Use of Nervonic Acid and Long Chain Fatty Acids for The

~™ mi ii ii ii ii mill illinium (19) J European Patent Office

Office europeen des brevets (1 1 ) EP 0 455 783 B1

(12) EUROPEAN PATENT SPECIFICATION

(45) Date of publicationation and mention (51) Int. CI.6: A61 K 31/20, A61K 31/23 of the grant of the patent: 13.03.1996 Bulletinulletin 1996/11 (86) International application number: PCT/GB90/01870

(21) Application number: 90917506.9 (87) International publication number: WO 91/07955 (13.06.1991 Gazette 1991/13) (22) Date of filing: 30.11.1990

(54) USE OF NERVONIC ACID AND LONG CHAIN FATTY ACIDS FOR THE TREATMENT OF DEMYELINATING DISORDERS VERWENDUNG VON NERVONSAURE UND LANGKETTIGER FETTSAUREN ZUR BEHANDLUNG DEMYELINISIERENDER ERKRANKUNGEN UTILISATION D'ACIDE NERVONIQUE ET D'ACIDES GRAS A LONGUES CHAINES DANS LE TRAITEMENT DES TROUBLES PROVOQUES PAR LA DEMYELINISATION

(84) Designated Contracting States: (56) References cited: AT BE CH DE DK ES FR GB GR IT LI LU NL SE EP-A- 0 071 357 GB-A-2 218 334 US-A- 4 505 933 (30) Priority: 30.11.1989 GB 8927109 30.07.1990 GB 9016660 • Proc. Natl. Acad. Sci. USA, vol. 86, June 1989, R.T. Holman et al.: "Deficiencies of polyunsaturated (43) Date of publication of application: fatty acids and replacement by nonessential fatty 13.11.1991 Bulletin 1991/46 acids in plasma lipids in multiple sclerosis", pages 4720-4724 (73) Proprietor: CRODA INTERNATIONAL pic • Prog. Lipid. Res., vol. 25, 1986, Pergzamon Goole North Humberside DN14 9AA (GB) Journals Ltd, (GB), L.S. Harbige et al.: "Dietary intervention studies on the phosphoglyceride Inventors: (72) fatty acids and electrophoretic mobility of • COUPLAND, Keith erythrocytes in multiple sclerosis", pages 243- YorkY04 3UX(GB) 248 Alan • LANGLEY, Nigel, • Z. Neurol., vol. 202, 1972, Springer- Verlag, B. York Y04 5LW (GB) Gerstl et al.: "Sphingolipids and their precursors in human brain (Normal and MS)", 104-120 (74) Representative: Wain, Christopher Paul et al pages • Journal of vol. 1970, A.A. THORNTON & CO. Neurochemistry, 17, B. Gerstl et al.: Northumberland House Pergamon Press, (GB), "Lipids and proteins in multiple sclerosis white matter", 303-306 High Holborn 677-689 London WC1V7LE(GB) pages • Chemical Abstracts, vol. 109, 1988, (Columbus, Ohio, US), D.J. Murphy et al.: "Biosynthesis of very long chain monounsaturated fatty acids by subcellular fractions of developing seeds", see page 264 • Chemical Abstracts, vol. 104, 1986, (Columbus, Ohio, US), K.D. Mukherjee et al.: "Lipids containing very long chain monounsaturated acyl moieties in seeds of lunaria annua", see CO page 407 CO CO r»- LO LO Note: Within nine months from the publication of the mention of the grant of the European patent, any person may give notice to the European Patent Office of opposition to the European patent granted. Notice of opposition shall be filed in o a written reasoned statement. It shall not be deemed to have been filed until the opposition fee has been paid. (Art. Q_ 99(1) European Patent Convention). LU Printed by Rank Xerox (UK) Business Services 2.9.12/3.4 EP 0 455 783 B1

Description

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. 5 GB-A-2218334 describes the topical application of a composition comprising cyclosporin, in admixture with long chain fatty acids, for use as an immuno-suppresive agent. We have now realised that the provision of a pharmacologically acceptable source of long chain fatty acids would be useful in the treatment of 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 10 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 15 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 G7 or GM4), 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: 20 TABLE 1 Long Chain Fatty Acids in Myelin Lipids Fatty Acid* Ganglioside G/(GM4)** Cerebroside** 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 * The total number of carbon atoms: number of double bonds " Derived from human purified myelin (see Ledeen, R.W., et al J. Neurochem., 21829-839, 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 40 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 G7 or GM4) in MS white brain matter and demyelinated plaque (see 45 Yu.R.K. etal, 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 G7 (GM4) prior to injection it significantly 50 reduces the encephalogenic potential of the MBP in guinea pigs. (See Mullin, B.R., etal, 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, 55 particularly Ganglioside G7 (GM4) which is known to inhibit the myelinogenic propensity of MBP. According to the present invention, therefore, there is provided a pharmaceutical composition comprising nervonic acid (cis-tetracos-15-enoic acid) or a functional derivative thereof in a physiologically acceptable form, in the absence of cyclosporin, and optionally a carrier or diluent therefor.

2 EP 0 455 783 B1

The invention also provides a pharmaceutical composition comprising nervonic acid or a functional derivative thereof as the principal active component of the composition, and optionally a carrier or diluent therefor. According to a further aspect of the present invention, there is provided a composition for use in the treatment of demyelinating diseases such as multiple sclerosis by enteric and intravenous administration, said composition compris- 5 ing nervonic acid in a physiologically acceptable form. 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 10 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 15 shown in Table 2.

TABLE 2 Fatty Acid Distribution in L.biennis Seed Oils*

20 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 25 C18:1 oleic acid 23.4 23.3 C18:2 linoleicacid 4.8 5.4 C18:3 linolenic acid 1.0 0.8 -***** 30 C20:0 eicosanoic acid tr*** C20:1 eicosenoic acid 1.6 0.5 C22:0 behenicacid 0.2 0.2 C22:1 erucicacid 45.3 45.1 35 C22:2 docosandienoic acid 0.1 0.2 C24:0 tetracosanoic acid 0.2 0.1 C24:1 nervonic acid 21.8 22.8 4n 100.00 100.00 * 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 45 ***** not detected

In addition to the various natural sources, examples of which are listed above, it is also possible to provide nervonic so acid by a synthetic procedure. The starting point for such synthesis could be, for example, the readily available erucic acid (cis-docosa-1 3-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 55 and decarboxylation to yield nervonic acid. This synthesis has some advantages, particularly in the preparation of iso- topically 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 etseq. 4th edition, Pub. 1982, John Wiley & Sons Inc.). These methods include: dry rendering, wet rendering, batch pressing, continuous pressing, solvent extrac-

3 EP 0 455 783 B1

tion and extraction with super-critical gases such as carbon dioxide. In practice the most efficient processes involve continous pressing or super-critical 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 5 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, w 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. 15 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. Alter- 20 natively one or more than 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 25 like, salts such as sodium salts, potassium salts, calcium salts, amino acid salts and the like. The acids and their func- tionally active derivatives nay 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 30 acid or functional derivatives thereof, are administered in a suitable pharmaceutical^ 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. Further- more, 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 35 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 - 50g per day, especially in the range 0.1g - 10g, and preferably 0.5g - 5g 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, adre- 40 noleukodystrophy (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 quan- tities of erucic acid. Erucic acid and its derivatives are effective methods of reducing the concentration of accumulated toxic metabolites characteristic of ALD. Thus, these preferred compositions of the present invention can also be used 45 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, so 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

55 We describe hereafter, by way of illustration only, the treatment of an oil obtained from the seeds of Lunaria biennis (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.

4 EP 0 455 783 B1

The typical composition of the crude oil is as follows:

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 (1mol) with an excess of absolute alcohol (5mol) in the presence of sodium ethoxide (0.01 mol) at 80°C for 3 hours. The reaction mixture is then cooled to 30°C and the lower glycerol layer is separated off. The product is washed three times with water at 80°C and then dried under vacuum up to a temperature of 1 10°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 EP 0 455 783 B1

(B)

Purification of Ethyl Removal of Saturated Fatty Esters Acids

Saturated fatty acid ethyl esters are removed from the Honesty ethyl esters by refluxing with an excess of urea dissolved in absolute alcohol for 2 hours. On cooling with stirring over 1 4 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 once, at 85-90°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°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% EP 0 455 783 B1

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

20 (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°C under vacuum, (b) adding a further 7% and reacting for 3 hours at 220°C. (c) adding a further 4% and 25 reacting for 3 hours at 220°C. (d) adding the final 4% and reacting for 5 hours at 220°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°C. 30 (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 1 10°C. The GTN is filtered in vacuo at 70°C to give a pale yellow liquid which solidifies on standing at 35 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

55 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 gelatin capsules containing about 1g of oil. The dose will depend on the patient's circumstances but we believe it

7 EP 0 455 783 B1 will normally be from 5 to 10g 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.

Claims

Claims for the following Contracting States : AT, BE, CH, DE, DK, FR, GB, IT, LI, LU, NL, SE

1. A pharmaceutical composition comprising nervonic acid (cis-tetracos-15-enoic acid) or a functional derivative thereof in a physiologically acceptable form, in the absence of cyclosporin, and optionally a carrier or diluent therefor.

2. A pharmaceutical composition as claimed in claim 1 comprising nervonic acid (cis-tetracos-15-enoic acid) or a functional derivative thereof in a physiologically acceptable form as the principal active component of the composition, and optionally a carrier or diluent therefor.

3. A composition for use in the treatment of demyelinating diseases such as multiple sclerosis by enteric and intravenous administration, said composition comprising nervonic acid (cis-tetracos-15-enoic acid) in a physiologically acceptable form.

4. A composition as claimed in claim 1 ,2 or 3, further comprising at least one additional long chain fatty acid (C16 to C26) in a physiologically acceptable form.

5. A composition as claimed in claim 1 ,2,3 or 4, wherein the or each acid is present in the form of its ester.

6. A composition as claimed in claim 5, wherein the or each ester is a triglyceride ester.

7. A composition as claimed in any of the preceding claims, wherein nervonic acid in physiologically acceptable form comprises at least 20 wt% of the therapeutically active component of the composition.

8. A composition as claimed in any of the preceding claims, wherein the composition comprises the oil from a plant or micro-organism in a substantially purified form.

9. A composition as claimed in claim 8, wherein the oil is the seed oil of Lunaria biennis or Lunaria annua.

1 0. The use of a composition as claimed in any of the preceding claims for the manufacture of a medicament for the treatment of demyelinating diseases such as multiple sclerosis.

1 1 . The use of nervonic acid for the manufacture of a medicament for the treatment of demyelinating diseases such as multiple sclerosis.

1 2. A method of making a composition as claimed in any of claims 1 to 9, comprising the steps of providing plant seeds which contain long chain fatty acids, extracting the oils therefrom by pressing or supercritical gas extraction, purifying the oil by adsorption chromatography and optionally selectively removing components other than long chain fatty acids or esters or salts thereof.

13. A method as claimed in claim 12, wherein seeds of Lunaria biennis or Lunaria annua are provided.

14. The use of a composition comprising nervonic acid in a physiologically acceptable form or a pharmaceutical preparation thereof in the manufacture of a medicament to treat demyelinating diseases, for example multiple sclerosis.

Claims for the following Contracting State : ES

1. A method of making a composition for use in the treatment of demyelinating diseases such as multiple sclerosis, said composition comprising nervonic acid (cis-tetracos-15-enoic acid) in a physiologically acceptable form, which method comprises the steps of providing plant seeds which contain nervonic acid, extracting the oils therefrom by pressing or supercritical gas extraction, purifying the oil by adsorption chromatography and optionally selectively removing components other than long chain fatty acids or esters or salts thereof, and formulating the purified oil to provide a composition comprising nervonic acid as such or as a derivative thereof in a physiologically acceptable form.

8 EP 0 455 783 B1

2. A method as claimed in claim 1 , wherein seeds of Lunaria biennis or Lunaria annua are provided.

3. A method as claimed in claim 1 or 2, wherein the composition comprises at least one additional long chain fatty acid (C16 to C26) in a physiologically acceptable form. 5 4. A method as claimed in claim 1 ,2 or 3, wherein the or each acid is present in the form of its ester.

5. A method as claimed in claim 4, wherein the or each ester is a triglyceride ester.

10 6. A method as claimed in any one of the preceding claims, wherein nervonic acid in physiologically acceptable form comprises at least 20 wt% of the therapeutically active component of the composition.

Claims for the following Contracting State : GR

15 1 . The use of a composition comprising nervonic acid (cis-tetracos-1 5-enoic acid) in a physiologically acceptable form, for the manufacture of a medicament for the treatment of demyelinating diseases such as multiple sclerosis.

2. The use as claimed in claim 1 , wherein said composition further comprises at least one additional long chain fatty acid (C16 to C26) in a physiologically acceptable form. 20 3. The use as claimed in claim 1 or claim 2, wherein in the composition the or each acid is present in the form of its ester.

4. The use as claimed in claim 3, wherein in the composition the or each ester is a triglyceride ester.

25 5. The use as claimed in any one of the preceding claims, wherein in the composition the nervonic acid in physiologically acceptable form comprises at least 20 wt% of the therapeutically active component of the composition.

6. The use as claimed in any one of the preceding claims, wherein the composition comprises the oil from a plant or micro-organism in a substantially purified form. 30 7. The use as claimed in claim 6, wherein the oil is the seed oil of Lunaria biennis or Lunaria annua.

8. The use of nervonic acid for the manufacture of a medicament for the treatment of demyelinating diseases such as multiple sclerosis. 35 9. A method of making a composition for the use claimed in any one of claims 1 to 7, comprising the steps of providing plant seeds which contain nervonic acid, extracting the oils therefrom by pressing or supercritical gas extraction, purifying the oil by adsorption chromatography and optionally selectively removing components other than long chain fatty acids or esters or salts thereof. 40 10. A method as claimed in claim 9, wherein seeds of Lunaria biennis or Lunaria annua are provided.

Patentanspruche

45 Patentanspruche fur folgende Vertragsstaaten : AT, BE, CH, DE, DK, FR, GB, IT, LI, LU, NL, SE

1 . Eine pharmazeutische Zusammensetzung, bestehend aus Nervonsaure (cis-1 5-Tetracosensaure) Oder einem funktionellen Derivat derselben in physiologisch akzeptabler Form, ohne Cyclosporin, sowie gegebenenfalls einer Tra- gersubstanz oder einem Verdiinnungsmittel derselben. 50 2. Eine pharmazeutische Zusammensetzung nach Anspruch 1 , bestehend aus Nervonsaure (cis-1 5-Tetracosensaure) oder einem funktionellen Derivat derselben in vom physiologischen Standpunkt aus akzeptabler Form als Haupt- aktivbestandteil der Zusammensetzung und gegebenenfalls einer Tragersubstanz oder einem Verdiinnungsmittel derselben. 55 3. Eine Zusammensetzung zur Verwendung bei der Behandlung von Entmarkungskrankheiten wie der multiplen SWe- rose durch enterische oder intravenose Verabreichung, wobei die besagte Zusammensetzung Nervonsaure (cis- 1 5-Tetracosensaure) in vom physiologischen Standpunkt aus akzeptabler Form enthalt.

9 EP 0 455 783 B1

4. Eine Zusammensetzung nach Anspruch 1 , 2 oder 3, welche auBerdem mindestens eine weitere langkettige Fett- saure (von C16 bis C26) in vom physiologischen Standpunkt aus akzeptabler Form enthalt.

5. Eine Zusammensetzung nach Anspruch 1,2,3 oder 4, wobei die oder jede Saure in Form ihres Esters vorliegt. 5 6. Eine Zusammensetzung nach Anspruch 5, wobei der bzw. jeder Ester ein Triglyceridester ist.

7. Eine Zusammensetzung gemaB einem der vorgehenden Anspriiche, wobei die Nervonsaure in vom physiologischen Standpunkt aus akzeptabler Form zu mindestens 20 Gew.-% aus dem therapeutisch aktiven Bestandteil der Zusam- 10 mensetzung besteht.

8. Eine Zusammensetzung gemaB einem der vorhergehenden Anspriiche, wobei die Zusammensetzung aus dem Ol einer Pflanze oder eines Mikroorganismusses in im wesentlichen gereinigter Form besteht.

15 9. Eine Zusammensetzung gemaB Anspruch 8, wobei das Ol das Samenol der Lunaria biennis oder Lunaria annua ist.

10. Anwendung der Zusammensetzung gemaB einem der vorhergehenden Anspriiche zur Herstellung eines Arznei- mittels fur die Behandlung von Entmarkungskrankheiten wie der multiplen Sklerose.

20 11. Anwendung von Nervonsaure zur Herstellung eines Arzneimittels fur die Behandlung von Entmarkungskrankheiten wie der multiplen Sklerose.

12. Methode zur Herstellung einer Zusammensetzung gemaB einem der Anspriiche 1 bis 9, bestehend aus den Ver- fahrensstufen der Bereitstellung von langkettige Fettsauren enthaltenden Pflanzensamen, Extraktion des Ols aus 25 diesen durch Pressen oder Extraktion mit Gasen im superkritischen Zustand, Reinigung des Ols durch Adsorpti- onschromatographie und gegebenenfalls selektive Entfernung von Bestandteilen, bei denen es sich nicht urn langkettige Fettsauren oder Ester oder Salze derselben handelt.

13. Methode nach Anspruch 12, wobei die Samen der Lunaria biennis oder der Lunaria annua zur Verfiigung gestellt 30 werden.

14. Anwendung einer Zusammensetzung, bestehend aus Nervonsaure in einer vom physiologischen Standpunkt aus akzeptablen Form oder einer pharmazeutischen Zubereitung derselben zur Herstellung eines Arzneimittels fur die Behandlung von Entmarkungskrankheiten wie der multiplen Sklerose. 35 Patentanspruche fur folgenden Vertragsstaat : ES

1. Methode zur Herstellung einer Zusammensetzung zur Anwendung fur die Behandlung von Entmarkungskrankheiten wie der multiplen Sklerose, wobei die Zusammensetzung aus Nervonsaure (cis-1 5-Tetracosensaure) in vom phy- 40 siologischen Standpunkt aus akzeptabler Form besteht, wobei die Methode die Verfahrensstufen der Bereitstellung von Nervonsaure enthaltenden Pflanzensamen, Extraktion des Ols aus diesen durch Pressen oder Extraktion mit Gasen im superkritischen Zustand, Reinigung des Ols durch Adsorptionschromatographie und gegebenenfalls selektive Entfernung von Bestandteilen, bei denen es sich nicht urn langkettige Fettsauren oder Ester oder Salze derselben handelt, und Konfektionierung des gereinigten Ols zur Bereitstellung einer Zusammensetzung, beste- 45 hend aus Nervonsaure als solcher oder einem Derivat derselben in vom physiologischen Standpunkt aus akzeptabler Form umfaBt.

2. Methode nach Anspruch 1 , wobei Samen der Lunaria biennis oder der Lunaria annua bereitgestellt werden. so 3. Methode nach Anspruch 1 oder 2, wobei die Zusammensetzung mindestens eine weitere langkettige Fettsaure (von C16 bis C26) in vom physiologischen Standpunkt aus akzeptabler Form enthalt.

4. Methode nach Anspruch 1 , 2 oder 3, wobei die bzw. jede Saure in Form ihres Esters vorliegt.

55 5. Methode nach Anspruch 4, wobei der bzw. jeder Ester ein Triglyceridester ist.

6. Methode gemaB einem der vorgehenden Anspriiche, wobei die Nervonsaure in vom physiologischen Standpunkt aus akzeptabler Form mindestens zu 20 Gew.-% aus dem therapeutisch aktiven Bestandteil der Zusammensetzung besteht.

10 EP 0 455 783 B1

Patentanspruche fur folgenden Vertragsstaat : GR

1 . Anwendung einer Zusammensetzung, bestehend aus Nervonsaure (cis-1 5-Tetracosensaure) in einer vom physiologischen Standpunkt aus akzeptablen Form zur Herstellung eines Arzneimittels fur die Behandlung von Entmar- 5 kungskrankheiten wie der multiplen Sklerose.

2. Anwendung nach Anspruch 1 , wobei besagte Zusammensetzung auBerdem mindestens eine weitere langkettige Fettsaure (von C16 bis C26) in vom physiologischen Standpunkt aus akzeptabler Form enthalt.

10 3. Anwendung nach Anspruch 1 oder Anspruch 2, wobei die bzw. jede Saure in der Zusammensetzung in Form ihres Esters vorliegt.

4. Anwendung nach Anspruch 3, wobei der bzw. jeder Ester in der Zusammensetzung ein Triglyceridester ist.

15 5. Anwendung gemaB einem der vorhergehenden Anspriiche, wobei die Nervonsaure in vom physiologischen Stand- punkt aus akzeptabler Form in der Zusammensetzung mindestens zu 20 Gew.-% aus dem therapeutisch aktiven Bestandteil der Zusammensetzung besteht.

6. Anwendung gemaB einem der vorhergehenden Anspriiche, wobei die Zusammensetzung aus dem Ol einer Pf lanze 20 oder eines Mikroorganismusses in im wesentlichen gereinigter Form besteht.

7. Anwendung nach Anspruch 6, wobei das Ol das Samenol der Lunaria biennis oder Linaria annua ist.

8. Anwendung von Nervonsaure zur Herstellung eines Arzneimittels fur die Behandlung von Entmarkungskrankheiten 25 wie der multiplen Sklerose.

9. Methode zur Herstellung einer Zusammensetzung fur die Anwendung gemaB einem der Anspriiche 1 bis 7, bestehend aus den Verfahrensstufen der Bereitstellung von Nervonsaure enthaltenden Pflanzensamen, Extraktion des Ols aus diesen durch Pressen oder Extraktion mit Gasen im superkritischen Zustand, Reinigung des Ols durch 30 Adsorptionschromatographie und gegebenenfalls selektive Entfernung von Bestandteilen, bei denen es sich nicht urn langkettige Fettsauren oder Ester oder Salze derselben handelt.

10. Methode nach Anspruch 9, wobei Samen der Lunaria biennis oder Linaria annua bereitgestellt werden.

35 Revendications

Revendications pour les Etats contractants suivants : AT, BE, CH, DE, DK, FR, GB, IT, LI, LU, NL, SE

1. Une preparation pharmaceutique comportant de I'acide nervonique (cis-tetracos-15-acide enoi'que) ou un derive 40 fonctionnel de cet acide, sous une forme physiologiquement admissible, en I'absence de cyclosporine et, en option, un vehicule ou un diluant pour ledit acide.

2. Une preparation pharmaceutique selon la revendication 1 comportant de I'acide nervonique (cis-tetracos-15-acide enoi'que) ou un derive fonctionnel de cet acide, sous une forme physiologiquement admissible comme composant 45 actif principal de la preparation et, en option, un vehicule ou un diluant pour ledit acide.

3. Une preparation a utiliser pour le traitement de maladies demyelinisantes telles que la sclerose en plaques, par administration enterique et intraveineuse, ladite preparation comportant de I'acide nervonique (cis-tetracos-1 5-acide enoi'que) ou un derive fonctionnel de cet acide, sous une forme physiologiquement admissible. 50 4. Une preparation selon la revendication 1 , 2 ou 3, comportant en outre au minimum un acide gras a longue chame (C16 a C26) additionnel sous une forme physiologiquement admissible.

5. Une preparation selon la revendication 1, 2, 3 ou 4, dans laquelle I'acide ou chaque acide est present sous forme 55 de son ester.

6. Une preparation selon la revendication 5, dans laquelle Tester ou chaque ester est un ester de triglyceride.

11 EP 0 455 783 B1

7. Une preparation selon I'une quelconque des revendications precedentes, dans laquelle I'acide nervonique sous une forme physiologique admissible comporte au minimum 20% en poids du composant therapeutiquement actif de la preparation.

5 8. Une preparation selon I'une quelconque des revendications precedentes, dans laquelle la preparation comporte I'huile provenant d'une plante ou d'un microorganisme, sous une forme substantiellement purifiee.

9. Une preparation selon la revendication 8, dans laquelle I'huile est I'huile de graines de Lunaria biennis ou de Lunaria annua. 10 10. L'usage d'une preparation telle que revendiquee dans I'une quelconque des revendications precedentes, pour la fabrication d'un medicament pour le traitement de maladies demyelinisantes telles que la sclerose en plaques.

1 1 . L'usage de I'acide nervonique pour la fabrication d'un medicament pour le traitement de maladies demyelinisantes 15 telles que la sclerose en plaques.

12. Une methode de realisation d'une preparation comme revendique dans I'une quelconque des revendications 1 a 9, comportant les etapes d'obtention des graines/fruits de plantes contenant des acides gras a longue chame, d'extraction des huiles de ces graines par pressage ou extraction avec un gaz supercritique, de purification de I'huile par 20 chromatographie d'adsorption sur colonne et, en option, d'extraction selective de composants autres que les acides gras a longue chame, ou leurs esters ou leurs sels.

13. Une methode selon la revendication 12, dans laquelle les graines de Lunaria biennis ou de Lunaria annua sont fournies. 25 14. L'usage d'une preparation comportant de I'acide nervonique sous une forme physiologiquement admissible ou une preparation pharmaceutique derivee de ladite preparation pour la fabrication d'un medicament destine au traitement de maladies demyelinisantes, la sclerose en plaques par exemple.

30 Revendications pour I'Etat contractant suivant : ES

1. Une methode de realisation d'une preparation destinee au traitement de maladies demyelinisantestelles que la sclerose en plaques, ladite preparation comportant de I'acide nervonique (cis-tetracos-1 5-acide enoi'que) sous une forme physiologiquement admissible, methode qui regroupe les etapes d'obtention de graines de plantes contenant 35 de I'acide nervonique, d'extraction des huiles contenues dans ces graines par pressage ou extraction avec un gaz supercritique, de purification de I'huile par chromatographie d'adsorption sur colonne et, en option, extraction selective de composants autres que les acides gras a longue chame ou leurs esters ou leurs sels et I'elaboration de I'huile purifiee pour produire une preparation comportant de I'acide nervonique proprement dit sous une forme physiologiquement admissible d'un derive de I'acide nervonique. 40 2. Une methode selon la revendication 1 , dans le cadre de laquelle des graines de Lunaria biennis ou de Lunaria annua sont fournies.

3. Une methode selon la revendication 1 ou la revendication 2, dans le cadre de laquelle la preparation comporte au 45 minimum un acide gras a longue chame (C16 a C26) additionnel sous une forme physiologiquement admissible.

4. Une methode selon les revendications 1 , 2 ou 3, dans le cadre de laquelle I'acide ou chaque acide est present sous forme de son ester. so 5. Une methode selon la revendication 4, dans le cadre de laquelle Tester ou chaque ester est un ester de triglyceride.

6. Une methode selon Tune quelconque des revendications precedentes, dans le cadre de laquelle I'acide nervonique sous une forme physiologiquement admissible comporte au minimum 20% en poids du composant therapeutiquement actif de la preparation. 55

12 EP 0 455 783 B1

Revendications pour I'Etat contractant suivant : GR

1 . L'usage d'une preparation comportant de I'acide nervonique (cis-tetracos-1 5-acide enoi'que) sous une forme physiologiquement admissible, pour la fabrication d'un medicament destine au traitement de maladies demyelinisantes telles que la sclerose en plaques.

2. L'usage, selon la revendication 1 , dans le cadre duquel ladite preparation comporte par ailleurs au minimum un acide gras a longue chame (C16 a C26) additionnel sous une forme physiologiquement admissible.

3. L'usage, selon la revendication 1 ou la revendication 2, dans le cadre duquel I'acide ou chaque acide est present dans la preparation sous forme de son ester.

4. L'usage selon la revendication 3, dans le cadre duquel Tester ou chaque ester present dans la preparation est un ester de triglyceride.

5. L'usage, selon Tune quelconque des revendications precedentes, dans le cadre duquel I'acide nervonique physiologiquement admissible contenu dans la preparation comporte au minimum 20% en poids du composant therapeutiquement actif de la preparation.

6. L'usage, selon Tune quelconque des revendications precedentes, dans le cadre duquel la preparation comporte I'huile provenant d'une plante ou d'un microorganisme sous une forme substantiellement purifiee.

7. L'usage selon la revendication 6, dans le cadre duquel I'huile est de I'huile de graines de Lunaria biennis ou Lunaria annua.

8. L'usage de I'acide nervonique pour la fabrication d'un medicament destine au traitement de maladies demyelinisantes telles que la sclerose en plaques.

9. Une methode de realisation d'une preparation pour l'usage revendique dans Tune quelconque des revendications 1 a 7, comportant les etapes d'obtention de graines de plantes contenant de I'acide nervonique, d'extraction des huiles contenues dans ces graines par pressage ou extraction avec un gaz supercritique, de purification de I'huile par chromatographie d'adsorption sur colonne et, en option, d'extraction selective de composants autres que les acides gras a longue chame ou leurs esters ou leurs sels.

10. Une methode selon la revendication 9, dans le cadre de laquelle des graines de Lunaria biennis ou de Lunaria annua sont fournies.