LU87488A1 - PHARMACEUTICAL COMPOSITIONS ACTING IN PARTICULAR ON THE CARDIAC AND CARDIOVASCULAR SYSTEM AND PROCESS FOR PREPARING THEM - Google Patents

Description

Pharmaceutical compositions acting in particular on the cardiac and cardiovascular system and method for preparing them.

The present invention relates to pharmaceutical compositions which act in particular on the thromboembolic states of the cardiac and cardiovascular system.

According to another aspect of the invention, there is provided a process for the preparation of said compositions.

We know that the fatty acids 3ο - 3 unsaturated in G18-24 have advantageous biological properties. Among these compounds, eicosapentaenoic acid (EPA) and Docosahexaenoic Racide (DHA) are remarkable, Dyerberg et al, / "The Lancet 15, 117 (1978) ^ 7 have emphasized the importance and the multiple biological effects of these two acids.

The effects of the important role of poly-saturated fatty acids, in particular EPA and DHA, in hyperlipidemia and thrombotic conditions have been reviewed by Goodnight et al, /".Arteriosclerosis 2, 87 (1982) Review 7 ·

Pharmaceutical compositions containing EPA and DHA as active ingredients have been described, for example, in the specification of German Patent No. 3,438,630 for Lowering Blood Cholesterol Levels, and further in the published Japanese Patent Applications under Nos. 58.08037 and 60, 49097 against cerebral sclerosis and to prevent the formation of thrombus in patients suffering from heart disease.

Several articles have been devoted to the inhibitory effect of platelet aggregation, therefore to the inhibitory effect of thrombus formation of EPA and DHA / "spencer and Garaega: Prostagl, Leukotria and Med, 23, 129 (l986) in Knapp et al, s New Engl. Journ. of Med, 314, 937 (1986) ^ 7 · The antiviral action of EPA and DHA is described in the specification of the patent for U, S, A, No. 4,513,008,

The active ingredients in fish oil, for example EPA and DHA, are precursors of biosynthesis of the PG-3 series and they inhibit the formation of harmful metabolites such as TXA2 and TXB2 from the "waterfall". * arachidonic acid ", so called, which is a chain of complicated biochemical processes starting from 1 * arachidonic acid *

Apart from their various favorable effects, polyunsaturated fatty acids have the disadvantageous pathological property of being subject to spontaneous decomposition by oxidation in the human body, thus giving rise to the formation of active aldehydes, such as malonodialdehyde which is harmful to the organism. These aldehydes are capable of reacting mainly with the connective tissues in a physiologically harmful way which can lead to the "lipofus-cinose cerolde", so called, of the central nervous system *

Research has started on the biological effects of microelements and trace elements in the last decade. It is thus now known that selenium is one of the most important and essential substances for life * The favorable action of selenium is mainly due to its activating effect towards the enzyme glutathione peroxidase, because selenium is an essential constituent of the prosthetic group of the enzyme glutathione-peroxidase. This enzyme is the most important endogenous inhibitor of pernicious peroxidation processes.

Selenium, in itself, has a hypotensive effect, improves ischemic, hypoxic and infarctic states of the heart and inhibits the lipofuscinosis cerolde, and, moreover, it also exerts an advantageous effect on periodontitis. It has a notable anticancer activity and decreases the probability of progression of cancerous diseases. In addition, it is considered to be a mutagenesis-inhibiting agent.

Selenium does not accumulate in the organism, so it must be supplemented continuously · Until then, selenium has been mainly Introduced into the organism in the form of inorganic compounds, for example dioxide selenium and sodium selenite · A number of pathological alterations or conditions, such as hepatic necrosis, muscle necrosis, destruction of the erythrocyte membrane, interstitial lesions, elevation of SX in the ECG, Mulberry heart syndrome, kwashiorkor syndrome (protein malnutrition) and multiple sclerosis have been shown to be respectively triggered by selenium deficiency.

A comprehensive review of the biological effects of selenium has been published by Thressa et al. / Nutrition Review 35, 7 (1977) ^ /, Shamberger / "j. Of Env · Path, and Tox. 4, 305 (1980) 7 as well as Masu- a * ·» kawa et al * / Experientia 39, 405 ( 1983) 7mt ^ ·

The organic selenium compounds can be obtained by synthetic methods described, for example, by Klaiman and GUnther in their work entitled "Organic Selenium Compounds, Their Chemistry and Biology", John Wiley and Sons, Inc. tTew-York, 1973 · In addition, organic selenium compounds can also be prepared using microorganisms · According to this latter process, microorganisms are cultivated on media enriched with inorganic selenium compounds. The organisms which are suitable for this purpose fix selenium and, by introducing it into their metabolism, they bind it to organic substances, most frequently to amino acids or fats. These possibilities are described by Danch and Chmielow-sky./"Pr. Nauk. Univ. Slask Katowich 1 ^ 57 (1985) ^ 7 as well as by Genni-ty et al, / "Biochem. Biophys, Res, Commun, 118, 176 (1984) /. m m

We know a certain number of micro-organisms which are able to collect some elements, such as selenium, in their environment and to accumulate them in their own organism.

The preparation of selenium-enriched yeast is described in the specification of U.S. Patent No. 4,530,846 and in Japanese Patent Applications published under Nos. 60.224451 and 57.174098. The enrichment in selenium by other micro-organisms, such as bacteria, actinomycetes and hyphomycetes, in addition to yeast, is described in the Japanese patent application published under No. 78.148587.

According to the Japanese patent application published under No. 58.129954, the use of yeast containing selenium, mixed with vegetable oils, is proposed for treating senile diseases.

The object of the present invention is to provide a new pharmaceutical composition acting on the cardiac and cardiovascular system, which makes it possible to combine the advantageous properties of microorganisms containing nascent organic selenium with those of DHA and EPA and which simultaneously eliminates the disadvantageous properties of polyunsaturated fatty acids. The invention is based on the discovery that the abovementioned objective can be fully achieved by virtue of a composition, useful for eliminating ceroid lipofuscinosis, which can be obtained by using a microorganism containing selenium, efcansfr 'nontoxic from the point of for human use, together with polyunsaturated fatty acids.

According to a characteristic of the invention, a pharmaceutical composition acting on the cardiac and cardiovascular system is thus proposed, which comprises 0.5 to 50% by mass of a microorganism containing selenium as well as 99.5 to 5% by weight. mass of fatty acids in c ^ ge24 containing at least two unsaturated bonds and / or their derivatives, optionally in admixture with vehicles and / or additives commonly used in the pharmaceutical industry and optionally with antioxidants.

Furthermore, the invention relates to a process for preparing the above-mentioned composition, which consists in mixing 0.5 to 50% by mass of a microorganism containing selenium as well as 99.5 to 50% by mass. * C fatty acids containing at least two unsaturated 1 1 · 2μ · bonds and / or their derivatives, optionally with vehicles and / or additives, commonly used in the pharmaceutical industry, and optionally with antioxidants, d * a manner commonly known in the pharmaceutical industry,

The composition according to the invention suitably comprises eicosapentaenoic acid (EPA) and docosahexaenolic acid (DHA) as well as a yeast containing selenium.

As raw materials of the unsaturated CO-3 fatty acids in C ^ g ^ representing one of the components of the composition of the invention, use may be made of oils which can be obtained from various marine fish and fresh water, mainly from mackerel, cod, herring, sardines, or molluscs such as squid, as well as from the liver of said fish, for example cod liver oil and cod liver oil shark.

In addition to EPA and DHA, fish oils contain a large amount of saturated fatty acids as well as partially unsaturated fatty acids and other non-hydrolyzable constituents. The elimination of these constituents is very important, otherwise the administration of the therapeutic compositions prepared from fish oils would have the effect of appreciably increasing the caloric quantity introduced as well as the blood level of triglycerides. In addition, the non-hydrolyzable 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 body, so that a prolonged treatment, necessary for the desired effect, would be impossible using a composition containing these vitamins. Consequently, the abovementioned constituents, such as saturated and partially unsaturated fatty acids as well as the non-hydrolyzable constituents, for example cholesterol and vitamins A and D, must be eliminated from fish oil. the total amount of EPA and DHA in fish oil is enriched by more than 50%.

It is suitable to use strains of microorganisms which are not toxic towards the human organism and which are useful for human consumption. Genera widely used in biotechnology, such as Lactobacillus, Leukonostoc, Pediococcus, Acetobacter, Streptococcus, Torula, Kluyveromyces, Candida, Brettano-myces, Brevibacterium, Saccharomyces, Torulopsis, Pichia, Hansenula, Oidium, Rhodotorula, Rhodotorula, Trichosporula, Trichosporula , Monas-cus, Aspergillus, among others, which are useful for human consumption, are very suitable for this purpose.

To inhibit the oxidation of the composition of the invention, it is suitable to use -tocopherol (vitamin E), glutathione or common antioxidants such as butylhydroxy-toluene, as active preservatives.

As a vehicle or carrier, substances commonly used in the pharmaceutical industry, such as lactose, starch or magnesium stearate, can be used.

Pharmaceutical research has demonstrated that the composition of the invention is devoid of harmful health side effects, since no modification of the enzymatic micro-somal system of the liver is observed in rats using a hundred times the amount of the current dose · The amount of lipofuscin accumulated in the body is significantly lowered by comparison with the control a-pres treatment with the composition of the invention · According to examinations carried out using the composition of the invention in rats Wistar for 6 weeks, there is an inhibitory effect on platelet aggregation, free of ambiguity.

The optimal daily effective dose (calculated for an average body weight of 70 kg) of the composition of the invention is 0.1 to 5 g. The oil component contains 22% EPA and 43% DHA on average. The effective daily dose is 100-300 ^ xg of selenium.

The main advantages of the composition of the invention can be summarized as follows: 1. The advantageous properties of EPA and DHA as well as those of selenium and yeast are combined. 2. The damaging effects of known compositions containing fish oils, which are caused by constituents such as sterols as well as vitamins A and D, are reduced or eliminated. 3. There is no possibility of the appearance of "ceroid lipofuscinosis" as in the consumption of polyunsaturated fatty acids. 4. The composition of the invention contains selenium as a nascent substance enriched in a microorganism such as bacteria, fungi or yeasts. Selenium administered together with the specific microorganism is better absorbed and preferably exerts its favorable effect. 5. Selenium bound in organic compounds can be economically prepared in large quantities, specifically enriched in microorganisms, mainly in yeasts. 6. The composition exerts a favorable therapeutic action in the case of atopic disorders and is useful for the preventive treatment of eczema, asthma, allergic symptoms, allergic rhinitis and / or atopic disorders such as migraine, Crohn's disease, ulcerative colitis, otitis media, nephrotic syndrome and diabetes. It is particularly useful for the treatment of disorders of the cardiovascular system: for apoplectic manifestations, thromboembolic states such as ictus, infarction and Keshan syndrome in young patients, as well as for the prevention of abnormal conditions. The invention is illustrated in detail by the following examples, given by way of indication, but in no way limiting.

The polyunsaturated fatty acids 3 used in the composition of the invention can be prepared in accordance with Example 1, while the selenium-enriched microorganism can be prepared as described in Examples 2 to 6. The pharmaceutical compositions according to invention are illustrated by Examples 7 to 9,

Example 1

8 kg of a 40% sodium hydroxide solution, 50-60 ° C in 24 kg of mackerel oil dissolved at 60 ° C are poured in 16 liters of methanol with stirring. Then the mixture is stirred at 60 ° C for an additional 45 minutes. After adding 20 kg of 15% hydrochloric acid to the solution at approximately 60 ° C., the layers are separated and the organic phase is washed with 10 kg of 15% hydrochloric acid, then with 180 liters of water from the hot tap, until neutral. The phases are again separated, 10C liters of acetone are added to the oily phase which is then heated to approximately 45 ° C. and 3.8 kg of lithium hydroxide dissolved in 30 liters of water are added. stirring for 30 minutes, the mixture is left to stand overnight, then it is filtered and the ace-tonic filtrate is evaporated. The residue is acidified by adding approximately 8 kg of 15% hydrochloric acid, it is extracted 3 times with hexane and the hexane solution is evaporated. A nitrogen atmosphere is used during the entire purification operation. 6.4 kg of purified fish oil are thus obtained.

1 kg of the purified mackerel oil as described above is poured dropwise at 60 ° G into a solution containing 3 kg of urea in 9 liters of methanol. The mixture is stirred at the same temperature for 2 hours, then cooled. It is left standing at -10 ° G overnight, then filtered and the filtrate is evaporated. 2.5 liters of hydrochloric acid III are added to the residue and the mixture is stirred for 15 minutes. 0 After extraction with hexane, the hexane phase is washed with water until neutral, it is dried over sulphate. anhydrous sodium and evaporated to obtain 0.34 kg of bi-unsaturated fatty acids having an iodine number of 315 and containing 247% of EPA and 42% of DHA.

Example 2

2 liters of a culture medium with liquid malt extract, sterilized, are poured into glass vials with a volume of 5 liters each time and the medium is enriched by adding 3% by mass of glucose. After the addition of 5 µg / ml of sodium selenite, it is inoculated with Saccharomyces çerevisiae, then cultivated under aeration at 32 ° C for 72 hours. An additional 5 μg / ml of sodium selenite is added to the culture at the 48th hour of the culture operation.

After filtration, the cells are washed several times with water and dried at 68-70 ° C. After micronization, the selenium content is determined by atomic absorption, obtaining a value of 2600 μg / g.

Example 3

Each time 100 ml of a culture medium with the liquid yeast extract are poured into Erlenmeyer flasks with a volume of 500 ml under sterile conditions. After the addition of 5 μg / r.il of sodium selenite, the medium is inoculated with the mold Aspergillus sojae, then the cultures are shaken at 28-30 ° C. An additional 5 µg / ml sodium selenite is added to the culture on the 3rd day of the culture operation. After 5 days, the dry matter is collected by filtration, washed and dried as described in Example 20. After micronization, the selenium content of a sample is determined by atomic absorption and it is found to be 3000yug / g.

Example 4

The procedure described in Example 3 is followed, except that the yeast Torulopsis utilis is used, instead of mold. The microbial mass obtained during the culture contains 1200 μg / g of selenium.

Example 5

Streptococcus thermophilus is cultured at 45 ° C. in a culture medium prepared as described in 1 * Example 3. After the culture and the subsequent treatment, the microbial powder contains 4200 yug / g of selenium.

Example 6

The Lactobacillus mesenteroides bacterium is cultivated as described in Example 3, except that the medium is supplemented by the addition of 1%, glucose and 2% calcium carbonate, obtaining a bacterial mass containing 3000 μg / g of selenium.

Example 7

10 g of a yeast powder containing selenium (selenium content: 2600yug / g) are added to 150 g of mackerel oil (containing 24% EPA and 42% DHA) enriched at 65%, · After homogenization, the mixture is preserved by adding 0.15 g of vitamin E. It is filled with the active agent thus obtained, soft gelatin capsules which are packaged in blister sheets *

Example 8

The procedure described in Example 7 is followed, except that the following starting substances are used: 400 g * cod liver oil enriched at 65%, having an EPA content of 22% and a DHA content 43%; 100 g of yeast powder with selenium content of 1200 jug / g; and 0.4 g of vitamin E. Capsules capable of receiving 500 mg of active agent are filled with 1 * homogenate.

Example 9

The procedure described in Exeraple 8 is followed, except that a fish oil containing 400 gd * is used as a fish oil component at 30% (containing 18%, EPA and 12% e of DHA) (produced by Martens and Go, Bergen, Norway)

Example 10 i I I tmmim

The procedure described in Example 8 is followed, except that 400 g of a fish oil containing 9.2% of EPA and 10.4% of DHA are used as the fish oil component.

Example 11

Tablets having the following composition are prepared, using known pharmaceutical devices and methods:

Cod liver oil enriched in EPA and DHA and containing 0.1% vitamin E (with 43% DHA content and 22% EPA content) 200.0 mg

Yeast powder containing selenium (selenium content 1500 ^ ig / g) 86.0 mg

Lactose 140.0 mg

Starch 60.0 mg

Polyvinyl pyrrolidone 3.5 mg

Magnesium stearate 3.5 mg

If desired, the tablets can be coated with sugar or another substance in a tablet making machine

It is understood that the invention can be implemented according to numerous variants and modifications, without however departing from its scope and spirit.

Claims (6)

1. Pharmaceutical compositions acting on the cardiac and cardiovascular system, characterized in that they comprise 0.5 to 507. by mass of a microorganism containing selenium as well as 99.5 to 507. by mass of fatty acids in Cig_24 containing at least two unsaturated bonds and / or their derivatives, optionally in admixture with vehicles and / or additives commonly used in the pharmaceutical industry and possibly with antioxidants. 2. Composition according to claim 1, characterized in that it comprises 5 to 35% by mass of the microorganism containing selenium and 95 to 65% by mass of the unsaturated fatty acids or their derivatives. 3. Composition according to claim 1, characterized in that it comprises 15 to 25% by mass of the microorganism containing selenium and 85 to 757. by mass of the unsaturated fatty acids. 4. Composition according to claim 1, characterized in that it comprises fatty acids extracted from oil of marine fish as unsaturated fatty acids. 5. Composition according to claim 1, characterized in that it comprises 5,8,11,14,17-eicosapentaenoic acid and 1 ”4,7,10,13,16,19-docosahexaenoic acid as unsaturated fatty acids. 6 · Process for the preparation of a pharmaceutical composition acting on the cardiac and cardiovascular system, characterized in that 0.5 to 507 by mass of a microorganism containing selenium is mixed as well as 99.5 to 507 by mass of C 18-24 fatty acids containing at least two unsaturated bonds or their derivatives with vehicles and / or additives commonly used in the pharmaceutical industry and possibly with antioxidants, in a commonly known manner in 1 * pharmaceutical industry * 7 "Process according to claim 6, characterized in that 5 * 35% by mass of the microorganism containing selenium is mixed with 95 to 657" by mass of unsaturated fatty acids * 8 # Process according to claim 6, characterized in that 15 to 25% by mass of the microorganism containing selenium are mixed with 85 to 75% by mass of the unsaturated fatty acids, 9 "Process according to claim 6, characterized in that l * fatty acids extracted from marine fish oil are used as unsaturated fatty acids 10, A method according to claim 6, characterized in that it comprises the use of 5,8,11,14,17-eicosapenta-enolque acid and 4,7,10,13,16,19 acid -docosahexaenoic as * unsaturated fatty acids.