Note: Descriptions are shown in the official language in which they were submitted.
2~61~88
Reyword: Sydnon~ imine/hirudin combination
Inventors: Dr. Just
Dr. Sch~nafinger
Ref. 3473
Dr. My/Bu Bu0084
Combination of active su~stances comprisinq
a sydnone imine and a hirudin
The present invention relates to a combination of active
substances comprising a sydnone imine and a hirudin and to the
use thereof.
Sydnone imines are compounds whose ~harmacology has now
been known for a long time (Chemie in unserer Zeit, volume 13,
page 51 (1984)), and besides the vasodilating, in particular also
the antithrombotic effects have been described (J. Cardiovasc.
Pharmacol. l9B9, 14 (Suppl. ll), page 129). Sydnone imines can be
used as agents to prevent and treat disorders of the cardio-
vascular system and are commercially available as such.
Hirudin is a ~ eptide which can be isolated from the
medicinal leech (Hirudo medicinalis) and is known as thrombin
inhibitor (Die Pharmazie 36, 653 (1981)). It has recently become
possible to prepare by genetic engineering processes adequate
amounts of recombinant hirudin (r-hirudin) (MUnch med. Wschr.
127, 16 (1985)). However, a large number of synthetic hirudins is
also known.
It has now been found, surprisingly, that there is mutual
enhancement of the antithrombotic effects of sydnone imines and
hirudins.
The present invention accordingly relates to a combi-
nation of active substances comprising a sydnone imine and a
hirudin.
The term sydnone imine means all pharmacologically active
sydnone imines. In particular, these are the sydnone imines
embraced in the following publications: German Offenlegungs-
schrift 1,620,501; German Offenlegungsschrift 1,670,127; German
Offenlegungsschrift 1,695,897; EP-A-23,343; EP-A-59,356;
EP-A-76,952, EP-A-210,474; EP-A-276,710; EP-A-312,773;
EP-A-324,408; EP-A-346,684; EP-A-346,694; EP-A-367,036;
EP-A-406,659 and EP-A-406,661.
Preferred sydnone imines are those of the general
206 ~88
fornlu1a
R - N ~ -R
N - = N--R (I)
n
or a pharmacologically acceptable salt thereof, where
R1 denotes an amino group of the formula
R" ~H3 CH~
R5~N-- R C~3 CH,
COt)H
4 ~
~ J or x N--
02S~ `--
R2 denotes hydrogen, alkyl, cycloalkyl, arylalkyl, aryl-X-alkyl,
alkoxyalkyl, alkylthioalkyl or alkenylthioalkyl;
R3 denotes hydrogen or the group -CoR7;
R4 denotes alkyl, cycloalkyl, alkyl-X-alkyl, arylalkyl, hydroxy-
alkyl;
Rs denotes hydrogen or has one of the meanings of R4;
R6 denotes hydrogen or methyl;
R7 denotes aryl, aryl radical mono-, di- or trisubstituted by 1 to
3 halogen atoms and/or 1 to 3 alkyl radicals and/or 1 to 3 alkoxy
radicals andJor 1 or 2 nitro groups, or oR2, or has one of the
meanings of R2;
X denotes NR4, NSO2R8, NCO2alkyl, S(O)D~ O, CH2 or a single bond;
n is 0, 1 or 2, and
R8 denotes alkyl, aryl, alkylaryl or dialkylamino.
Alkyl radicals and alkenyl radicals can be straight-chain
or branched and preferably have 1 to 6 C atoms, particularly
preferably 104 C atoms. This also applies when they are present
in conjunction with other groups, for example as alkoxy, aryl-
alkyl etc.
Examples of alkyl radicals are methyl, ethyl, propyl, isopropyl,
butyl, sec.-butyl, and tert.-butyl.
Cycloalkyl preferably has 5 to 7 C atoms and particularly
~061~8~
preferably denotes cyclopentyl and cyclohexyl.
Aryl preferably has 6 to 10 C atoms and particul~rly pre~erably
denotes ~- or ~-naphthyl or phenyl.
Arylalkyl i~ preferably benzyl and phenylethyl.
~ryl-X-alkyl i8 preferably phenoxymethyl and phenoxyethyl.
The aryl radical~ representing R7 can be mono-, di- or
trisubstituted, but it is possible for a maximum of only 2 nitro
groups to be present even in the case of trisubstLtution, such
as, for example, 2-methyl-4,6-dinitrophenyl and 2-chloro-
6-methyl-4-nitrophenyl. Examples of suitable halogen substituents
for the aryl radicals are chlorine and bromine atoms. Aryl radi-
cals representing R7 which should be particularly mentioned are:
methylphenyl (= tolyl), nitrophenyl and chlorophenyl, especially
4-nitrophenyl and 4-chlorophenyl.
Preferred R1 radicals are morpholino, 3,3-dimethyl-thio-
morpholino, cis-2,6-dimethylpiperidino and 1,1-dioxo-3,3-di-
methylthiomorpholino.
R2 preferably denotes hydrogen.
R3 preferably denotes hydrogen, ethoxycarbonyl, propionyl or
p-anisoyl.
The sydnone imines according to the claims form acid
addition salts with inorganic or organic acids. Examples of
suitable acids are hydrogen chloride, hydrogen bromide,
naphthalenedisulphonic acids, especially naphthalene-1,5-di-
sulphonic acid, phosphoric, nitric, sulphuric, oxalic, lactic,tartaric, acetic, salicylic, benzoic, formic, propionic, pivalic,
diethylacetic, malonic, succinic, pimelic, fumaric, maleic,
malic, sulphamic, phenylpropionic, gluconic, ascorbic, isonico-
tinic, methanesulphonic, p-toluenesulphonic, citric or adipic
acid. Pharmacologically acceptable addition salts are preferred.
The hydrochlorides are particular preferred.
Examples of sydnone imines according to the claims are:
N-ethoxycarbonyl-3-morpholino-sydnone imine (molsidomine);
3-morpholino-sydnone imine hydrochloride (SIN-l); N-~4-methoxy-
benzoyl)-3-(cis-2,6-dimethylpiperidino)sydnone imine;
N-propionyl-3-(3,3-dimethyl-thiomorpholino)sydnone imine;
3-(1,1-dioxo-3,3-dimethyl-thiomorpholino)sydnone imine
hydrochloride; 3-(3,3-dimethyl-thiomorpholino)sydnone imine
hydrochloride; 3-(cis-2,6-dimeth~lpiperidino)sydnone imine
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hydrochloride; 3-(tert.-butyl-2-hydroxyothyl-amino)sydnone Lmine
hydrochloride; 3-(tert.-butyl-(2-dii~opropyl~mlno-
ethyl)amino)sydnone imine dihydrochlorlde; 3-(tert.-butyl-
(2-morpholinoethyl)~mino)sydnone lmine hydrochloride;
3-(3,3-dimethylmorpholino)sydnone imine hydrochloride;
3-~2,2-dimethylpiperidino)sydnone imine hydrochloride;
3-(4-isopropyl-2,2-dimethyl-piperazino)sydnone Lmine
dihydrochloride; N-p-anisoyl-3-~4-isopropyl-2,2-dlmethyl-
piperazino)sydnone imine dihydrochloride; 3-(2/2l6/6-tetramethyl-
4-isopropyl-piperazino)sydnone imine dihydrochloride;
3-(2,6-dimethylp~peridino)-4-hexyl-sydnone imine hydrochloride;
4-benzyl-3-(2,6-dimethylpiperidino)sydnone imine hydrochloride;
3-(2,6-dimethylpiperidino)-4-(2-phenylethyl)sydnone imine hydro-
chloride; 3-(2,6-dimethylpiperidino)-4-phenylthiomethyl-sydnone
imine hydrochloride; N-benzoyl-4-benzylthiomethyl-3-(2,6-di-
methylpiperidino)-sydnone imine ; 3-(2,6-dimethylpiperidino)-
4-phenoxymethylsydnone imine hydrochloride; 3-(2-hydroxyethyl-
amino)-sydnone imine hydrochloride; 3-ethylamino-sydnone imine
hydrochloride; 3-cyclohexylamino-sydnone imine hydrochloride;
3-(2-hydroxycyclohexyl-amino)sydnone imine hydrochloride;
3-(3,3-dimethyl-1,4-tetrahydrothiazin-4-yl l,1-dioxide)sydnone
imine hydrochloride; 3-(3,3-dimethyl-perhydro-1-oxo-1,4-thiazin-
4-yl)sydnone imine hydrochloride; N-p-anisoyl-3-dicyclohexyl-
amino-sydnone imine; N-pivaloyl-3-dicyclohexylamino-sydnone
imine; 3-(2-carboxypiperidino)sydnone imine hydrochloride;
3-(2,2-dimethyl-4-methanesulphonyl-piperazino)sydnone imine
hydrochloride; 3-(2,2-dimethyl-4-toluenesulphonyl-
piperazino)sydnone imine hydrochloride; 3-(2,2-dimethyl-
4-dimethylaminosulphonyl-piperazino)sydnone imine hydrochloride;
N-isobutyroyl-3-(2,2-dimethyl-4-methanesulphonyl-
piperazino)sydnone imine; 3-(N-tert.-butyl-N-methylamino)sydnone
imine hydrochloride; 3-(N-tert.-butyl-N-butylamino)sydnone imine
hydrochloride.
The term hirudins means all pharmacologically active
hirudins. ~hese can be of natural or synthetic, but also of
genetic engineering origin. Compounds with N- or C-terminal
extensions beyond the active amino-acid sequence are embraced
just as much as pharmacologically active part-sequences and
fragments. Compared with the natural sequence, it is also
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2061~,~8
possible for one or more amino acids to be modi~ied or replaced
by other amino acids, speclfically those occurring naturally as
well as those not occurring naturally.
Covered by the term hirudins are, in particular, the
compounds specified in the following publications:
DE-A-3,445,532; EP-A-142,860; EP-A-158,564; EP-A-158,986;
EP-A-168,342; EP-A-171,024; EP-A-173,619; EP-A-193,175;
EP-A-200,655; EP-A-209,061; EP-A-225,633; EP-A-227,938;
EP-A-236,330; EP-A-252,854; EP-A-273,800; EP-A-324,712;
EP-A-345,616; EP-A-364,942.
Particularly pre~erred hirudins are recombinant
desulphato-hirudins (r-hirudin) which are obtained from E. coli,
yeast of B. subtilis and differ from natural hirudin in 2 amino
acids. One example of an r-hirudin of this type is HBW 023
lS (r-desulphato-Ile1-Thr2-hirudin1~65; Drugs of the Future I5, 267-280
(1980)).
The ratio by weight between sydnone imine and hirudin in
the combination of active substances according to the invention
can vary within wide limits.
In particular, the sydnone imine : hirudin ratio by
weight is 1:(0.025 to 40), preferably 1:(0.1 to 10).
The combination of active substances according to the
invention can be prepared by mixing the individual active sub-
stances in the specified ratios by weight.
It is normally expedient to administer the combination of
active substances in the form of a pharmaceutical preparation.
These preparations can be administered orally, for example in the
form of pills, tablets, lacquered tablets, sugar-coated tablets,
hard and soft gelatin capsules, solutions, syrups, emulsions or
suspensions or aerosol mixtures. However, administration can also
take place rectally, for example in the form of suppositories, or
parenterally, for example in the form of injection solutions, or
percutaneously, for example in the form of ointments or
tinctures.
Pharmaceutically inert inorganic or organic vehicles can
be used to produce the pharmaceutical preparations. Examples
which can be used for the production of pills, tablets, sugar-
coated tablets and hard gelatin capsules are lactose, maize
starch or derivatives thereof, talc, stearic acid or salts
2061~8
thereof, etc. Examples of vehicles for soft gelatin capsules and
suppositories a~e fats, waxes, semi-~olid and liquld polyols,
natural or hardened oils, etc. Suitable examples of vehicleq for
the production of solutlons and syrups are water, sucrose, invert
sugar, glucose, polyols, etc. Suitable examples of vehicles for
the production of injection solutions are water, alcohols, gly-
cerol, polyols or vegetable oils.
The pharmaceutical preparations can contain, besides the
active substances and vehicles, also additives such as, for
example, fillers, extenders, disintegrants, binders, lubricants,
wetting agents, stabilisers, emulsifiers, preservatives, sweet-
eners, colorants, flavourings or aromatising agents, buffer
substances, also solvents or solubilisers or agents to achieve a
depot effect, as well as salts for altering the osmotic pressure,
coating agents or antioxidants. They can also in each case con-
tain two or more sydnone imines and hirudins as well as other
therapeutically active substances.
Examples of other therapeutically active substances of
this type are: ~-receptor blockers such as, for example, pro-
pranolol, pindolol, metoprolol; vasodilators such as, for
example, carbochromen; sedatives such as, for example, barbituric
acid derivatives, 1,4-benzodiazepines and meprobamate; diuretics
such as, for example, chlorothiazide; cardiac tonics such as, for
example, digitalis products; agents lowering blood pressure, such
as, for example, hydralazine, dihydralazine, ramipril, prazosin,
clonidine, rauwolfia alkaloids; agents which lower the level of
fatty acids in the blood, such as, for example, bezafibrate,
fenofibrate; agents for thrombosis prophylaxis, such as, for
example, phenprocoumon.
In a particular embodiment of the present invention, the
combination of active substances according to the invention is
administered not in the form of a mixture, that is to say in a
pharmaceutical preparation, but separately from one another, that
is to say in two separate pharmaceutical preparations, simul-
taneously or in quick succession.
In this case, the pharmaceutical preparations contain
either sydnone imine or hirudin, but otherwise correspond to the
preparations described above.
In a further embodiment of the present invention, a
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preparation which contains sydnone imine and hirudin i~ admini-
stered and, sLmult~neously or shortly thereafter, ~ prepc~rAtion
which contains sydnono imino or hlrudin. ~h~ p~eparatlons corres-
pond to those describ~d above in thi~ cas~ too.
The combinations of active sub~tances ~ccording to the
invention can be used in humans to control or prevent disorders
of the cardiovascular system, in particular as antithrombotic
medicines.
The dosage can vary within wide limits and should be
suited to the individual circumstances in each individual case.
In general, on oral administration, a daily dose of about 0.5 to
lO0 mg, preferably 1 to 20 mg, of combination of active sub-
stances is appropriate per human individual. Because of the good
absorption of the active substances, the daily dose with other
types of administration is also in similar ranges of amounts,
that is to say generally likewise 0.5 to 100 mg/person. The daily
dose is normally divided into several, for example 2 to 4, part
administrations.
Surprisingly, there is mutual enhancement of the anti-
thrombotic effects of sydnone imine and hirudin in the combi-
nations of active substances according to the invention. It is
thus possible to achie~e a very strong effect even at dosages at
which neither sydnone imine nor hirudin alone has an effect.
This effect has been determined in the following test:
A coronary thrombosis was produced by the method of Folts
et al. (Circulation lg76, 5~; 365-70~ in male domestic pigs about
4 months old. The animals were deeply anaesthetised and the
thorax was opened; the left descending coronary artery was
exposed and provided with an electromagnetic blood flow sensing
element. The coronary vessel was damaged distal of the sensing
element by brief (l s) pinching with a vessel clamp. A plastic
constrictor (internal diameter 1.7-2.8 mm, length 2 mm) which
stenosed the vessel was placed at this point. These experimental
conditions resulted in the occurrence of regular cyclic changes
in blood flow (CFVs), caused by the repeated formation and
embolisation of thrombi at the damaged stenosed site. The thrombi
were quantified by determining the frequency (number per 60 min)
of the cyclic changes in blood flow. After cycles had occurred
regularly for 60 min, the animals received one of the following
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206 1~'8
substances:
&roup 1 (n = 6) N-ethoxycarbonyl-3-mo~pholino-sydnone imine
(molsidomine), 0.1 mg/kg :intr~venously as
bolus in;ection;
Group 2 (n = 6) recombinant desulphato-hirudin (r-hirudin) in
a total dose of 0.2 mg/kg divided into intra-
venous bolus injection of 0.1 mg/~g followed
by a continuou~ intraven~u~ inf~sion of
16.67 ~gJkg min (- 1 mg/kg 60 mln);0 Group 3 (n = 6) molsidomine + r-hirudin, i.e. 0.1 mg.kg
molsidomine i.v. together Wit}l 0.1 mg/kg
r-hirudin i.v., followed by 0.1 mg/kg-60 min
r-hirudin infusion.
In prelLminary experiments, these single doses of
molsidomine and r-hirudin were found to be just without anti-
thrombotic effect.
The experLment was terminated 1 hour after the end of the
infusion or 2 hours after bolus injection of the substances. The
frequencies of the cyclic changes in blood flow in the hour
before and in the first and second hour after injection of the
test substances were a~eraged for each experimental group. The
average frequencies of the cycles before and after administration
of the substances were compared statistically (Student's t test
for connected samples). A p value of < 0.05 was regarded as a
25 significant difference. The results are to be found in the
following table:
Experimental group Number of thrombotic flow cycles
(n/60 min)
1st hour 1st hour 2nd hour
preliminary after injection of
period substances
1 16 ~ 2 19 + 3 #
(molsidomine)
2 12 + 1 11 + 2 12 + 1
(r-hirudin)
3 12 + 1 8 + 2 2 + 2
# no effect; number of cycles could not be determined because
continuous occlusion occurred in some cases
* p < O.01 compared with preliminary phase.
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2 0 ~
In addition, the systolic and diastolic peripheral
arterial blood pressure (mm Hg) of all the animals was measured.
In group 2, r-hirudin infusion dld not change the blood pres~ure.
Molsidomine (group 1) at the dose administered lowered the
S systolic blood pressure by a maximum of 18 + 5 mm Hg tp ~ 0.05)
and the diastolic blood pressure not significantly by
10 + 5 mm Hg. In combination with r-hirudin (group 3), mol-
sidomine lowered the systolic blood pressure by 21 + 4 (p ~ 0.05)
and the diastolic blood pressure not significantly by
11 + 3 mm Hg. This lowering effect of molsidomine on blood
pressure was the same in both experimental groups.
The results show that the combination of active sub-
stances according to the invention in these doses leads to an
almost complete inhibition of experimental thrombi. The anti-
thrombotic effects of these two substances with a different
mechanism of action accordingly potentiate each other without
leading to an increase in the haemodynamic effect.
The advantage of the combination of active substances
according to the invention is that low doses of both substances
are possible. This reduces the potential for side effects and the
combination can also be used in those patients whose cardio-
vascular status does not permit a lowering of blood pressure.
Another advantage of the combination according to the invention
is that the simultaneous influencing of two independent mechan-
isms of thrombosis means that a particularly effective treatment
and prophylaxis of thrombo-embolic disorders is possible. On the
one hand, the effect of thrombin is abolished by the hirudin.
This inhibits fibrin formation and platelet activation. Thrombin
is regarded as the principal platelet activator in arteries too
(Proc. Natl. Acad. Sci. USA 1988; 85: 3184-88). On the other
hand, the sydnone imine activates one of the two endogenous
counter-regulations of platelet stimulation by NO-mediated
increase in cyclic guanosine monophosphate. This results in inhi-
bition of platelet adhesion and aggregation (Circulation 1989;
79: 657-65). The two mechanisms together potentiate one another
in the antithrombotic effect.
The following examples illustrate the combination of
active substances according to the invention:
2 U ~ 8
Example l
N-Ethoxycarbonyl-3-morpholino-9ydnone Lmine (mol~ldomine) and
r-hirudin in the ratio 1~2 by weight.
Example 2
3-Norpholino-sydnone imine hydrochloride (SIN-l) and r-hirudin in
the ratio of 1:2 by weight.
Example 3
N-(4-Methoxybenzoyl)-3-(cis-2,6-dimethylpiperidino)~ydnone imine
and r-hirudin in the ratio of 5:2 by weight.
Example 4
N-Propionyl-3-(3,3-dimethylthiomorpholino)sydnone imine and
r-hirudin in the ratio of 1:2 by weight.
Example 5
3-(1,1-Dioxo-dimethyl-thiomorpholino)sydnone imine hydrochloride
and r-hirudin in the ratio of 1:1 by weight.
Example 6
3-(3,3-Dimethyl-thiomorpholino)sydnone imine hydrochloride and
r-hirudin in the ratio of 1:3 by weight.
ExamPle 7
3-(cis-2,6-Dimethylpiperidino)sydnone imine hydrochloride and
r-hirudin in the ratio of 1:1 by weight.
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