CA1239587A

CA1239587A - Combined fatty acid composition for lowering blood cholestrol and triglyceride levels

Info

Publication number: CA1239587A
Application number: CA000465531A
Authority: CA
Inventors: David Rubin
Original assignee: Century Laboratories Inc
Current assignee: Century Laboratories Inc
Priority date: 1983-10-24
Filing date: 1984-10-16
Publication date: 1988-07-26
Also published as: GB8426765D0; SE8405308L; GB2148713B; DE3438630A1; FR2553662A1; IT1178170B; GB2148713A; SE462701B; SE8405308D0; IT8449063A0; CH661209A5; IT8449063A1; FR2553662B1

Abstract

ABSTRACT OF THE DISCLOSURE A pharmaceutical composition includes a combination of eicosapentaenoic acid and/or docosahexaenoic acid together with one or more of dihomo-.gamma.-linolenic acid, cis-linoleic acid and .gamma.-linolenic acid. This particular combination of fatty acids may also be administered in the form of a food product such as mar-garine or cooking oil. The composition causes lowering of blood cholesterol and triglyceride levels. The fatty acids used in this composition may be separated from mixtures thereof or from natural sources thereof by iodinating the double bonds in the starting fat or oil, saponifying, extracting the iodinated fatty acids, methyl-ating and separating by column chromatography, and then deiodin-ating.

Description

~239'~37 cor1BINED FATTY ACID COMPOSITION FOR LOWERING BLOOD
CHOLESTEROL AND TRIGLYCERIDE LEVELS

weld of the Invention The present invention relates to pharmaceutical combo-sessions and food products, and more particularly, to such combo-sessions or food products containing a specific combination of fatty acids which can be used for the treatment of a human being or other mammal in order to lower the blood cholesterol and in-glyceride levels of the subject.

Background of the Invention It is known that Greenland Eskimos rarely suffer from atherosclerotic cardiovascular diseases. This fact has been at-tribute to the consumption of high amounts of fish oil. The active ingredients in fish oil are (all-7)-5,~,11,]4,17-eicosa-pentaenoic acid, sometimes designated 20:5~3 fatty acid (herein-after referred to as "EPA") and (all-Z)-4,7,10,13,16,19-docosa-hexaenoic acid, sometimes designated 22:6~3 fatty acid (herein-after referred to as Ala EPA and HA are known to he pro-cursors in the biosynthesis of the prostaglandin PGE3.
The above alternate designations, such as 20:5~3, refer to the total number of carbon atoms in the chain, before the colon: the number of unsaturated bonds, after the colon and the number of carbon atoms from the end opposite the carboxylic acid at which the first unsaturation appears, following the omega.
Members of a given omega series of fatty acids, e.g. I can usually be converted to acids of differing lengths and total numb bier of unsaturations by normal bodily enzymes, but it is generally impossible to change a compound from one omega series to another, e.g. I to I This is because bodily enzymes generally cause changes of length and unsaturation to occur starting from the carboxylic acid end of the chain.
It is disclosed in British patents 1,604,554 and

2,033,745 that EPA can be used to treat effectively, or provide effective prophylaxis against, thromboembolic conditions such as myocardial infarctions, strokes, or deep vein thrombosis during surgical operations. They disclose the extraction of EPA from ~L239~8'7 fish oil, such as cod liver oil or menhaden oil. The EPA may be administered by replacing butter or ordinary margarine by a special margarine formulated so that in normal usage the recipient would receive the required amount of the EPA.
This process has not achieved widespread attention, ales-pile the fact that it uses a natural substance which can readily be incorporated into the daily diet. One reason may be due to the difficulty of efficiently separating EPA from natural fish oils to obtain a pure product at reasonable cost. Another reason may be that the effects of administration of EPA are not as dramatic as had been anticipated.

summary of the Invention It is an object of the present invention to eliminate the above-discussed deficiencies in the prior art.
It is another object of the present invention to pro-vise improvements in compositions of the type of British patents 1,604,554 and 2,033,7~5.
It is a further object of the present invention to pro-vise a composition which has superior therapeutic effects compared to those of the prior art.
It is yet another object of the present invention to provide a therapeutic composition containing naturally obtainable fatty acids which will serve to reduce blood cholesterol and triglyceride levels.
It is still another object of the present invention to provide a therapeutic composition which will increase the Peel :
PGE2 ratio in the patient and increase the absolute amount of PIE
in the system.
These and other objects are obtained through the Somali-Tunis administratiorl of one or more of EPA and HA, together with one or more of clihomo-y-linolenic acid (ekes-trunk acid), i.e., 20:3~6 fatty acid, (hereinafter referred to as "DHLA"), cis-lino]eic acid ((Z,Z)-9,12-octadecadienoic acid), ire, 13:2~6 fatty acid, and y-]inolenic acid, ((Z,Z,Z-6,9,12-octa-c]ecatrienoic acid), i.e., 18:3~6 fatty acid, either in the form of a pharmaceutical dosage or in the form of a food product such as margarine or cooking oil, or in the form of skin ointments or 395~7 lotions for topical administration.

Detailed Description of Preferred Embodiments The prostaqlandins are a family of substances showing a wide diversity of biological effects. Prostag]andins of the 1-, 2- and 3-series, respectively, incorporate one, two or three double bonds in their basic 20-carbon carboxylic fatty acid starlike-lure which includes a 5-member cyclopentene ring.
The l-series of prostaglandins are strong vasodilators and inhibit cholesterol and kilojoule biosynthesis, as well as platelet aggregation. On the other hand the 2-series priest-gland ins are known to enhance platelet aggregation, cholesterol and collagen biosynthesis, and also to enhance endothelial cell proliferation. The main effect of the 3-series prostaglandins, particularly PGÆ3, is the suppression of the 2-series priest-gland ins.
The precursor of the 2-series prostaqlandins is Karachi-tonic acid ((all Z)-5,8,11,14-eicosatetraenoic acid), i.e., 20:4~6 fatty acid. DHLA is the precursor for the l-series prostaglan-dins, and, as indicated hereinabove, EPA and DOW are the precut-sons for the 3-series prostaglandins.
It is believed that the effectiveness of EPA and DOW in preventing atherosclerotic cardiovascular diseases lies both in their effect as a precursor for prostaqlandin PGF3, which sup-presses the 2-series prostaglandins, as well as the fact that the SPA and/or Diva itself competes with arachidonic acid on the same enzymatic system and thus inhibits the biosynthesis of 2-series prostalandins. This inhibition of the 2-series prostaglandins results in an increase of the ratio of Peel : PGE2.
In order to improve the effects of the administration of SPA and/or DIVA alone, by further increasing the Peel : PGÆ2 ratio, as well as effecting an increase in the absolute amount of Peel in the system, Della should be administered simultaneously with the pure SPA and/or Ella. Since cis-linoleic acid and y-linolenic acid hot form Della metabolically within the body, either or both of these fatty acids may be substituted, in whole or in part, for Della.
It has been found that the combination of EPA (and/or DOW) and DHLA (and/or cis-linoleic acid and/or -linolenic acid) causes a substantial reduction in hood cholesterol and trimly-derides. Recent research has definitely linked blood cholesterol levels with incidence of coronary heart disease (JAM, 251, 351-36~ (198~) and JAM 251, 365 -374 (198~)). Additionally, it is expected that such a combination will have other beneficial there-peptic properties. For example, it is known that in Chihuahuas-frown, rheumatoid arthritis and outlawry collagen and auto-immune diseases, as well as in some forms of cancer, there are evidences of extremely low levels of Peel and high levels of PIE Thus, it its 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 cortico-steroids and the pain killing effect of aspirin are believed to be due to their suppressing effect of PGE2 formation. Thus, the use of the combination of the present invention can be expected to be a natural and most effective anti-inflammatory pain killing agent.
The dose of the composition of the present invention, comprising a combination of SPA (and/or DOW) and DOYLE (and/or is-linoleic acid and/or y-linolenic acid), needed for therapeutic or prophylactic effect will vary with the route of administration and the nature of the condition being treated, but will generally be at least 1 gram, preferably from 1.5 to 3 grams, per clay. This is the dose for an average 70 kg man, and the dose for other men or animals will vary pro fate according to their weight, i.e. about 20-40 mg/kg.
The relative amounts of EPA Andre Ala and ALA
(and/or cis-linoleic acid and/or ~-linolenic acid) in the come position of the present invention is preferably 1:1, although the ratio may vary from 3:1 to 1:3.
The EPA (and/or DOW) and DOYLE (and/or cis-linoleic acid and/or ~-linolenic acid) need not be administered as the acids themselves but may he used as their pharmaceutically acceptable salts, esters or asides. Esters or asides which can be converted in viva to the acid and other pharmaceutically acceptable pro-ducts may be used, the preferred ester being the ethyl ester. The preferred salts are the sodium or potassium salts, or any other pharmaceutically acceptable solid salt, as these are suitable for 3L~3~

making into tablets.
While it is preferred to administer the composition of the present invention orally, as this is a convenient route for routine administration, the active compounds may be administered by any route by which it may be successfully absorbed, e.g., parenterally (i.e. subcutaneously, intramuscularly or intro-venously), rectally or vaginal, or topically, for example as a skin ointment or lotion.
While it is possible for the active compounds to be administered as such, as a simple mixture of components, it is preferable to present them as a pharmaceutical formulation. The formulations, both for veterinary and for human medical use, of the present invention comprise the active compounds as defined, together with one or rrlore pharmaceutically acceptable carriers therefore and, optionally, other therapelltic ingredients, although other unsaturated fatty acids should be avoided, particularly arachidonic acid. The carrier(s) must be "pharmaceutically accept table" in the sense of being compatible with the other ingredients of the formulations and not deleterious to the recipient thereof.
Formulations include those suitable for oral, rectal, vaginal, intrapulmonary or parenteral (including subcutaneous, intro-muscular and intravenous) administration. Formulations for oral administration, such as tablets or capsules are preferred.
The EPA (and/or HA) - DHLA (and or cis-linoleic acid and/or y-linolenic acid) combination may also be administered by replacing butter and/or ordinary margarine by a special margarine, e.g. of the emulsion type, formulated so that in normal usage the recipient would receive the required amount of the combination.
Cooking oils and fats may also be similarly formulated to contain the composition of the present invention.
The EPA (and/or HA) and DHLA (and/or cis-linoleic acid and/or y-linolenic acid) used in the compositions of the present invention should be as pure as possible. EPA and/or ha cannot be used in the form of fish oil Directly, as the use of the amount of fish oil necessary in order to provide the desired amount of EPA
and/or HA would provide excessive calories and potential toxic amounts of vitamins A and D. Thus, pure EPA and/or HA should be extracted from the fish oil. The presence of unsaturated fatty ~3~5~7 acids other than EPA, HA, THEA, cis-linoleic acid and y-linolenic acid should be avoided.
Substantially pure EPA and/or HA may be extracted from fish oil, such as cod liver oil, by means of the process set forth, for example, in U.S. Patent ~,377,526. Alternatively, the separation may be accomplished by a novel process of the present applicant involving iodination of the double bonds of the unset-unrated fatty acids in the starting fat or oil. Such iodination permits protection of the fatty acids from oxidation during further processing, and increases the resolution of the fatty acids upon eventual column chromatography. After iodination, the fat or oil is saponified and the iodinated fatty acid extracted from the saponification mixture. The iodinated fatty acids are then methylated and separated by column chromatography, after which the desired fractions are cleiodinated. This process can be used not only for the separation of EPA and Delia from fish oils, but also for the separation and extraction of other unsaturated fatty acids, such as c~-linolenic acid and y-linolenic acid from the triglyceride forms in which they naturally occur in, for example, soybean oil, cottonseed oil, safflower oil, oil of eve-nine primrose, etc. The separation of any unsaturated fatty acid can be facilitated by means of the present iodination process.
The starting material in the present process can be a natural fat or fatty oil in which the first step is iodination followed by saponification. Ivory, the starting material may also be any mixture of unsaturated fatty acids which are difficult to separate, in which the first step will be iodination but no saponification will be required as the starting material is not a triglyceride.
Iodination takes place by adding iodine, in an organic solvent, preferably 20% ethanolic solution, slowly to the starting material until the color fails to disappear in the starting material. This reaction takes place at room temperature under continuous stirring.
The saponification step can take place in any convent tonal manner such as, for example, with a 20% ethanolic solution of KOCH for two hours.
The iodinated fatty acid is extracted from the supine-5~37 ligation mixture by means of any conventional procedure, for example, extraction with ether.
The next step is the methylation of the iodinated fatty acids to prepare them for column chromatography. Again, this is a conventional step and may be done, for example, with 56 hydrogen chloride in methanol.
Finally, the fatty acids are separated by means of column chromatography. The column chromatography is carried out in a known manner with a conventional elusion mixture. While resolution among the various fatty acids it very poor in the con-ventional processes, the resolution is greatly improved when the fatty acids are iodinated at the time of column chromatography.
The column may be packed with silica gel as is conventional and the elusion solution may be any conventional solution, such as hexane-ether-acetic acid (85-10-5).
After the fractions are obtained from the column, the fatty acids are deiodinated using, for example, silver nitrate.
While specific reagents and process conditions are set forth for the various steps of the present process, it should be understood that those skilled in the art will readily be aware of other reagents and conditions in order to carry out the steps once the desirability of each step is known. The critical factor is the concept of iodination prior to chromatography in order to increase the resolution and to protect the fatty acids from ox-ration.
Furthermore, although the separation is accomplished ho column chromatography in the above description, it should be understood that other means of separation may be used as, for example, high speed centrifugation. The resolution will also be improved by iodination in such other separation means.
The following is an example of a method for the spear-anion of EPA and HA from cod liver oil in accordance with this process.

Preparative Example:
A 2nQ6 ethanolic solution of iodine is added slowly to 300g of cod liver oil. The iodine is added as long as its color disappears in the oil. The reaction takes place at room temper-~2~5~

azure under continuous stirring. When iodination is completed, the iodinated oily solution is saponified with 20% ethanolic soul-lion of KIWI for two hours. The iodinated fatty acid, 260 g, is extracted with ease from the saponification mixture.
The iodinated fatty acids are then methylated with 5%
hydrogen chloride in methanol. The EPA and the HA are separated by column chromatography (silica-gel 1,500 g, Kieselgel* 70-230 mesh, Merck). The elusion is done with 5 liters hexane-ether-acetic acid (35-10-5). The first fraction to be extracted is the iodinated HA. The second fraction is iodinated EPA.
Once the suh~tantially pure methylated and iodinated fatty acid mixture is obtained, it may also be separated by other conventional techniques, such as high speed centrifugation or distillation. Deiodination takes place by shaking the iodinated Mud and Me-EPA, separately, with 10% aqueous solutions of sit-Yen nitrate. Precipitates of silver-iodine appears and the organ nix phases are separated. The same procedure is repeated until no more precipitation occurs. Microanalysis, HPLC and NOR proved that the desired products are obtained. The yield is above 90%, the purity 96-100%. There is no need to carry out the procedure under nitrogen since the fatty acids are saturated with iodine, thus preventing oxidation from taking place.
The following clinical tests illustrate the synergistic effects which are obtained when using the combination of the pros-en invention as compared to the effects of each of the components administered alone.

Therapeutic Example:
Thirty-six outpatients, ages 35-75, males and females, were divided into three groups of twelve. Each group added to their normal diet 5 cc/day of free fatty acids for 45 days. Group I added S cc/day of substantially pure EPA. Group II added to their diet 5 cc/day of substantially pure cis-linoleic acid, and group III added to their diet 3 cc/day of substantially pure EPA
and 2 cc/ day of substantially pure cis-linoleic acid. sty the term "substantially pure" is meant a purity of about 96-100%.
Blood cholesterol and triglycerides were tested one day before the treatment began and after 45 days of treatment. The * Trade mark ~3g58~

results of these treatments are set forth in Tables I, II and III
hereinbelow:

Table I: 5 cc/day Pure SPA

Patient age sex Total Total Triglycerides Triglycerides No. cholesterol cholesterol day after 45 1 day after buffer days before days treatment treatment my% my% my% my%

3 47 M 230 210 102 90

4 73 M 270 240 95 90 a 52 F aye 250 115 100 12 35 M 200 190 90 90+7 Average: 256.9+43.2227.9+24.4111.8+18.6 94.95+
% reduction: 11.2% 15~, Table II: 5 cc/day Pure cis-Linoleic Acid Patient age sex Total Total Triglycerides Triglycerides No. cholesterol cholesterol day after 45 1 day after buffer days before days treatment treatment my% my% my% my%

9 62 F 310 300 lay 140 Average: 265.3+61.75250+50112+22.8 109.16+19.4 % reduction: 5% 2%

~23~S~7 Table III: 3 cc/day Pure EPA and 2 cc/day Pure cis-Linoleic Acid Patient age sex Total Total Triglycerides Triglycerides Jo. cholesterol cholesterol day after 45 1 day after 45 before days before days treatment treatment McKee my% my% my%

Average: 270.1~67.5217.9+24.499.5 59.16+16 % reduction: 19.3% 40.5 While the administration of 5 cc/day of SPA alone pro-voided a reduction in serum cholesterol and triglyceride levels during the 45 days of treatment, i.e. an average reduction of 11.2% for total cholesterol and 15~ for triglycerides, the effect of the administration of pure cis-linoleic acid alone for 45 days is almost insignificant. In fact, in many patients the chores-twirl level actually rose.
A definite synergism, however, is observed by ad minis-traction of the combination of 3 cc EPA plus 2 cc cis-linoleic acid per day. By use of the combination, a very significant reduction of serum cholesterol (an average of 19.3% decrease) and serum triglycerides (an average of 40.5~ decrease) is observed.
It will be obvious to those skilled in the art that various changes may be made without departing from the scope of the invention and the invention is not to be considered limited to what is described in the specification.

Claims

WHAT IS CLAIMED IS:

1. A pharmaceutical composition for causing a reduction of blood cholesterol and triglyceride levels, consisting essen-tially of an effective amount of a combination of a first com-ponent selected from the group consisting of 5,8,11,14,17-eicosa-pentaenoic acid, 4,7,10,13,16,19-docosahexaenoic acid and a com-bination thereof, and a second component selected from the group consisting of dihomo-.gamma.-linolenic acid, cis-linoleic acid, .gamma.-lino-lenic acid and combinations thereof, said first and second com-ponents being present in relative amounts of 3:1 to 1:3.

2. A composition in accordance with claim 1, further including a pharameceutically acceptable excipient.

3. A composition in accordance with claim 1, wherein said composition is substantially free of other unsaturated fatty acids.

4. A composition in accordance with claim 1, wherein said first component is 5,8,11,14,17-eicosapentaenoic acid.

5. A composition in accordance with claim 1, wherein said second component is cis-linoleic acid.

6. A composition in accordance with claim 4, wherein said second component is cis-linoleic acid.

7. A food product containing a substantial amount of at least one fatty acid, characterized in that said at least one fatty acid present in said food product consists essentially of a combination of a first component selected from the group consis-ting of 5,8,11,14,17-eicosapentaenoic acid, 4,7,10,13,16,19-do-cosahexaenoic acid and a combination thereof, and a second com-ponent selected from the group consisting of dihomo-.gamma.-linolenic acid, cis-linoleic acid, .gamma.-linolenic acid and combinations there-of, said first and second components being present in relative amounts of 3:1 to 1:3.

8. A food product in accordance with claim 7 which is substantially free of other unsaturated fatty acids.

9. A food product in accordance with claim 7, wherein said first component is 5,8,11,14,17-eicosapentaenoic acid.

10. A food product in accordance with claim 7 wherein said second component is cis-linoleic acid.

11. A food product in accordance with claim 9, wherein said second component is cis-linoleic acid.