Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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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
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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
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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
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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
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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
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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-
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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-
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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
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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%
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
F 280 240 115 US
6 56 M 265 260 120 105
7 54 F 215 205 95 90
a 52 F aye 250 115 100
9 63 M 300 250 110 95
64 M 350 260 160 105
11 55 M 190 190 95 80
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%
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
F 240 210 90 100
6 37 M 270 260 105 95
7 40 M 220 230 80 80
8 45 M 400 350 165 150
9 62 F 310 300 lay 140
54 M 230 220 115 115
11 52 M 260 250 110 110
12 61 F 215 215 130 125
Average: 265.3+61.75250+50112+22.8 109.16+19.4
% reduction: 5% 2%
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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%
1 48 M450 260 160 90
2 60 M310 240 70 40
3 45 M257 210 106 50
4 40 M305 250 98 45
54 M210 200 95 55
6 35 F210 190 95 45
7 40 M290 240 100 70
8 61 F270 220 116 45
9 45 F240 215 95 I
50 F210 190 75 60
11 64 M300 220 130 80
12 64 M190 180 55 50
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.
