Note: Descriptions are shown in the official language in which they were submitted.
1319607
PHARMACEUTICAL COMPOSITIONS ACTING PARTICUIARLY ON THE
HEART AND CARDIOVASCULAR SYSTEM AND PROCESS F02 PREPA~ING
SAME
This invention relates to pharmaceutical compositions
acting particularly on the thrombo-embolic states of the
heart and cardiovascular system.
According to an other aspect of the invention,
there is provided a process for the preparation of these
compositions.
It is known that C18_24 3 unsaturated f y
acids possess advantageous biological properties. Of these
compounds eicosapentaenoic acid (EPA) and docosahexaenoic
acid (DHA) are outstanding. Dyerberg et al. / The Lancet
15, 117 (197a)7 pointed at the importance and multifold
biological effects of both acids.
The effects connected with the important role of
polyunsaturated fatty acids, particularly of EPA and DHA,
in the hyperlipidaemia and thrombotic diseases have been
summarized by Goodnight et al. / Arteriosclerosis 2, 87
(1982) Review7.
Pharmaceutical compositions containing EPA and DHA
as active ingredients have been described e.g. in the
German patent specification No. 3,438,630 for lowering the
blood cholesterol level, further in the published Japanese
patent applications Nos. 58.08037 and 6D.49D97 against
A 4419-5603 PT/Gi
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cerebral sclerosis and for preventing the thrombus formation
in patients suffering from heart diseases.
Several papers have been devoted to the platelet-
-aggregation inhibiting and thus the thrombus-formation
inhibiting effect of EPA and DHA / Spencer and Caraega:
Prostagl. Leucotrienes and Med. 23, 129 (1986); Knapp et
al.: New Engl. Journ. of Med. 314, 937 (19B6)7.
The antiviral action of EPA and DHA is described
in the United States patent specification No. 4,513,008.
The active ingredients of the fish-oil, e.g. EPA
and DHA, are precursors of the biosynthesis of the PG-3
series and they inhibit the formation of harmful metabolites
such as TXA2 and TXB2 arising from the so-called "arachidonic
acid cascade" which is a chain of complicated biochemical
processes starting from arachidonic acid.
In addition to their several favourable effects,
the polyunsaturated fatty acids have the disadvantageous
pathologic property that they are subjected to a spontaneous
oxidative decomposition in the human organism and thus give
rise to the formation of active aldehydes such as malondi-
aldehyde which is harmful to the organism. These aldehydes
are capable to react mainly with the connective tissues
in a physiologically harmful way which may lead to the
so-called "ceroidal lipofuscinosis" of the central nervous
System-
Investigations on the biological effects of micro-
elements and trace elements have been started in the last
decade. Thus, it became known that selenium is one of the
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most important and indispensable substances of life. The
beneficial action of selenium is mainly based on its
activating effect directed to the glutathione-peroxidase
enzyme as selenium is an indispensable constituent of the
prosthetic group of the glutathione-peroxidase enzyme. This
enzyme is the most important endogenic inhibitor of the
harmful peroxidation processes.
Selenium in itself has hypotensive effect, improves
the ischaemic, hypoxic and infarction states of the heart
and inhibits the ceroidal lipofuscinosis, furthermore, it
also exerts a beneficial effect on periodontitis. It has
a significant anticancer activity and diminishes the
probability of the development of cancer diseases; further-
more, it is considered to be a mutagenesis-inhibiting agent.
Selenium is not accumulated in the organism, thus
it should continuously be supplemented. Hitherto, selenium
has mainly been introduced to the organism in the form of
inorganic compounds, e.g. selenium dioxide and sodium
selenite. A number of pathologic alterations or diseases,
respectively, such as liver necrosis, myonecrosis, destruc-
tion of the erythrocyte membrane, interstitial laesions,
ST-elevation in the ECG, Mulberry's heart syndrome,
kwashiorkor syndrome (protein malnutrition) and multiplex
sclerosis proved to be induced by selenium deficiency.
A comprehensive report on the biological effects
of selenium was published by Thressa et al. / Nutrition
Review 35, 7 (1977)7, Shamberger / J. of Env. Path. and
1 31 9607
Tox. 4, 305 (1980)7 as well as Masukawa et al. / Experientia
_ , 405 (198~)7.
Organic selenium compounds can be obtained by
synthetic methods described e.g. by;<laiman and Gunther in
their book entitled "Organic Selenium Compounds, Their
Chemistry and Biology", Oohn Wiley and Sons, Inc. New York,
1973; besides, organic selenium compounds can be prepared
also by using microorganisms. According to the latter
method, the microorganisms are cultivated on media enriched
with inorganic selenium compounds. Organisms which are
suitable to this purpose take up selenium and, introducing
it to their metabolism, they bind it to organic substances,
most frequently to amino acids or fats. Such possibilities
are published by Danch and Chmielowsky / Pr. Nauk. Univ.
Slask Katowich 1, 57 (1985)7 as well as by Gennity et al.
/ Biochem. Biophys. Res. Commun. 118, 176 (19a4)7.
A number of microorganisms are known which are
capable to collect some elements such as selenium from their
environment and to accumulate them in their own organism.
The preparation of yeast enriched with selenium is
described in the United States patent specification No.
4,530,846 and in the published Japanese patent applications
Nos. 60.224451 and 57.17409B.
The enrichment of selenium by other microorganisms
such as bacteria, actinomycetes and hyphomycetes, besides
the yeast, is described in the published Japaness patent
application No. ~ .148587.
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According to the published Japanese patent applica-
tion No. 58.129954 selenium-containing yeast in an admixture
with vegetable oils is suggested to use for treating senile
diseases.
The aim of the present invention is to provide a
novel pharmaceutical composition acting on the heart and
cardiovascular system which renders possible to combine the
advantageous properties of native organic selenium-containing
microorganisms with those of OHA and EPA
and which si~ultaneously eliminates
the disadvantageous properties of polyunsaturated fatty acids.
The invention is based on the recognition that the
above aim can be fully achieved and a composition, useful
to eliminate ceroidal lipofuscinosis, can be
obtained by using a selenium-containing microorganism,
being non-toxic from the viewpoint of human use, together
with polyunsaturated fatty acids.
Thus, the present invention relates to a pharmaceutical
composition acting on the heart and cardiovascular system,
which comprises 0,5 to 50% by mass of a selenium-containing
microorganism as well as 99.5 to 50% by mass of Cla 24 fatty
acids containing at least two unsaturated bonds and/or the
derivatives thereof, optionally in admixture with carriers
and/or additives commonly used in the pharmaceutical industry
and optionally with antioxidants.
Furthermore, the invention relates to a process for
preparing the above composition, which comprises admixing
1319607
0,5 to 50% by mass of a selenium-containing microorganism
as well as 99.5 to 50% by mass of D18 24 fatty acids contain-
ing at least two unsaturated bonds and/or derivatives there-
of, optionally with carriers and/or additives, commonly
used in the pharmaceutical industry, and optionally with
antioxidants, in a manner commonly known in the pharmaceutical
industry.
The composition according to the invention suitably
comprises eicosapentaenoic acid (EPA) and docosahexaenoic
acid (DHA) as well as selenium-containing yeast.
As raw material of the C18 24 ~~3 unsaturated
fatty acids representing one component of the composition,
oils obtainable from various marine and fresh-water fishes,
mainly from mackerel, cod-fish, herring, sardine, squid
as well as from the liver of these fishes, e.g. cod-liver
oil and shark-liver oil, can be used.
In addition to EPA and OHA, the fish-oils contain
a large amount of saturated fatty acids as well as fatty
acids unsaturated to a low extent and other nonhydrolyzable
components The removal of these constituents is very
important since the dosis of the therapeutical compositions
prepared from the fish-oils would sensitively enhance the
introduced calory quantity as well as the triglyceride
level of the blood. Besides, the nonhydrolyzable constituents
may contain steroids such as cholesterol; vitamin D (and
its provitamin) and/or vitamin A. Vitamins D and A are
accumulated in the human organism whereby a prolonged treat-
ment, needed to the desired effect, would be impossible by
1 31 9607
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using a composition containing these vitamins. Therefore,
the components mentioned above such as the saturated and
partly unsaturated fatty acids as well as the nonhydro-
lyzable constituents, e.g. cholesterol, vitamins A and 0,
have to be removed from the fish-oil. In this way the
total amount of EPA and DHA in the fish-oil is enriched to
more than 50%.
It is suitable to use microorganism strains which
are non-toxic to the human organism and are useful for
human consumption. Genera widely used in the biotechnology
such as Lactobacillus, Leukonostoc, Pediococcus, Aceto-
bacter, Streptococcus, Torula, Kluyveromyces, Candida,
Brettanomyces, Srevibacterium, Saccharomyces, Torulopsis,
Pichia, Hansenula, Oidium, Rhodotorula, Trichospora,
Penicillium, Rhizopus, Mucor, Monascus, Aspergillus, etc.
which are useful for human consumption, are most convenient
for this purpose.
For inhibiting the oxidation of the composition
according to the invention, it is suitable to use ~-
-tocopherol (vitamin E), glutathione or the traditional
antioxidants such as butylhydroxytoluene as active preserv-
ing agents.
Materials commonly used in the pharmaceutical
industry such as lactose, starch or magnesium stearate
can be used as vehicle or carrier.
Pharmaceutical investigations proved that the
composition is free from side effects damaging the health
1 3 1 9607
as no change was observed in the microsomal enzyme system
of the rat liver by using a hundred-fold quantity of the
usual dose. The amount of lipofuscine accumulated in the
organism was significantly decreased as compared to the
control after treatment with the composition. ~ased on
investigations carried out by using the composition of the
invention on female Wistar rats for 6 weeks, an unambiguous
platelet-aggregation inhibiting effect was observed.
The optimum effective daily dose (calculated for
an average body-weight of 70 kg) of the composition is from
0.1 to 5 9. The oil component contains 22% of EPA and 43
of OHA as an average. The effective daily do~e amounts
to lOO 300 /ug of selenium.
The main advantages of the composition according to
the invention can be summarized as follows:
1. The advantageous properties of EPA and DHA
as well as of selenium and the yeast are combined.
2. The harmful effects of the known fish-oil contain-
ing compositions, which are induced by saturated lipid
components such as sterols as well as vitamins A and D,
are diminished or eliminated.
3. The possibility of "ceroidal lipofuscinosis"
occurring on the consumption of polyunsaturated fatty acids
is removed.
4. The composition contains selenium as native
substance enriched in a microorganism such as bacteria, fungi
or yeast; the selenium administered together with the
specific microorganism is better resorbed and exerts more
1319607
preferably its favourable effect.
5. Selenium bound in organic compounds can cheaply
be prepared in large amounts specifically enriched in micro-
organisms, mainly in yeast.
6. The composition develops a favourable therapeutic
action in case of atopic disturbances and is useful for
the preventive treatment of eczema, asthma, allergic
symptoms, allergic rhinitis and/or atopic disturbances
such as migraine, Crohn's disease, ulcerative colitis, otitis
l~ media, nephrotic syndrome and diabetes. It is particularly
useful for the treatment of disturbances of the cardio-
vascular system: for apoplectic manifestations, thrombo-
-embolic states such as stroke, infarction and Keshan
syndrome of young patients as well as for the prevention
of abnormal conditions.
The invention is illustrated in detail by the follow-
ing non limiting Examples.
The ~-3 polyunsaturated fatty acids used in the
composition according to the invention can be prepared
according to Example 1 whilst the microorganism enriched
with selenium can be prepared as described in Examples 2 to
6. The pharmaceutical compositions according to the invention
are illustrated by Examples 7 to 9.
Example l
8 kg of 40% sodium hydroxide solution are dropped
at 50 to 60 C to 24 kg of mackerel oil dissolved at 60 C
in 16 litres of methanol under stirring. Then the mixture
is stirred at 60 C for additional 45 minutes. After adding
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20 kg of 15% hydrochloric acid to the solution at about
60 C, the layers are separated and the organic phase is
washed with 10 kg of 15% hydrochloric acid and then with
180 litres of hot tap water until neutral. The phases are
again separated, 100 litres of acetone are added to the
oily phase which is then heated to about 45 C and 3,8 kg
of lithium hydroxide dissolved in 30 litres of water are
added. After stirring for 30 minutes the mixture is left
to stand overnight, then filtered and the acetone filtrate
is evaporated. The residue is acidified by adding about 8 kg
of 15% hydrochloric acid, extracted 3 times with hexane and
the hexane solution is evaporated. Nitrogen atmosphere is
used during the whole purifying operation. Thus 6.4 kg of
purified fish-oil are obtained.
lS One kg of the mackerel oil purified as described
above is dropped at 60 C to a solution containing 3 kg of
urea in 9 litres of methanol. The mixture is stirred at
the same temperature for 2 hours and then cooled. It is left
to stand at -10 C overnight, then filtered and the filtrate
is evaporated. 2.5 litres of 1:1 hydrochloric acid are added
to the residue and the mixture is stirred for 15 minutes.
After extraction with hexane, the hexane phase is washed
with water until neutral, dried over anhydrous sodium sulfate
and evaporated to give 0.34 kg of ~-3 unsaturated fatty
acids with an iodine number of 315 and containing 24% of
EPA and 42~ of OHA.
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Example 2
2 litres of sterilized, liquid malt extract culture
medium each are poured into glass bottles of 5 litres
volume and the medium is enriched by adding 3% by mass of
glucose. After adding 5 /ug/ml of sodium selenite, it is
inoculated with Saccharomyces cerevisiae and then cultivated
under aeration at 32 C for 72 hours. An additional amount
of 5 /ug/ml of sodium selenite i5 added to the culture in
the 48th hour of cultivation.
After filtration the cells are washed several times
with water and dried at 68 to 70 C. After micronization,
the selenium content is determined by atomic absorption to
give a value of 2600 /ug/g.
ExamPle 3
100 ml of liquid yeast extract culture medium each
are poured in Erlenmeyer flasks of 500 ml volume under
sterile conditions. After adding 5 /ug/ml of sodium selenite,
the medium is inoculated with the fungus Aspergillus sojae,
then the cultures are shaken at 28 to 30 C. An additional
amount of 5 /ug/ml of sodium selenite is added to the
culture on the 3rd day of the cultivation. After 5 days
the dry material is filtered off, washed and dried as
described in Example 2. After micronization, the selenium
content of a sample determined by atomic absorption is found
to be 3000 /ug/g.
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Example 4
The process described in Example 3 is followed,
except that the yeast Torulopsis utilis is used, instead
of the fungus. The microbial mass obtained during the
cultivation contains 1200 /ug/g of selenium.
Example 5
Streptococcus thermophilus is cultivated at 45 C
in a culture medium prepared as described in Example 3.
After cultivation and working up, the microbial powder
contains 4200 /ug/g of selenium.
Example 6
The bacterium Lactobacillus mesenteroides is
cultivated as described in Example 3, except that the medium
is supplemented by adding 1% of glucose and 2% of calcium
carbonate to give a bacterial mass containing 30nO /ug/g
of selenium.
Example 7
10 p of a selenium-containing yeast powder (selenium
content: 2600 /ug/g) are added to 150 9 of 65% enriched
mackerel oil (containing 24% of EPA and 42% of DHA). After
homogenization the mixture is preserved by adding 0.15 9 of
vitamin E. The thus-obtained active agent is filled into
soft gelatine capsules and packaged into blister foils.
Example a
The process described in Example 7 is followed,
except that the following starting materials are used: 400 9
of 65% enriched cod-liver oil with an EPA content of 22%
and DHA content of 43%; 100 9 of yeast powder with a selenium
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content of 1200 /ug/g; and 0.4 9 of vitamin E. The
homogenizate is filled into capsules capable of taking
. up 500 mg of active agent each.
Example 9
The process described in Example 8 is followed,
except that 400 9 of 30% fish-oil (containing 18% of EPA
and 12% of DHA) (produced by Martens and Co., Bergen,
Norway) are used as fish-oil component.
Example lO
The process described in Example 8 is followed,
except that 400 9 of fish-oil containing 9.2 % of EPA and
10.4 % of DHA are used as fish-oil component.
Example ll
Tablets with the following composition are prepared
by using known pharmaceutical devices and processes:
Cod-liver oil enriched with EPA and DHA
and containing 0.1 % of vitamin E
(with a DHA content of 43% and EPA
content of 22%) 200.0 mg
Selenium-containing yeast powder
(selenium content: 1500 /ug/g)86.0 mg
Lactose 140.0 mg
Starch 60.0 mg
Polyvinyl pyrrolidone 3.5 mg
Magnesium stearate 3.5 mg
If oesired, the tablets may be coated with sugar or
with an other substance in a tabletting machine.
