINE, NIMODIPINE AND
SOLVENTS IN FE~LE MICE
EXAMPLE 1 was followed except as de8cribed hereinafter. Co~nercially
available pharmaceutical preparations of nifedipine and ninodipin~ were
used in their respective ~olvent6 l~ee index 5) and ?) of TABLE 21.
Groups of female mice were irradiated with B.5 ay (dose rate 0.9
Gy/min). Their radiation LDsO was approximately 7.75 GY. The co~pound~
were intraperitoneally injected 30 min before irradiation. For a better
detection of their relative contributions, the calcium antagonists and
their 601vents were applied only at half of their optimal dose.
The results are compiled in TABLE 2 and FIGURES 2~ and 2B.
TABLE 2: Survival rate of female (f) C3H mice treated with nifedipine
or nimodipine in their respective solvents or with the aolventa alone
after lethal irradiation with a ~cobalt aource
Group dose (mg/kg) adm.1) time2) ~ex ~urv.rate3)
,
11 1.5 nifedipine4) i.p. -30 min f 100
12 0.15 ml~lOg solvent5) i.p. -30 min f 61~
13 2.0 nimodipine5) i.p. -30 min f 83%
14 0.1 ml/lOg solvent7~ i.p. -30 min f 42
.
1), 2) and 3) as in EXAMPLE 1
4) in 0.15 ml/10 g of nolvent 5)
' ' " ' ! ' 1,- ,, , ~ ~ ,
~J 93/0267U ~ PC~/EPgl/01410
5) 50 ml solvent of nlfedipine contain: 7.5 9 of ethanDl 96~, 7.5 9 of
polyethyleneglycol 400, and 35 g of dlstllled water
6) in 0.1 mlllO g of solvent 7)
7) 50 ml solvent of nimodipine contain: 10 g ethanol 968, 8.5 9 of
polyethyleneqlycol 400, 0.1 9 of tertiary ~odium citrate, 0.015 9 of
cltrlc acid, and 31,265 9 of di~tilled water
Similar do~e dependent survival wa~ observed wlth nifedipine and
nimodipine in lethally irradiated mice. However, for parenteral
admini~tration, these calcium antagonist were admini~tered in their
aolvents defined under 5) and 7) of TABLE 2. In order to avoid
confounding the additive or ~ynergistic effects of the solvents with
tho~e of nifedipine and nimodipine, it was neces~ary to differentiate
between the relative contributions of the solvents and the calcium
antagonists. As shown in TABL~ 2 the survival of lethally irradiated
mice waa 100~ with 1.5 mg/kg of-nifedipine and 613 with its ~olvent
alone, and B2~ with nimodlpine and only 42~ with its ~olvent alone.
This indicates that the solvents alone offer also ~ome radioprotection.
The differeaces of tha survival rates bstwePn the calcium antagonists
and their respective solvents are ~tatistically ~ignificant.
Accordingly, after subtraction of the solvent effect6, th~ residual
radioprotection of 39~ with nifedipine and 40~ with nimodipine must be
ascribed to the calciu~ antagoni~ts.
FIGURE 2A shows the survival rates up to 30 days of Groups 11 ~) and
12 (y) and of the control group (c).
FIGURE 2B ahows the survival rate~ up to 30 day~ of Groups 13 (0) and
14 (~)and af the control gr~up (c~.
It is assumed that the protective activity of the ~olvents is due to
the wellknown radioprotective efect of the ethanol component, if
adminiEtered in very high quantlties. ~his ~s~umpticn was corroborated
by the fact that the solvents of the calcium antagonists as compared to
the respective amounts af ethanol in the solvents afforded
radioprotection to a ~imilar and ~tati~t~cally not sigificantly
differing extent.
~4
EXAMPLE 3: RADIOPROTECTIVE EFFECTS OF NIFEDIPINE, NIMODIPINE A~DE~HANOL IN MALE MICE.
Wlth exeption of the ~ollowing, the procedure of EXAMPLE 1 was
repeated. The lntrln~lc radioprotection by the calcium antagonist~ wa~
al~o clearly displayed in a further experlment designed to compare
nifedipine and nimodipine wlth ethanol in more radio~en~itive male C3H
mice irradiated with 8.1 Gy ~Groups 15-18; FIGURE 3A) or the
~upralethal dose of 9.0 Gy (Groups 19-22; FIGURE 3B)
The procedure i5 analogous to that of EXAMPLE 1. The radiation L350 in
these mice was 6.35 Gy (dose rate 0.9 Gy/min). The test compounds were
intraperitoneally injected 30 min. before the ~tart of the irradiation.
The mice recelved either nifedipine or nimodipine ~ith thelr solvents,
or ethanol at the do~e corresponding to the quantity present in the
~olvents. The results are compiled in TABLE 3 and FIGURES 3A and 3B.
TA~LE 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 ~ource
_
Groupe dose (mg/kg)4) adm.1) tlme2) ~ex surv.ra~e3) Gy
4 nimodipine i.p. -30min m75~ 8.1
16 0.2ml/lOg ethanol i.p. -30min m 8~ 8.1
(23.7~)7)
17 3 nifedipine i.p. -30min m67~ 8.1
18 0.3ml/lOg ethanol i.p. -30min m 25~ 3.1
(18~)7)
19 4 nimodipine i.p. -30min m55~ g.o
0.2ml/lOg ethanol i.p. ~30min m O~ g.O
(23.7~)7)
21 3 nifedipine i.p. -30min m 58~ 9.0
22 0.3ml/10 ethanol i.p. -30min m 0~ 9.0
~18~)7)
_
1), 2), 3) an 4) ~ee EXAMPLES 1 and 2
7) amoun~ of ethanol correRponding to the amount of ethanol in the
solvents of nifedipine and nimodipine.
8UE3STITUTE SHEET
`~ 93/026l~ 2 ~ $ ~ PCT/EPgl/0l410
If administered i.p. with their solvents 30 min before irradiation with8.1 Gy, nifedipine afforded a survival rate of 67 % and nimodipine of
75 ~, as compared to a ~urvival rate of 25 % and 8 %, respectively,
provided by ethanol adminiatered i.p. in dose~ corresponding to the
quantities contained in the solvent (FIGU~E 3~). Mice similarly treated
but irradiated with 9.0 Gy showed a survival of 58 ~ with nifedlpine
and 55 % wLth nimodipine applied in their solvents while the
corresponding do~es 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 day~ (FIGURE 3B).
Treatments were as follow~:
- Controls: 0.2ml dist. water/i0g body weight (c~;
- nifedipine, 3mg/kg in 0.3ml solvent/10 g (1-7 and 21t;
- 0.3mljl0g of an 18 ~ ethanol soiution 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/10~ of an 23.7 % ethanol ~olution (4050mg ethanol/kg) in
dist. water (16 and 20).
For reference reasons, less radiosensitive female mice were treated as
6tated for group 17, 18, 19 and 20. Survlval rates were 83~, 28%, 100
and 39%, respectively.
A further attempt has be~n made in order to distlnguish the effect~ of
nifedipine and nimodipine from those due to their 301vents by
determining the average weight of th~ mice used ln the test. The weight
105~ after irradiation with 9.75 Gy in groups of female C3H mice and
treatment with nimodipine and nifedipine was from day ~ to day 29
wlth a difference of 2 to ~ g signiLicantly lower than in groups
treated with the re~pective solvents.
EXAMPLE 4: RADIOPROTECTIVE EFFECTS OF ISRADIPINE AND NITRENDI~INE
With exception of the following, the procedure of EXAMPEE 1 w3s
repeated. The result~ are summari~ed in Table 4.
~ll~ ~r~-r!'^ ~ ~
~ 16
TA~LE 4: Survival rate of f emAle ~f) C3H mlce tre~ted with L~radipins
(in an oLly ~olutlon) or nitrendLpine (su3pended in water) after lethal
irradiation with a ~cobalt ~ource
-
Groupe dose ~mg/kg) adm,l) time2) sex aurv.rate3)
2~ 27.5 isradipine i.p. - 30~in f 58
24 27.5 i~radipine i.p. -120mln f 28~
27.5 i3radipine i.p. -240min f 17%
26100 nitrendipine i.p. - 30mln f 33
27100 nitrendipine i.p. -120min f 30
2850 nitrendipine i.p. - 30min f 25
2959 nitrendipine i.p. -120min f 17
1), 2~ and 3) as in EXAMPLE 1
EXAMPLE 5: SYNERGISTIC R~DIOPROTECTIVE EFFECTS OF A COMBINATION OF
DILTIAZEM AND ZINC ASP~RTATE, A COMBINATION OF DILTIAZEM
AND NIFEDIPIN~, AND A COMBINATION OF NIT~ENDIPINE AND ZINC-
ASPARTATE.
With exception of the ~ollowing, the procedure of EX~MPLE 1 was
repeated. Isobolograms were used to determine 6ynergistic effects by
the combination of two radioprotector6 which alone allowed at optimal
dosage 3urvival of not less than 50 % against the radiation LD1oo
Isoeffective do6es of each compound were marked on the coordinates Isee
FIGURE 4 ~zinc a6partate and diltlazem) and FIGUR~ S (nifedipine and
diltiazem~]. A hyphenated line wa~ drawn between the lowest tested
do~es of two compounds wich confered each survival of not less than 50
(lowest effective do~e, LED). Lowest effective doses were as follows:
diltiazem: 110 mglkg
nifedipine: 3 mgtkg
~inc a~partate. 3~ mg/kg
~he dose combinations lying on the hyphenated line drawn between the
LE~ of the two combined compounda ~epresent the hypothetical amount6 of
both compound~ reguired to allow not lcag than 50 ~ 3urvlv~1 If the
interactiona were additive. Point3 at the left of thi~ line indica~e
synergistic interaction between two compounds and can be u~ed to
0 93/02670 ~ ~ 9 ~ PCI/EP91/01410
17
con~truct the concave iaobol~. 80th compound~ were teated in n checker-
board fa~hion and ln general a~ serially twofold reduced fractlons.
S.nergi~m between two compounds i6 ~aid do occur from a combinatlon
where each compound ls used at a do~e of less than 1/2 I,ED or at lea~t
one compound being u~ed at the 1/2 LED an the other below the 1/2 LED.
Accordingly, all do~e combinatlons providing ~urvival of not le~ than
50 ~ and ~ituated left of the line representing additive interaction~
indicate ~ynergi6m.
The ~ynergistic effect can al~o be demonstrated by the te~t re~ulta
~hown in TABLE 5 and TABLE 6.
Table 5: Survival of C3H mice irradiated with 10.5 Gy and treated with
diltiazem, zinc a~partate or combinations of both.
_
- ¦diltiazem (mg/kg)
lo.oo 13.75 27.50 41.25 55.00 110.00
Zn aspartate
(mg/kg) ¦ 0~ 17~ 33% 44% 91~
2.5 ¦o~ n.d. 17~ 17~ 42~ n.d.
¦o~ n.d. 25~ 8~ 92~3) n.d.
¦o~ nOd. 5B~2) n.d. 75~1) n.d.
¦o% 0~ 50~1) 100~2)92~ n.d.
¦92~ n.d. n.d. n.d. n.d. n.d.
1) pc0.005; 2) p~0.02; 3) p~0.01; n.d.= not done
1), 2) and 3) indicate aynergism and the atati0tical 0ignlficance.
In combinations of diltiazem with zinc aspartate, ~urvival of 44 ~
obtained with 55 mg/kg of diltiazem alone was increased by combining it
with the alone ineffectivP dose6 of 5 or 10 mg/kg of zinc a~partate to
92~ and 75%, respectively. Simllarly, survival of 17~ with 27,5 mg/kg
of diltiazem wa6 enhanced to 50 ~ with 15 mg/kg of zinr a6partate and
to 58~ with 10 mg/kg of zinc a~partata.
18
Table 6: Survival of C3H mice irradiated with 10.5 GY and tr~ated with
diltiazem, nifedlpine and combination~ of hoth:
¦diltiazem (mg/kg~
lo.oo 13.75 27.50 55.00 110.00
nifedlpine
(mg/kg) ¦ 0~ 17% 44% 91%
0.375 ¦n.d. 0% 42~ 83~1) n.d.
0.75 ¦o% 0% 50~ n.d. n.d.
1.5 140~ 0% n.d. n.d. n.d~
3.0 179% n.d. n.d. n.d. n.d.
I
1) p<0.05; n.d. = not done
1) indicates the occurrence of synergism and the statistLcal
qigniflcance.
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 % ~een 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 a~partate anhancPd the
survival affordad by 15 mg/kg of zinc a3partate from 0~ to 100% by
add~ng 50 mg/kg of nitrendipine.
EXAMPLE 6: RADIOPROTECTIVE EFFECT OF DILTIAZEM BEFORE ~ND AFTER
IRRADIATION.
The procedure according EXAMPLE 1 wa~ repeated with the following
exceptions. The survival rate of C3H mice was measured when ixradiatad
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
~ignificant survival rate of 56~, or ~ubcutaneously 10 min after
irradiation, which stlll allows a ~urvlval rate of slgnificant 42%.
Thus, diltiazem shows also curative effect.
SUBSTITUTE~ SHEE7
'0 93/02670 2
19
EXAMPLE 7: RADIOPROTECTIVE EFF~CTS OF DILTIAZE~, NIFEDIPIN~, NIHODIPINE
AND EFFECT ON TUMOR GROWTH DELAY
~ale C3H mice weighing 25-30 g and fed with Nafng pellets and water ad
llbitum were used~ Groups of mice were irradlated according to the
procedure of EXAMPLE l. Total body irradiation was given with sublethal
doses in general at day 12 after transplantLng a human xenograft.
Three different human tumors were used, namely nn Ewing'~ carcoma and
two adenocarcinomas of the colon, and transplanted into immuno~up-
pressed mice.
The first tumor measu~ement 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 ~topped when
necrosis and exulceration of Large tumors made reliahle measurements
impossible. The mean values of the tumor values for ~ach group were
tabulated and entered into the FIGURES.
Tumor volumes not ~ignificantly larqer than in the irradiated,
untreated control group indicated that the used agent failed to protect
the tumor from radiation-induced regression. Smaller tumors ~han in the
irradiated controls indicated sensiti~ation 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 containlng 25 mg of diltia~em and 150 mg of
mannitol was dissolved in distilled water and 0.1 ml/10 9 was
administered to the mice ~mannitol alone did not show any
radioprotective effect). Nlfedipin and nimodipine were u~ed in their
respective solvents (see EXAMPL~ 2). Nitrendipine was injected as
suspension in distilled water (0.1 ml/lO g). Nifedipine solvent and 18
ethanol were applied at 0.3 ml/lO 9, ~nd nlmodipine solvent and 23.7
ethanol at 0.2 mlllO g body wei~ht.
Ths drugs were administered intraperitoneally 30 min before irradiationr~tarted, with the exce~tion of dlltiazem whlch was aubcutaneously
administered 15 min before the start of irradiation. In all experiments
the difference ~etween tumor volume of the unirradiated contro1s and
the group which was only irradlated was significant from a few days
after the irradiation to the end of the experiment. On the other hand,
the dlfference between tumor volume of the irradiated control and the
pretreated irradiated groups was at no lnstance signiflcant, lndicating
that the calcium antagoni~t failed to protect the human tumors from
radlation induced regression.
~he result3 can be taken from FIGURES 6 to 9.
FIGURE 6: The changes of the average volumes (mm3) of an Ewing'0
sarcoma irradiated at day 12 with 4.875 Gy from a 60cobalt source up to
30 days are shown. No significant differences can be seen between the
irradiated controlrJ (1) and the groups pretreated with 110 mq/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 control~,
and curve (6) those obtained with 3 mg/kg of the so1vent of nifedipine.
Asterisks indicate statistical significance of differences between
unirradiated and irradiated controls.
FIGURE 7: The changes of the average volumes (mm3) of an adenocarcinomaof the colon irradiated a day 12 with 5.625 Gy are shown. Pretreatment
with nitrendiplne led to tumor volumes comparable with the irradiated
controls. In the group pretreated ~ith 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
control6, 100 rng/kg of nitrendipine, 3mg/kg of nifedipine, and
unirradiated controlr;, 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 8hown where groups of
rnice carrying an adenocarcinoma of the colon wer~ irradlated as
described under F~GURE 7. The re~ults indicate that 110 mg/kg of
S~J~35T~l1T1~ 5~ T
~o ,~ 2 ~ 0
21
diltiazem (2), 3 mg/kg of nifsdipine (3~ or 4 mg/kg of nimodipine (4)
did not reduce radiation-induced tumor growth delay. Curve~ nd (5)
~how the re~ults of unirradiated and irradiated control~, re~pectively.
Asteri~ks indicate ~tatistical differences a~ explained above.
FICURE 9: Slmilar re~ultD a~ deccribed in FIGURE 8 were obtained when
groups of mice were irradiated at day 12 with 3.5 Gy aftes pretreatment
with ~ mg/kg of nifedipine (2) or 4 mq/kg of nimodipine (3). Curves (1)
and (4) ~how irradlated and unlrradiated controla, recpectively.
EXAMPLE 8: TA~LETS
Tablets co~tainig each 180 mg of diltiazem are manufactured by admixingcarefully lB kg of diltiazem with 90 kg of mannitol (or lactose) and
pre~sing the mixture into 105 tablets.
In a ~imilar manner tablet~ with the de~ired amount of another active
ingredient can be manufactured.
EXA~PLE 9: DRY A~POULES
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 ~imilar manner ampoules with the desired amount~ o~ another activeingredient can be manufactured.
EXAMPLE 10: AMPOULES FOR INJECTION
Ampoules for injection each containing 100 mg ~f nifedipine are
manufactured by di~olving 10 kg of nL~edipine in 103 kg of a ~olution
compri~ing 150 kg of ethanol (96%), 150 kg of polyethylen~glycol 400
and 700 kg of di~tilled water, ~terllizing thi~ mlxture and fllllng lt
in 105 nterilized ~mpoule~ each compri~ing 100 mg of active compound in
10 9 of solution.
~2 ~2~
r In u ~imll~r manner ~mpoule~ wlth th~ des~red ~mount of another ~ct~ve
ln~redlent can be manufactured.
