Use of nervonic acid and long chain fatty acids for the treatment of demyelinating disorders

Composition containing nervonic acid (cis-tetracos-15-enoic acid) in a physiologically acceptable form are useful in the treatment of demyelinating disease, such as multiple sclerosis. Additional long chain fatty acids of chain length C16 to C26, especially erucic acid, or their esters, may be included in such compositions.

This invention is concerned with pharmaceutical compositions containing 
long chain fatty acids such as nervonic acid, or derivatives thereof, for 
the treatment of demyelinating diseases such as multiple sclerosis. 
Multiple sclerosis (MS) is a disease affecting the mature central nervous 
system (CNS). The disease is characterised by successive periods of CNS 
demyelination followed by periods of remission. Although the aetiology of 
MS is not fully understood a number of factors appear to be important 
including a genetic predisposition, viral infection and an auto immune 
response to some antigen resulting in demyelination of the CNS 
particularly brain, optic nerve and spinal cord. 
Regardless of the causes of MS, the pattern of demyelination and 
remyelination (remission) involves both loss of and reassimilation of 
myelin components. Since myelin comprises about 60% of its dry weight as 
lipid, the important lipid components, the fatty acids, have received 
considerable attention over the years, in particular the long chain fatty 
acids, since they are the most abundant of fatty acids in many important 
complex lipids. Long chain fatty acids in the context of this invention 
are defined as those mono-carboxylic acids having a carbon chain length 
greater than C22. The complex lipids in the context of this invention 
include gangliosides (particularly Ganglioside G.sub.7 or G.sub.M4), 
cerebrosides, sulphatides and sphingomyelin. These lipids contain 
significant amounts of long chain fatty acids (particularly nervonic 
acid-cis-tetracos-15-enoic acid) in their structures. Typical compositions 
of normal myelin are tabulated below: 
TABLE 1 
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Long Chain Fatty Acids in Myelin Lipids 
Fatty Ganglioside 
Cere- 
Acid* G.sub.7 (G.sub.M4)** 
broside** 
Sulphatide** 
Sphingomyelin*** 
______________________________________ 
C24:1 24.1 45.7 48.0 35.0 
C24:0 11.1 15.8 14.3 -- 
C25:1 5.0 7.8 10.2 2.3 
C25:0 1.8 2.3 3.4 -- 
C26:1 4.1 4.5 9.1 1.0 
C26:0 0.8 0.3 1.1 -- 
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*The total number of carbon atoms: number of double bonds 
**Derived from human purified myelin (see Ledeen. R. W., et al J. 
Neurochem., 21 829-839, 1973). 
***Derived from human white brain matter (see Gerstl, B., et al Z. 
Neurol., 202 104-120, 1972). 
Destruction of myelin during active MS results in significant loss of these 
myelin components. Additionally, their replacement requires the 
availability of their specific component fatty acids. This can arise by 
two distinct routes, namely in-vivo synthesis (the more important route) 
and exogenously from the diet. Long chain fatty acids are rare trace 
components in most modern diets and in-vivo synthesis is not efficient and 
may be impaired in MS. Several workers have shown that there is a 
significant depletion of total lipid in MS tissue (brain, spinal cord). 
More significantly, perhaps, is the absence of specific gangliosides 
(Ganglioside G.sub.7 or G.sub.M4) in MS white brain matter and 
demyelinated plaque (see Yu, R. K. et al, J. Neurochem., 23 169-174, 
1974). 
Myelin destruction not only depletes CNS tissue of vital lipids but also 
releases myelin basic protein (MBP). MBP is known to be antigenic to 
myelin and also induces experimental allergic encephalomyelitis (EAE) when 
injected into laboratory animals. EAE has some neuropathological features 
in common with MS and is widely used as an animal model for the diseases. 
However, when MBP is incubated with Ganglioside G.sub.7 (G.sub.M4) prior 
to injection it significantly reduces the encephalogenic potential of the 
MBP in guinea pig. (See Mullin, B. R., et al Brain Research, 296 174-176, 
1984). 
We have now realised that the provision of a pharmacologically acceptable 
source of long chain fatty acids in MS patients would provide a pool of 
material vital to the assembly of myelin components for reassembly of the 
myelin sheath. Moreover the ready availability of these long chain fatty 
acids would enable the biosynthesis of important gangliosides, 
particularly Ganglioside G.sub.7 (G.sub.M4) which is known to inhibit the 
myelinogenic propensity of MBP. According to the present invention, 
therefore, there is provided a pharmaceutical composition comprising one 
or more long chain fatty acids, in a physiologically acceptable form, and 
a carrier or diluent therefor. Preferably, the compositions include 
nervonic acid as the, or one of the, fatty acids, e.g. in the form of an 
ester thereof. 
Although the long chain fatty acids such as nervonic acid 
(cis-tetracos-15-enoic acid) are rare or insignificant in normal diets, 
they do exist in a small number of plants and micro-organisms. These 
include the seed oils of Cardamine gracea, Heliphila longifola, Thlaspi 
perfoliatum, Tropaeolum speciosum, Lunaria biennis, Lunaria annua and 
Malania oleifera; the moulds Neocallismastix frontalis, Erysiphe graminis 
and Sphaerotheca humuli; the bacterium Pseudomonas atlantica; the yeast 
Saccharomyces cerevisiae and the marine diatom Nitzschia cylindrus. 
A preferred source is the seed oil of plants known to contain significant 
amounts, i.e. greater than 10%, of nervonic acid in the triglyceride 
lipid. Clearly other sources containing less than 10% are of lower value 
since additional steps would have to be taken to concentrate the active 
components. Of particular value is the seed oil of Lunaria biennis or 
Lunaria annua since they contain over 20% nervonic acid in the 
triglyceride. A detailed typical composition of the oil is shown in Table 
2. 
TABLE 2 
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Fatty Acid Distribution in L.biennis Seed Oil* 
Fatty 
Acid Name Amount (%)** 
Amount (%)**** 
______________________________________ 
C16:0 palmitic acid 1.2 1.1 
C16:1 oleopalmitic acid 
0.2 0.1 
C18:0 stearic acid 0.2 0.2 
C18:1 oleic acid 23.4 23.3 
C18:2 linoleic acid 4.8 5.4 
C18:3 linolenic acid 
1.0 0.8 
C20:0 eicosanoic acid 
tr*** --***** 
C20:1 eicosenoic acid 
1.6 0.5 
C22:0 behenic acid 0.2 0.2 
C22:1 erucic acid 45.3 45.1 
C22:2 docosandienoic acid 
0.1 0.2 
C24:0 tetracosanoic acid 
0.2 0.1 
C24:1 nervonic acid 21.8 22.8 
100.00 100.00 
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*Analysed by gas chromatography 
**The triglycerides ester converted to the corresponding methyl ester 
***trace amount, usually less than 0.1% 
****second determination on a different sample 
*****not detected In addition to the various natural sources, examples 
of which are listed above, it is also possible to provide nervonic acid by 
a synthetic procedure. The starting point for such synthesis could be, for 
example, the readily available erucic acid (cis-docosa-13-enoic acid). A 
typical synthesis, but by no means the only possibility, has been 
described by Carrol, K. K. (see Canadian J. Chem., 35 757-760, 1957). This 
synthesis involves the conversion of erucic acid to its methyl ester by 
esterification with methanol, reduction to erucyl alcohol using lithium 
aluminium hydride, conversion of the alcohol to its alkyl bromide by 
reaction with phosphorous tribromide, reaction of the erucyl bromide with 
diethyl malonate and decarboxylation to yield nervonic acid. This 
synthesis has some advantages, particularly in the preparation of 
isotopically labelled nervonic acid, but suffers from the lengthy 
procedure and cost. 
The various methods of extracting seed oils from the oil bearing seeds are 
well known to those skilled in the art (see "Baileys Industrial oil and 
Fat Products", ed. D. Swern, Vol. 2, pages 175 et seq. 4th edition, Pub. 
1982, John Wiley & Sons Inc.). These methods include: dry rendering, wet 
rendering, batch pressing, continuous pressing, solvent extraction and 
extraction with supercritical gases such as carbon dioxide. In practice 
the most efficient processes involve continuous pressing or supercritical 
extraction with or without secondary solvent extraction of the oil seed 
cake. 
Extracted oils free from solvent may also contain undesirable impurities 
which can detract from the value of the oil as a pharmaceutical. 
Undesirable impurities or contaminants may be removed by various refining 
processes. Refining is defined as any purifying treatment designed to 
remove free fatty acids, phosphatides, gums or other major impurities. 
The oil may be further improved by bleaching and deodorisation. Bleaching 
is defined as any process designed to reduce the colour of the oil. 
Various methods are used and are well known to those skilled in the art. 
Deodorisation is defined as any process designed to remove trace 
contaminants that give rise to flavour and odour. 
Bleaching and deodorisation are described in detail in "Baileys Industrial 
Oil & Fat Products" ed. D. Swern, Vol. 2, 4th edition, pages 253 et seq, 
Pub. 1982, John Wiley & Sons Inc. 
A particularly valuable purification process which has the advantage of 
refining, bleaching and deodorisation in one step is by adsorption 
chromatography. 
As can be seen from Table 2, natural oils contain a large number of 
component fatty acids in addition to the long chain fatty acids. If 
desired, the long chain fatty acids may be concentrated by selectively 
removing other components. Suitable methods include conversion of the 
triglyceride to the free fatty acid or lower alkyl ester, particularly 
their methyl or ethyl esters. Concentration may then be effectively 
performed by fractional distillation, crystallisation, solvent extraction, 
urea clathration or chromatography to yield nervonic acid rich fractions. 
In some cases, it may be desirable to use combinations of these 
techniques. 
The compositions of the invention may comprise nervonic acid as the only 
therapeutically active substance. Alternatively one or more other active 
materials may be present. 
A further embodiment of the invention involves the use of a functional 
derivative of the long chain fatty acids, in particular functional 
derivatives of nervonic acid. As used herein the term "functional 
derivative" is defined as any of those derivatives of long chain fatty 
acids herein defined, particularly nervonic acid, which contain the intact 
nervonyl acyl group. Examples of these functional derivatives include 
esters, particularly glyceride esters, ethyl esters and the like, salts 
such as sodium salts, potassium salts, calcium salts, amino acid salts and 
the like. The acids and their functionally active derivatives may be 
prepared synthetically by processes described heretofore. These processes 
involve, however, a number of stages and high cost. It is especially 
preferred, therefore, that the materials be obtained from naturally 
occurring seed oils or micro-organisms. Particularly preferred are seed 
oils such as those described heretofore and especially the seed oil of 
Lunaria family. It is further preferred that the long chain fatty acids, 
particularly nervonic acid or functional derivatives thereof, are 
administered in a suitable pharmaceutically acceptable form. Many such 
forms are known and include oral administration of the oil itself, the 
free fatty acids or functional derivatives thereof. Additionally, the oil 
free fatty acids or functional derivatives may be administered as 
capsules, tablets or emulsions in water. Furthermore, the pharmaceutical 
composition may be administered where appropriate by injection, 
intravenous intubation or nasogastric intubation, for example. It is 
understood that the amount of material administered is defined as the 
amount of active therapeutic material required to achieve the required 
pharmacological effect. In general, the required dosage rate for an adult 
will range from 0.01 g-50 g per day, especially in the range 0.1 g-10 g, 
and preferably 0.5 g-5 g per day, of the nervonic acid containing oil, 
functional derivative thereof or pure nervonic acid or functional 
derivative thereof. 
As is well known MS is one of a group of demyelinating diseases including 
acute disseminated encephalomyelitis, Western Hurst disease, progressive 
multifocal leukoencephalopathy, idiopathic polyneuritis, diphtheric 
neuropathy, adrenoleukodystrophy (ALD) and adrenomyeloneuropathy. It is 
possible that one or more of these diseases could benefit from treatment 
with the nervonic acid containing preparations described above. The 
preferred compositions of the invention based on natural oils, 
particularly from the Lunaria plants all contain, in addition to nervonic 
acid, substantial quantities of erucic acid. Erucic acid and its 
derivatives are effective methods of reducing the concentration of 
accumulated toxic methabolites characteristic of ALD. Thus, these 
preferred compositions of the present invention can also be used for 
treating ALD. When used for this purpose, however, the oil should not 
contain any significant amounts of tetracosanoic acid or hexacosanoic 
acid. Removal of these components can be achieved by the methods well 
known to those skilled in the art and include the removal of all saturated 
fatty acids by urea clathration. Additionally when treating ALD it is 
often preferred to restrict the patients diet to exclude all dietary 
sources of undesirable fatty acids especially tetracosanoic acid and 
hexacosanoic acid. Rigorous restriction may, in some cases, also exclude 
fatty acids essential for growth, metabolism and health. Where this is the 
case the nervonic acid containing oils may be supplemented with suitable 
amounts of these fatty acids. These include gamma-linolenic acid, 
eicosapentaenoic acid and docosahexanenoic acid.

EXAMPLE 1 
We described hereafter, by way of illustration only, the treatment of 
Honesty oil to obtain a concentrate of ethyl nervonate, and the 
preparation therefrom of purified glyceryl trinervonate which is a useful 
physiologically acceptable derivative of nervonic acid. 
The typical composition of the crude oil is as follows: 
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Fatty Acid Amount (%) 
______________________________________ 
C16:0 1.2 
C16:1 0.2 
C18:0 0.2 
C18:1 23.4 
C18:2 4.8 
C18:3 1.0 
C20:0 Trace 
C20:1 1.6 
C22:0 0.2 
C22:1 45.3 
C22:2 0.1 
C24:0 0.2 
C24:1 21.8 
______________________________________ 
(A) Preparation of ethyl esters 
Honesty oil is converted into its ethyl esters by reacting the oil (1 mol) 
with an excess of absolute alcohol (5 mol) in the presence of sodium 
ethoxide (0.01 mol) at 80.degree. C. for 3 hours. The reaction mixture is 
then cooled to 30.degree. C. and the lower glycerol layer is separated 
off. The product is washed three times with water at 80.degree. C. and 
then dried under vacuum up to a temperature of 110.degree. C. 
______________________________________ 
Fatty Acid Composition 
Fatty Acid Amount (%) 
______________________________________ 
C16:0 1.2 
C16:1 0.2 
C18:0 0.2 
C18:1 23.2 
C18:2 4.9 
C18:3 1.1 
C20:0 Trace 
C20:1 1.6 
C22:0 0.2 
C22:1 45.1 
C22:2 0.1 
C24:0 0.2 
C24:1 22.0 
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(B) Purification of Ethyl Ester--Removal of Saturated Fatty Acids 
Saturated fatty acid ethyl esters are removed from the Honesty ethyl ester 
by refluxing with an excess of urea dissolved in absolute alcohol for 2 
hours. On cooling with stirring over 14 hours to ambient temperature the 
saturated ethyl esters crystallise out as the urea-fatty acid inclusion 
compounds. 
The solid inclusion compounds and excess urea are removed by filtration 
leaving an alcohol solution of unsaturated ethyl esters. This is 
concentrated by vacuum distillation to remove the solvent. 
The crude "saturate free" ethyl esters are washed one, at 
85.degree.-90.degree. C. with dilute aqueous potassium hydroxide, and 
washed again with two portions of hot water to remove any traces of soap 
and urea present in the crude product. The product is then dried under 
vacuum at a temperature of 100.degree. C. 
______________________________________ 
Fatty Acid Composition 
Fatty Acid Amount (%) 
______________________________________ 
C16:0 Trace 
C16:1 0.4 
C18:0 Trace 
C18:1 23.5 
C18:2 5.0 
C18:3 1.2 
C20:0 Trace 
C20:1 1.7 
C22:0 Trace 
C22:1 45.4 
C22:2 0.2 
C24:0 Trace 
C24:1 22.6 
______________________________________ 
(C) Enrichment of Ethyl nervonate (C24:1) by Fractional Distillation 
the "saturate free" ethyl esters are enriched in C24:1 by fractional 
distillation to give a residue containing 50-90% C24:1). The crude ethyl 
nervonate is then further distilled under vacuum. 
______________________________________ 
Fatty Acid Composition 
Fatty Acid Amount (%) 
______________________________________ 
C18:1 Trace 
C18:2 Trace 
C18:3 Trace 
C20:1 0.3 
C22:1 8.5 
C22:2 1.1 
C24:1 90.1 
______________________________________ 
(D) Preparation of Glyceryl trinervonate (GTN) 
GTN is prepared by reacting stoichiometric amounts of the enriched ethyl 
nervonate (90%) with glyceryl (BP) using sodium methoxide (0.03 mol) as 
catalyst. 
The reaction is carried out in a stepwise manner by (a) adding 85% of the 
glycerol initially and reacting for 10 hours at 220.degree. C. under 
vacuum. (b) adding a further 7% and reacting for 3 hours at 220.degree. C. 
(c) adding a further 4% and reacting for 3 hours at 220.degree. C. (d) 
adding the final 4% and reacting for 5 hours at 220.degree. C. The total 
reaction time being 21 hours. 
This gradual addition of the glycerol is necessary to maximise the 
conversion of the ethyl ester to the triglyceride and to minimise the 
formation of mono and diglycerides. After the reaction period has elapsed 
the crude triglyceride is cooled to 40.degree. C. 
(E) Chromatographic Purification of GTN 
The crude GTN is dissolved in pure hexane and subjected to column 
chromatography using activated silicon dioxide as the absorbant. 
The combined column eluate is isolated and distilled to remove hexane. The 
residue is then steam distilled under vacuum at 110.degree. C. The GTM is 
filtered in vacuo at 70.degree. C. to give a pale yellow liquid which 
solidifies on standing at ambient temperature. 
______________________________________ 
Fatty Acid Composition 
Fatty Acid Amount (%) 
______________________________________ 
C18:1 Trace 
C18:2 Trace 
C18:3 Trace 
C20:1 0.3 
C22:1 8.4 
C22:2 1.1 
C24:1 90.2 
______________________________________ 
EXAMPLE 2 
One preferred composition of the invention comprises the glyceryl 
trinervonate oil (described in Example 1) as the active ingredient. The 
oil can be administered orally to patients either as such or in 
encapsulated form. Suitable capsules are gelating capsules containing 
about 1 g of oil. The dose will depend on the patient's circumstances but 
we believe it will normally be from 5 to 10 g per day per adult. The 
gelatin of the capsules would, of course, constitute a carbohydrate and 
protein source for the patient. 
The oil can also be prepared as an emulsion for oral administration if 
desired.