SE462701B - PHARMACEUTICAL COMPOSITION FOR REDUCING CHOLESTEROL AND TRIGLYCERIDE CONDITIONS IN THE BLOOD, AND USE THEREOF IN A FOOD PRODUCT - Google Patents

Description

D20 25 30 35 462 701 2 This process has not received any widespread attention, despite the fact that it uses a natural substance, which can be easily incorporated into the daily diet. One reason may be the difficulty of effectively separating EPA from natural fish oils to obtain a pure product at a reasonable cost. Another reason may be that the effect of taking EPA is not as dramatic as one would expect.

It is an object of the invention to eliminate the shortcomings of the prior art discussed above.

It is a further object of the invention to provide improvements in such compositions known from British Patents 1,604,554 and 2,333,745.

It is a further object of the present invention to provide a composition which has superior therapeutic effects over the prior art compositions.

It is a further object of the present invention to provide a therapeutic composition containing naturally occurring fatty acids which serves to reduce blood cholesterol and triglyceride levels.

A further object of the present invention is to provide a therapeutic composition which will increase the PGEI PGE2 ratio in the patient and increase the absolute amount of PÜÉ1 in the system.

This and other objects of the invention are achieved by the simultaneous administration of one or more of EPA and DHA together with one or more of dihomo-γ-linolenic acid (8,11,14-eicosatrienoic acid), i.e. 2D: 3w6 fatty acid, hereinafter referred to as as "DHLA"), cislinic acid ((Z, Z) -9,12-octadecadienoic acid), i.e. 18: 2w6 fatty acid and) / - linolenic acid, ((Z, Z, Z% 6.9, 12-octadecatrienoic acid) , ie 18: 3w6 fatty acid, either in the form of a pharmaceutical dose or in the form of a food product, such as margarine or cooking oil, or in the form of a skin ointment or lotion for topical application. ma so ca - .. å The prostaglandins are a family of substances, which exhibit a wide range of biological effects.Prostaglandins of the 1st, 2nd, and 3rd series include one, two, or three double bonds in their basic 2U carbon carboxylic fatty acid structure, which includes a five-membered cyclopentene ring.

The 1-series prostaglandins are potent vasodilators and inhibit cholesterol and collagen biosynthesis, the formation of platelets. On the other hand, prostaglandins from the 2-series are known to increase the accumulation of platelets, cholesterol and collagen biosynthesis, and also to increase endothelial as well as accumulation. The main effect of prostaglandins from the 3-series, especially PGÉ3, is the suppression of prostaglandins from the 2-series.

The starting material for prostaglandins from the 2-series is arachidonic acid ((all Z) -5,8,11,14-eicosatetraenoic acid), i.e. 20: 4 6-fatty acid. DHLA is the starting material for prostaglandins of the L-series, and, as indicated above, EPA and DHA are starting materials for prostaglandins from the 3-series.

It is believed that the effectiveness of EPA and DHA in preventing atherosclerotic cardiovascular disease lies both in their effect as a starting material for prostaglandin PGE3, which suppresses 2-series prostaglandins, as well as the fact that EPA and / or DHA itself competes with arachidonic acid in the same enzyme system and thus inhibits the biosynthesis of prostaglandins from the 2-series. This inhibition of 2-series prostaglandins results in an increase in the PGE1: PGE2 ratio.

To enhance the effect of using EPA and / or DHA PGE2, as well as to achieve an increase in the absolute amount of the PGEI 1 system, DHLA should be used concomitantly with pure EPA and / or DHA. Since cislinic acid and I-linolenic acid both form alone by further increasing the PGEI DHLA ratio through the metabolism in the body, either or both of these fatty acids can be fully or partially substituted for DHLA. It has been found that the combination of EPA (and / or DHA) and DHLA (and / or cislinic acid and / or γ-linolenic acid) is blood cholesterol cerides. Recent companies' research has definitely shown a significant reduction in and triglyceride between iodine cholesterol levels and the incidence of coronary heart disease (JAMA, 251, 351-364 (1984) and JAMA, 251, 365-374 (1984)). ). In addition, it is believed that such a combination will have other beneficial therapeutic properties. It is known, for example, that in schizophrenia, rheumatoid arthritis and other collagen and autoimmune diseases_as well as in certain forms of cancer, there are signs of extremely low levels of PGE1 and high levels of PGE2. It is therefore expected that the combination of the present invention may be able to serve as an effective treatment for such conditions. Furthermore, the anti-inflammatory effect of corticosteroids and the analgesic effect of aspirin are thought to be due to their suppressive effect on PGE2 formation. Thus, the use of the combination of the present invention can be expected to be a natural and highly effective anti-inflammatory analgesic.

The dosage of the composition of the present invention, including a combination of EPA (and / or DHA) and DHLA (and / or cislinic acid and / or X-linolenic acid), required for therapeutic or prophylactic action, will of course vary with the route of administration and the nature of the condition being treated, but will usually be at least lg, preferably 1.5-3 g, per day. This is the dosage for an average 70 kg human, and the dosage for other humans or animals will of course vary proportionally to this in relation to their weight, i.e. about 20-40 mg / kg.

The relative amounts of EPA (and / or DHA) and DHLA (and / or cislinic acid and / or X-linolenic acid) in the composition of the present invention are preferably 1: 1, although the ratio may vary from 3: 1 to 1: 3.

EPA (and / or DHA) and DHLA (and / or cislinic acid and / or β-linolenic acid) need not be used as the acids themselves, but can also be used as their pharmaceutically acceptable salts, esters or amides. Esters or amides that can be converted in vivo to the acid and other pharmaceutically acceptable products can be used, with the preferred ester being the ethyl ester.

The preferred salts are sodium or potassium salts, or any other pharmaceutically acceptable solid salt, since such a salt is suitable for the manufacture of GV tablets.

While it is preferred to take the composition of the present invention orally, as this is a convenient route of routine use, the active compounds may be used in any manner by which they may be successfully absorbed, e.g., parenterally. (eg subcutaneously, intramuscularly or intravenously), rectally or vaginally, or topically, eg as a skin ointment or lotion.

While it is possible to use the active components as such, as a simple mixture of components, it is preferred to provide them as a pharmaceutical product. The products, both for veterinary and human medical use of the present invention, comprise the active compounds, as described, together with one or more pharmaceutically acceptable carriers thereof and, if desired, other therapeutic ingredients. although other unsaturated fatty acids should be avoided, especially arachidonic acid. The carrier or carriers must be "pharmaceutically acceptable" in the sense of being compatible with other ingredients of the products and not deleterious to the uptake thereof. The products include those suitable for oral, rectal, vaginal, intrapulmonary or parenteral (including subcutaneous, intramuscular and intravenous) use. Products for oral use, such as tablets or capsules, are preferred.

The EPA (and / or DHA) - DHLA (and / or r-cislinic acid and / or 7-linolenic acid) combination can also be used by replacing butter and / or regular margarine with a special margarine, e.g. of the emulsion type, compound, that in normal use the recipient receives the required amount of the combination. Cooking oil and cooking fat can also be formulated in a similar manner so that they contain the composition of the present invention.

The EPA (and / or DHA) and DHLA (and / or cislinic acid and / or iplinoleic acid) used in compositions of the present invention should be as pure as possible. EPA and / or DHA cannot be used directly in the form of fish oil, as the use of the necessary amount of fish oil to provide the desired amount of EPA and / or DHA would provide excessive amounts of calories and potentially toxic amounts of vitamins A and D. Thus, pure EPA and / or DHA should be extracted from the fish oil. The presence of unsaturated fatty acids other than EPA, DHA, DHLA, cislinic acid and ¿- linolenic acid should be avoided.

Substantially pure EPA and / or DHA can be extracted from fish oil, such as cod liver oil, by a process described in, for example, US-A 4,377,526. Alternatively, the separation can be accomplished by a novel process of the present invention, comprising iodizing the double bonds of the unsaturated fatty acids in the starting fat or oil. Such iodization provides protection of the fatty acids from oxidation during the continued process, increasing the separation sharpness of the fatty acids in the following column chromatography process. After iodization, the fat or oil is saponified and the iodized fatty acid is extracted from the saponified mixture. The iodized fatty acids are then methylated and separated by column chromatography, after which the desired fractions are deiodinated. This process can be used not only for the separation of EPA and DHA from fish oils, but also for the separation and extraction of other unsaturated fatty acids, such as M-linolenic acid and 5-linolenic acid from the triglyceride forms in which they occur naturally, eg soybean oil, cottonseed oil , sunflower oil, oil from "evening primrose" (Denothera biennis, fam. Ûnograceae), etc. The separation of any unsaturated fatty acid can be facilitated by the present iodination process.

The starting material in the present process may be a natural fat or a natural oil, in which process the first step is iodination followed by saponification. However, the starting material may also be any mixture of unsaturated fatty acids which are difficult to separate, the first step being iodination, but no saponification being required, since the starting material is not a triglyceride.

Iodization takes place by adding iodine in an organic solvent, preferably as a ZU% ethanol solution, at a slow rate to the starting material until the color no longer disappears in the starting material. This reaction takes place at room temperature with continuous stirring.

The saponification step ~ can take place in any conventional manner such as, for example, with a 20% ethanol solution of KÜH for two hours.

The iodized fatty acids are extracted from the saponified mixture by any conventional procedure, eg extraction with ether.

The next step is methylation of the iodized fatty acids to prepare them for column chromatography. This is again a conventional step and can be performed, for example, with 5% hydrochloric acid in methanol.

Finally, the fatty acids are separated by column chromatography.

The column chromatography is known in a known manner with a conventional elution mixture. While the separation sharpness between the various fatty acids is very poor in conventional processes, the separation sharpness is considerably improved when the fatty acids are iodized during the column chromatography. The column may be filled with silica gel in the usual manner and the elution solution may be any conventional solution, such as hexane-ether-acetic acid (85-110-5).

After the fractions have been obtained from the column, the fatty acids are deiodinated using, for example, silver nitrate. While specific reagents and process conditions have been described for the various steps of the present process, it is understood that those skilled in the art will readily recognize other reagents and conditions for performing the process steps, when desired. the heat of each step is known once. The critical factor is the iodination before the chromatography separation to increase the separation sharpness and to protect the fatty acids from oxidation. Although the separation is carried out by column chromatography according to what is described above, it is also understood that other means of separation can be used such as, for example, high-speed centrifugation. The separation sharpness will also be improved upon iodination in such other separation processes. SBI '. An example of the process for separating EPA and DHA from cod liver oil according to the present process is described below.

Example preparation: A 20% ethanol solution of iodine is slowly added to 300 g of cod liver oil. The iodine is added as long as its color disappears in the oil. The reaction takes place at room temperature with continuous stirring. When the iodination is complete, the iodized oily solution is saponified with a 20% ethanol solution of KOH for two hours. The iodized fatty acid, 260 g, is easily extracted from the saponified mixture.

The iodized fatty acids are then methylated with 5% hydrochloric acid in methanol. EPA and DHA are separated by column chromatography (silica gel 1,500 g, Kiesel gel 70-230 mesh, Merck). Eluerin- (ss-lo-5). The first extracted fraction is iodinated DHA. The second gene is performed with 5 liters of hexane-ether-acetic acid fraction is iodinated EPA.

Once substantially pure methylated and iodinated fatty acid mixture has been obtained, it can also be separated by other conventional techniques, such as high speed centrifugation or distillation. Deodising takes place by shaking the iodinated Me-DHA and Me-EPA separately, with a 10% solution of silver nitrate. Precipitation of silver iodide takes place and the organic phase is separated. The same procedure is repeated until no further precipitation takes place. Microanalysis, HPLC and NMR have shown that the desired products have been obtained.

The yield is over 90% and the purity 96-100%. There is no need to carry out the process in a nitrogen atmosphere, since the fatty acids are saturated with iodine, thus preventing oxidation from taking place.

The following clinical test shows the synergy effects obtained when using the combination of the present invention compared to the effects of using each of the components alone.

Therapeutic example: 36 outpatient patients, age 35-75, men and women, were divided into three groups of 12. Each group increased their normal diet by 5 cm3 / day of free fatty acids for 45 days. Group I ate 5 cm3 / day of substantially pure EPA. Group II increased their diet by 5 cm 3 / day of substantially pure cislinic acid and group III increased their diet by 3 cm 3 / day of substantially pure EPA and 2 cm 3 / day of substantially pure cislinic acid. By the term "substantially pure" is meant a purity of about 96-100%.

Blood cholesterol and triglyceride levels were tested one day before the start of treatment and after 45 days of treatment. The results of the treatments are reported below in Tables I, II and III: 462 7Ü1 10 Table I: 5 cm? / Day pure EPA Total co- Total lesterol cholesterol- Trigly- 1 day before sterol cerides Trigly- treated- after 1 day before oerider Patient lingen 45 days treated- by no Age Sex mg% mg% lingen 45 days 1 45 M 250 220 130 102 2 45 M 248 220 115 95 3 47 M 230 210 102 90 4 73 M 270 240 95 90 5 60 F 280 240 115 95 6 56 M 265 260 120 105 7 54 F 215 205 95 90 8 52 F 285 250 115 100 9 63 M 300 250 110 95 10 64 M 350 260 160 105 11 55 M 190 190 95 80 12 35 M 200 190 '90 90 Mean = 2se.9 + 43.2 227.9 + 2a.a 111.s + 1s.6 94.95 + 7% decrease: "T1_%" T5% Table II: 5 cm3 / day pure cislinolic acid Total co- Total lesterol cholesterol Trigly- 1 day before sterol cerides Trigly- treatment- after 1 day before cerides Patient lingen 45 days treatment- after no Age Gender mg% mg% _ lingen 45 days 1 47 M 190 200 95 95 2 75 F 350 340 90 95 3 60 F 200 205 110 105 4 60 F 220 220 115 100 5 55 F 240 210 90 100 6 37 M 270 260 1 05 95 7 40 M 220 230 80 80 8 45 M 400 350 165 150 9 62 F 310 300 140 140 10 54 M 230 220 115 115 11 52 M 260 250 110 110 12 61 F 215 215 130 125 Mean value: 265.3i61.75 250¿50 112i22.8 109.16i19.4 [Y / m decrease: 5 Ä 2% l0 15 20 462 701 ll Table 111; 3 Cm; / day pure EPA and 2 om3 / day pure cislinol Total co- Total lesterol cholesterol- Trigly- 1 day before sterol cerides Trigly- treat- after 1 day before cerides Patient lingen 45 days treated- after no Age Sex mg% mg % lingen 45 days l 48 M 450 260 160 90 2 60 M 310 240 70 40 3 45 M 257 210 106 50 4 40 M 305 250 98 45 5 54 M 210 200 95 55 6 35 F 210 190 95 45 7 40 M 290 240 100 70 8 6l F 270 220 ll6 45 9 45 F 240 215 95 80 l0 50 F 210 190 75 60 ll 64 M 300 220 130 80 12 64 M 190 180 55 '50 270.li67.5 2l7.9i24.4 99.5 59.l6il6 Mean value: 19.3% 40.5%% reduction: While the use of 5 cmz / day of EPA alone produced a reduction in serum cholesterol and triglyceride levels during 45 days of treatment, more specifically a mean reduction of ll, 2% for total cholesterol and 1% for triglycerides, the effect of using pure cislinic acid for 45 days was almost insignificant. In fact, for many patients, cholesterol levels even rose. However, a definitive synergy effect is observed when using a combination of 3 cm; EPA plus 2 cm; day. When using the combination, a very sigcislinic acid / significant reduction in serum cholesterol content (an average of 19, is observed). 3% reduction) and serum glyceride content (an average of 40.5% reduction).

It is obvious to the person skilled in the art that the embodiment described by OVGH by means of the GV example can be varied without deviating from the basic idea of the invention. The invention is therefore not limited to the examples described above but can be varied within the scope of the following claims without departing from the basic idea of the invention.

Claims (6)

462 701 IZ PATENTKRAV A pharmaceutical composition for lowering blood cholesterol and triglyceride levels, characterized in that it consists essentially of a combination of a first component consisting of 5, 8, 11, 14, 17-eicosapentaenoic acid and / or 4, 7, 10, 13, 16, 19-docosahexaenoic acid and a second component consisting of one or more of dihomo-X'-linolenic acid, cis-linolenic acid and X'-linolenic acid, said first and other components are present in relative proportions of 3: 1 - 1: 3. 0 A composition according to claim 1, characterized in that it comprises a pharmaceutically acceptable excipient. 3. A composition according to claim 1 or 2, characterized in that the composition is substantially free of other unsaturated fatty acids. Composition according to any one of claims 1 to 3, characterized in that the first component is 5, 8, 11, 14, 17-eicosapentaenoic acid. 5. A composition according to any one or more of claims 1-4, characterized in that said second component is cis-linoleic acid. Use of the pharmaceutical composition according to claim 1 in a food product.