EP 1 419 768 A1
`
`Fig.6
`
`2.0
`
`(R=-0.38) jo on 25
`HIPPOCAMPUSARACHIDONICACIDLEVEL(mg/g) ww °
`
`
`
`
`
`0
`
`60
`SO
`30
`20
`10
`Hit% OF PATH SWAM
`TIME REQUIRED TO: REACH
`" BY RATS (%)
`ESCAPE PLATFORM (s)
`eeee ee ee aeeee
`WATER MAZE LEARNING PARAMETERS
`(@: ARACHIDONIC ACID GROUP (n=5), O: CONTROL GROUP (n=5) )
`
`40
`
`7
`
`22
`
`RIMFROST EXHIBIT 1024
`
`RIMFROST EXHIBIT 1024 page 2251
`
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`
`

`

`EP 1 419 768 A1
`
`INTERNATIONAL SEARCH REPORT
`
`International application No.
`PCT/JP02/00671
`
`A. CLASSIFICATION OF SUBJECT MATTER
`Int.Cl’ A61K31/202, 31/232, A61P25/24, 25/28, B23L1/30, 2/52,
`A23D9/00
`
`Minimum documentation searched (classification system followed by classification symbols)
`Int.’
`AGIK31/202,
`31/232, AG1P25/24,
`25/28, A23L1/30,
`A23D9/00
`
`2/52,
`
`Documentation searched other than minimum documentation to the extent that such documentsare included in the fields searched
`
`
`Electronic data base consulted during the international search (name of data base and, where practicable, search terms used)
`CAPLUS (STN), MEDLINE(STN), EMBASE (STN)
`
`Relevant to claim No.
`
`A
`
`A
`
`WAINWRIGHT P. E. et al., Water maze performance is
`unaffected in artificially reared rats fed diets
`supplemented with arachidonic acid and
`docosahexaenoic acid, J. Nutr., 1999, Vol.129, No.5,
`pages 1079 to 1089
`
`WAINWRIGHT P. BE. et al., Arachidonic acid offsets the
`effects on mouse brain and behavior of a diet witha
`low {n-6):(n-3) ratio and very high levels of
`‘docosahexaenoic acid, J. Nutr. , 1997, Vol. 127, No.1,
`pages 184 to 193
`
`CI Further documentsare listed in the continuation of Box C. C] See patent family annex.
`Special categories of cited documents:
`“tT”
`later document published after the intemational filing date or
`document defining the general state of the art which is not
`priority date and not in conflict with the application but cited to
`considered to be of particular relevance
`understand the principle or theory underlying the invention
`earlier documentbut published on orafter the intemational fling
`documentof particular relevance; the claimed invention cannot be
`date
`considered novel or cannot be considered to involve an inventive
`document which may throw doubts on priority claim(s) or whichis
`step when the documentis taken alone
`cited to establish the publication date ofanothercitation or other
`documentofparticular relevance; the claimed invention cannot be
`special reason (as specified)
`considered to involve an inventive step when the documentis
`documentreferring to an oral disclosure, use, exhibition or other
`combined with one or more other such documents, such
`means
`combination being obviousto a person skilled in the art
`
`documentpublished priorto the international filing date butlater=“&” document member ofthe samepatentfamily
`than the priority date claimed
`Dateof the actual completion of the international search
`20 March, 2002 (20.93.02)
`
` “X"
`
`“y"
`
`C. DOCUMENTS CONSIDERED TO BE RELEVANT _
`Category*
`Citation of document, with indication, where appropriate,of the relevant passages
`LYNCH M. A. et al.,
`Impaired spatial memory in aged
`rats is associated with alterations in inositol
`phospholipid metabolism, Neuroreport, 1994, Vol.5,
`No.12, pages 1493 to 1497
`
`Japanese Patent Office Facsimile No.
`
`Date of mailing of the international search report
`O02 April, 2002 (42.04.02)
`
`Name and mailing address of the ISA/
`
`Authorized officer
`
`Form PCT/SA/210 (second sheet) (uly 1998)
`
`Telephone No.
`
`
`23
`
`RIMFROST EXHIBIT 1024
`
`RIMFROST EXHIBIT 1024 page 2252
`
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`
`

`

`EP 1 419 768 A1
`
`INTERNATIONAL SEARCH REPORT
`
`International application No.
`PCT/JIP02/00671
`
`BoxI Observations where certain claims were found unsearchable (Continuation of item 1 of first sheet)
`
`This international search report has not been established in respectof certain claims under Article 17(2)(a) for the following reasons:
`
`1.
`
`Claims Nos.: 33
`
`3. [7] Claims Nos.:
`because they are dependent claims and are not drafted in accordance with the second andthird sentences of Rule 6.4().
`
`Box If Observations where unity of invention is lacking (Continuation of item 2 of first sheet)
`This International Searching Authority found multiple inventions in this international application, as follows:
`
`1. CJ As all required additional search fees were timely paid by the applicant, this international search report covers all searchable
`claims,
`
`As all searchable claims could be scarched withouteffort justifying an additional fee, this Authority did not invite payment
`of any additional fee.
`
`3. C4 As only someof the required additional search fees were timely paid by the applicant, this international search report covers
`only those claims for which fees were paid, specifically claims Nos.:
`
`because they relate to subject matter not required to be searched by this Authority, namely:
`Claim 33 pertains to business activities and thus relates to a subject matter
`which this International Searching Authority is not required, under the
`provisions of Article 17(2)(a)(i) of
`the PCT and Rule 39 (iii) of
`the
`Regulations under the PCT,
`to search.
`2. Cc] Claims Nos.:
`because they relate to parts of the intemational application that do not comply withthe prescribed requirements to such an
`extent that no meaningful international search can be carried out, specifically:
`
`-) No protest accompanied the paymentof additional search fees.
`
`4. C] No required additional search fees were timely paid by the applicant. Consequently,this international search report is
`restricted to the invention first mentioned in the claims;it is covered by claims Nos.:
`
`Remark on Protest | The additional search fees were accompanied by the applicant's protest.
`
`Form PCT/ISA/210 (continuation of first sheet (1)) (July 1998)
`
`24
`
`RIMFROST EXHIBIT 1024
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`RIMFROST EXHIBIT 1024 page 2253
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`
`

`

`
`
`Europaisches
`Patentamt
`European
`Patent Office
`desbrevets
`
`(11)
`
`EP 1 385 500 B1
`
`(12)
`
`EUROPEAN PATENT SPECIFICATION
`
`(51) Int CL:
`(45) Date of publication and mention
`AGIK 31/23 (2006.01)
`A6bTK 31/22 (2006.04)
`of the grant of the patent:
`
`28.07.2010 Bulletin 2010/30 AGIK 31/23112006.91)=AGIK 31/0452006.01)
`AGIK 31/215 (2006.01)
`AG1K 31/25 (2006.01)
`
`(21) Application number: 02724588.5
`
`(22) Dateoffiling: 11.04.2002
`
`(86) International application number:
`PCT/AL2002/000294
`
`(87) International publication number:
`WO 2002/083122 (24.10.2002 Gazette 2002/43)
`
`(54) FATTY ALCOHOLS AND FATTY ACID ESTERS USEFUL FOR TREATMENTOF INFLAMMATION
`
`FETTALKOHOLE UND FETTSAUREESTER ZUR BEHANDLUNG VON ENTZUNDUNGEN
`
`ALCOOLS GRAS ET ESTERS D'ACIDES GRAS UTILES DANS LE TRAITEMENTD’ INFLAMMATION
`
`
`(84) Designated Contracting States:
`AT BE CH CY DE DK ES FIFR GBGRIEITLILU
`
`(74) Representative: Vossius & Partner
`Siebertstrasse 4
`
`EP1385500B1
`
`Note: Within nine months of the publication of the mention of the grant of the European patentin the European Patent
`Bulletin, any person may give notice to the European Patent Office of opposition to that patent, in accordance with the
`Implementing Regulations. Notice of opposition shall not be deemed to have beenfiled until the opposition fee has been
`paid. (Art. 99(1) European Patent Convention}.
`
`Printed by Jouve, 75001 PARIS RIMFROST EXHIBIT 1024
`
`RIMFROST EXHIBIT 1024 page 2254
`
`page 2254
`
`MC NL PT SE TR
`Designated Extension States:
`AL LTLV MK RO SI
`
`(30) Priority: 11.04.2001 IL 14253501
`
`(43) Date of publication of application:
`04.02.2004 Bulletin 2004/06
`
`(73) Proprietor: YEDA RESEARCH AND
`DEVELOPMENTCo., LTD.
`76100 Rehovot(IL)
`
`(72) Inventors:
`¢ COHEN, Irun, R.
`76354 Rehovot(IL)
`¢ SHINITZKY, Meir
`46910 Kfar Shmaryahu (IL)
`* MARGALIT, Raanan
`
`{IL}
`
`81675 Miinchen (DE)
`
`(56) Referencescited:
`WO-A-01/00139
`WO-A-02/083122
`US-A- 5 194 451
`US-B1- 6 280755
`US-B1- 6 365 628
`
`WO-A-99/04632
`US-A- 3 592 930
`US-B1- 6 210 700
`US-B1- 6 331 568
`
`¢ SNIPES WETAL: "Inactivation of lipid-containing
`viruses by long-chain alcohols."
`ANTIMICROBIAL AGENTS AND
`CHEMOTHERAPY.JAN 1977, vol. 11, no. 1,
`January 1977 (1977-01), pages 98-104,
`XP009034119 ISSN: 0066-4804
`« SANDS JET AL: "EXTREME SENSITIVITY OF
`ENVELOPED VIRUSES, INCLUDING HERPES
`SIMPLEX, TO LONG-CHAIN UNSATURATED
`MONOGLYCERIDES AND ALCOHOLS"
`ANTIMICROBIAL AGENTS AND
`
`CHEMOTHERAPY, AMERICAN SOCIETY FOR
`MICROBIOLOGY, WASHINGTON, DC, US, vol. 15,
`no. 1, January 1979 (1979-01), pages 67-73,
`XP009007372 ISSN: 0066-4804
`
`
`
`

`

`EP 1 385 500 B1
`
`Description
`
`FIELD OF THE INVENTION
`
`The presentinvention relates to anti-inflammatory agents and, more particularly, te fatty alcchols, esters thereof
`[0001]
`- Cg alkanoic acids or esters of fatty acids with alkanediols or glycerol which are useful in the treatment of
`with C,
`immunologically-mediated inflammation.
`[0002] Abbreviations: AA: adjuvantarthritis: CFA: complete Freund’s adjuvant; EAE: experimental autoimmune
`encephalomyelitis; GPSCH: guinea pig spinal cord homogenate; IFA: incomplete Freund’s adjuvant; OA: oleyl alcohol;
`PBS: phosphate-buffered saline; SC: subcutaneously.
`
`10
`
`BACKGROUND OF THE INVENTION
`
`[0003]=Inflammation is commonly divided into three phases: acute inflammation, the immune response and chronic
`15
`inflammation. Acute inflammation is the initial response to tissue injury and is mediated by the release of histamine,
`serotonin, bradykinin, prostaglandins and leukotrienes. The immune response, usually preceded by the acute inflam-
`mation phase, occurs when immunologically competent cells are activated in response to foreign organisms or antigenic
`substancesliberated during the acute or chronic inflammatory response. The outcome of the immune responsefor the
`host may be beneficial, as when it causes invading organisms to be phagocytosed or neutralized. However, the outcome
`may be deleteriousif it leads to chronic inflammation without resolution of the underlying injurious process as it occurs
`in rheumatoid arthritis.
`
`20
`
`Thetreatment of patients with inflammation envisagesthe relief of pain, which is the presenting symptom and
`[0004]
`the major continuing complaint of the patient, as well as the slowing or arrest of the tissue-damaging process.
`[0005] Anti-inflammateory agents are usually classified as steroidal or glucocorticoids and nonsteroidal anti-inflamma-
`tory agents (NSAIDs). The glucocorticoids are powerful anti-inflammatory agents but the high toxicity associated with
`chronic corticosteroid therapy inhibits their use except in certain acute inflammatory conditions. Therefore, the nonster-
`oidal anti-infammatory drugs have assumed a majorrole in the treatment of chronic conditions such as rheumatoid
`arthritis.
`
`[0006] Among the nonsteroidal anti-inflammatory agents are included derivatives of aminoarylcarboxylic acids, ary-
`lacetic acids, arylbutyric acids, arylcarboxylic acids, arylpropionic acids, pyrazole, pyrazolone, salicylic acid and some
`other derivatives of different chemical structure, including specific anti-arthritic/anti-rheumatic agents.
`[0007]
`Somefatty alcohols and esters of fatty acids have been described as solvents or emulsifiers for use in phar-
`maceutical compositions. For example, cetyl alcohol may be used in pharmaceutical compesitions as emulsifying and
`stiffening agent (The Merck Index, pp. 347-8, # 2037), oleyl alcohol may be used as a carrier for medicaments (The
`Merck Index, p. 1222, # 6900), and alkyl esters of oleic acid may be used as solvents for medicaments (The Merck
`Index, p. 6899, # 6898).
`[0008] A mixture of higher aliphatic primary alcohols, primarily isolated from beeswax, was described as having mod-
`erate anti-inflammatory activity. The composition of such a mixture was not disclosed (Rodriguez etal., 1998).
`[0009]
`Feeding laboratory animals with fish oil rich in the long-chain n-3 polyunsaturated fatty acids (PUFAs), eicos-
`apentaenoic acid (20:5n-3) and docosahexaenoic acid (22:6n-3), was described to reduce acute and chronic inflammatory
`responses, to improve survival to endotoxin and in models of autoimmunity and to prolong the survival of grafted organs,
`andit was therefore suggested that fish oil supplementation may be clinically useful in acute and chronic inflammatory
`conditions and following transplantation (Calder, 1998). A pharmaceutical preparation comprising eicosapentaencoic acid
`and/or stearidonic acid for treatment of schizophrenia is described in WO 98/16216 and US 6,331,568.
`[0010] Modified polyunsaturated fatty acids and derivatives thereof have been proposed for pharmaceutical uses. WO
`99/27924 and US 6,280,755 describe anti-inflammatory fatty acids uninterrupted by a methylene group for usein topical
`pharmaceutical and cosmetic compositions. WO 97/38688 and US 6,262,119 describe polyunsaturatedfatty acids having
`1 or 2 substitutions selected from oxa and thia in position beta or gammato the acyl group, for treating or ameliorating
`symptoms of T-cell mediated disease. WO 99/58122 and US 6,365,628 describe saturated fatty acids in which one or
`more methylene groups are substituted by O, S, SO, SQ,, or Se and alkyl esters thereof, for treatment or prevention of
`diabetes. US 5,019,383 describes synthetic vaccines comprising a peptide residue coupled to one or more alkyl or
`alkenyl groups of at least 12 carbon atomsor otherlipophilic substance, wherein said alkyl or alkenyl group may be a
`fatty acid residue coupled to one or more functional groups of a polyfunctional group which is bound to the N-terminal
`amino group and/or C-terminal carboxy group of the peptide residue.
`[0011] Thereis no description in the literature that isolated fatty alcohols or esters thereof with alkanoic acids may be
`used themselves as medicaments, and specifically not that they may be involved in immunomedulation of inflammation.
`
`25
`
`30
`
`35
`
`40
`
`45
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`5a
`
`55
`
`2
`
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`
`

`

`SUMMARYOF THE INVENTION
`
`EP 1 385 500 B1
`
`It has now been surprisingly found, in accordance with the present invention, that certain long-chain fatty
`[0012]
`alcohols, esters thereof with C, - Cg alkanoic acids, or certain esters of long-chain fatty acids with alkanediols or glycerol
`can suppress inflammation in experimental adjuvant arthritis (AA) and experimental autcimmune encephalomyelitis
`(EAE) models in rats and can prevent graft rejection in mice.
`[0013] Thepresent invention thus relates to pharmaceutical compositions for the treatmentof inflammation, particularly
`immunologically-mediated inflammation, comprising as active ingredient an immunomodulator selected from: (a} a sat-
`urated or cis-unsaturated C,,-Cj, fatty alcohol.
`[0014]
`In another embodiment, the invention relates to the use of an immunomodulator selected from: (a) a saturated
`or cis-unsaturated C,,- C4, fatty alcohol for the preparation of a pharmaceutical composition for the treatmentof inflam-
`mation, in particular immunologically-mediated inflammation.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`[0015]
`
`Fig. 1 shows the dose response effect of oleyl alcohol (OA) on adjuvant arthritis (AA). Different doses of OA were
`administered subcutaneously to rats once 14 days before induction of AA.
`Fig. 2 is a graph showing the disease profile of Lewis rats with experimental autoimmune encephalomyelitis (EAE)
`and treated with oleyl alcohol. Oleyl alcohol was administered to the rats 14 days before induction of EAE. Control
`group wastreated with incomplete Freund's adjuvant(IFA).
`Fig. 3 is a graph showing the disease profile of Lewis rats with EAE and treated with IFA. IFA was administered to
`the rats 14 days before induction of EAE. Contrel group was not treated.
`
`DETAILED DESCRIPTION OF THE INVENTION
`
`[0016] The present invention provides immunomodulators selected from: a saturated or cis-unsaturated C,)- C1. fatty
`alcohol.
`
`[0017] According to one preferred embodiment of the invention, the pharmaceutical composition comprises a long-
`chain saturated or unsaturated C,,.-C,.,, preferably C,¢-C1,, most preferably a C.,, fatty alcohol.
`[0018] Examples of C,,.-C,, saturated fatty alcohols that can be used according to the invention include, but are not
`limited to, decyl alcohol, lauryl alcohol, myristyl alcohol, stearyl alcohol and preferably cetyl alcohol (also known as
`palmityl alcohol).
`[0019] The unsaturated fatty alcohol according to the invention has preferably one or more double bonds in the cis
`form and 16-18 carbon atoms and may be, without being limited to, oleyl alcohol (cis-9-octadecenol), linoleyl alcohol
`(cis-9,12-octadecadienol), y-linclenyl alcohol (cis-6,9,12-octadecatrienol) and linolenyl alcohol (cis-9,12,15-octadeca-
`trienol). In preferred embodiments, the fatty alcohol used in the compositions of the invention is cetyl, linolenyl or, most
`preferably, oleyl alcohol.
`[0020]
`The C,,-C,, fatty acid is preferably a C,.-C,, most preferably a C,, fatty acid. In one embodiment, the C,>-C.g
`fatty acid is saturated such as, but without being limited to, capric acid, lauric acid, myristic acid, palmitic acid, stearic
`acid and arachidic acid. In another embodiment, the C,,.-C., fatty acid is a cis-unsaturated fatty acid such as, but without
`being limited to, palmitoleic acid (cis-9-hexadecenoic acid}, oleic acid (cis-9-octadecenoic acid), cis-vaccenic acid (cis-
`11-octadecenoic acid), linoleic acid (cis-9,12-octadecadienoic acid), y-linolenic acid (cis-6,9,12-octadecatrienoic acid},
`linolenic acid (cis-9,12,15-octadecatrienoic acid) and arachidonic acid (cis-5,8,11,14-eicosatetraencic acid).
`[0021] According to the invention, the alkanediol has 2 to 8, preferably 2 to 4, and more preferably, 2 carbon atoms,
`andis selected from, but not being limited to, 1,3-propanediol, 1,4-butanediol and, preferably, 1,2-ethylene glycol. An
`example of such an ester is 1,2-ethylene glycol monooleate.
`[0022] According to another embodimentof the invention, the active ingredient of the pharmaceutical composition is
`a mono-or diester of glycerol with the long-chain fatty acid. In one preferred embodiment, the monoglyceride is glycerol
`moncoleate. The diglycerides contain one free hydroxyl group and the other two hydroxyl groups may be both esterified
`with 2 molecules of the long-chain fatty acid, e.g. glycerol dicleate, or one of the hydroxyl groupsis esterified with one
`molecule of the long-chain fatty acid and a second hydroxyl group is esterified with a C,- Cg alkanoic acid such as acetic
`acid, propionic acid, butyric acid, valeric acid and caproic acid.
`[0023]
`Theimmunesystem, in both its innate and adaptive arms, is involved in regulating inflammation of every type,
`and inflammation is a key factor in processes such as wound healing, connective tissue re-modeling, angiogenesis,
`organ regeneration, neuroprotection, as well as in the adaptive immune responses seenin autoimmunity, allergies, graft
`rejection, and infection (see Cohen, 2000; Schwartz and Cohen, 2000). Therefore, anti-inflammatory agents that modulate
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`5a
`
`55
`
`3
`
`RIMFROST EXHIBIT 1024
`
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`
`page 2256
`
`

`

`EP 1 385 500 B1
`
`the inflammatory response such as those described here will be useful in a variety of conditions.
`[0024]
`Inflammatery disorders that can be treated with the immunomodulators of the present invention include, but
`are not limited to, immunologically-mediated chronic or acute inflammatory disorders selected from an autoimmune
`disease, severe allergies, asthma, graft rejection or for the treatment of chronic degenerative diseases such as Alzhe-
`imer's disease, and in neuroprotection, organ regeneration, chronic ulcers of the skin, and schizophrenia.
`[0025] Examples of autoimmune diseases that can be treated according to the invention are multiple sclerosis or a
`human arthritic condition, ¢.g. rheumatoid arthritis, reactive arthritis with Reiter's syndrome, ankylosing spondylitis and
`other inflammations of the joints mediated by the immune system. Other autoimmune diseases are contemplated and
`are presented in the following list in the context of the organ or tissue involved. Thus, according to the invention, the
`immunologically-mediated inflammatory disorder may be myasthenia gravis, Guillain-Barré syndrome, and otherinflam-
`matory diseases of the nervous system; psoriasis, pemphigus vulgaris and other diseases of the skin; systemic lupus
`erythematosus, glomerulonephritis and other diseases affecting the kidneys; atherosclerosis and otherinflammations
`of the blood vessels; autoimmune hepatitis, inflammatory bowel diseases, e.g. Crchn’s disease, pancreatitis, and other
`conditions of the gastrointestinal system; type 1 diabetes mellitus {insulin-dependent diabetes mellitus or IDDM), au-
`toimmune thyroiditis (Hashimoto’s thyroiditis}, and other diseases of the endocrine system.
`[0026] One ofthe models used to test the anti-inflammatory activity of the agents according to the invention is adjuvant
`arthritis (AA), an experimental disease of the joints inducible in some strains of rats by immunizing with Mycobacterium
`tuberculosis in complete Freund’s adjuvant (CFA). These animals develop an arthritis whose features are similar to
`those of rheumatoid arthritis in humans and thus serve as animal models of human arthritic conditions such as rheumatoid
`
`arthritis, reactive arthritis in Reiter’s syndrome, ankylosing spondylitis and other inflammationsof the joints which appear
`to be mediated by the immune system (Pearson, 1964). Adjuvant arthritis also serves as a modelof immune-mediated
`inflammation in general including cell-mediated autoimmune reactions, graft rejection and allergic reaction. For example,
`treatments which can suppress rheumatoid arthritis include immunosuppressive agents such as corticosteroids, cy-
`closporin A (Jaffee et al., 1989; Pollock et al., 1989}, azathioprine, and other immunosuppressive agents which are
`broadly used in the treatment of autoimmune diseases. Therefore, suppression of adjuvant arthritis by a therapeutic
`agent indicates that the agentis potentially useful as a broad anti-inflammatory agent.
`[0027] The pharmaceutical composition provided by the present invention may be in solid, semisolid or liquid form
`and mayfurther include pharmaceutically acceptable fillers, carriers or diluents, and other inertingredients and excipients.
`The composition can be administered by any suitable route such as, but not limited to, oral, topical, or parenteral e.g.
`by injection through subcutaneous,intravenous, intramuscular, or any other suitable route. Since many of the compounds
`are oily, they are preferably administered parenterally, more preferably subcutaneously. If given continuously, the com-
`poundsof the present invention are eachtypically administered by 1-4 injections per day or by continuous subcutaneous
`infusions, for example, using a mini-pump. The dosage will depend of the state of the patient and severity of the disease
`and will be determined as deemed appropriate by the practitioner.
`[0028]
`For parenteral administration, the compounds may be formulated by mixing each at the desired degree of
`purity, in a unit dosage injectable form (solution, suspension, or emulsion), with a pharmaceutically acceptable carrier,
`i.e., one thatis non-toxic to recipients at the dosages and concentrations employed andis compatible with other ingredients
`of the formulation. Generally, the formulations are prepared by contacting the compounds of the present invention each
`uniformly and intimately with liquid carriers or finely divided solid carriers or both. Then, if necessary, the productis
`shaped into the desired formulation. Preferably the carrier is a parenteral carrier, more preferably a solution that is
`isotonic with the blood of the recipient. Examples of such carrier vehicles include water, saline, Ringer’s solution, and
`dextrose solution. Non-aqueous vehicles such as fixed oils can be also useful, as well as liposomes. These preparations
`can be made by conventional methods knownte those skilled in the art, for example as described in "Remington’s
`Pharmaceutical Science”, A.R. Gennaro, ed., 17th edition, 1985, Mack Publishing Company, Easton, PA, USA.
`[0029]
`Theinvention will now beillustrated by the following non-limiting examples.
`
`EXAMPLES
`
`
`Example 1. Anti-inflammatory effect of oleyl alcohol and other agents-protection against adjuvant arthritis (AA)
`
`[0030] AA wasinduced by immunizing inbred 8-10-week old Lewis rats {Harlan-Olac Limited, Blackthorn, Oxon, UK),
`at the baseofthe tail with 1 mg/0.1 ml ofkilled Mycobacterium tuberculosis (Sigma) in IFA (Sigma) as described (Pearson,
`1956). Arthritis of the limbs was noted to develop 12-14 days later and was scored on a scale of 0-16 summing the
`severity of the inflammation of each of the 4 limbs on a scale of 0-4, as described (Holoshitz et al., 1983). The peak of
`the arthritis usually was observed around day 26 after immunization.
`[0031] Control rats were untreated or treated by injections of saline. A positive control of immunosuppression was
`obtained byincluding a group ofrats treated with the corticosteroid agent dexamethasone (200 yg} administered intra-
`peritoneally every other day beginning on day 12 after induction.
`
`10
`
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`20
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`
`

`

`EP 1 385 500 B1
`
`[0032] The immunomodulator of the invention (100 pl oleyl alcohol, glycerol mono-oleate, linolenyl alcohol or cetyl
`alcohol) was administered subcutaneously (SC} once 14 days before induction of AA or on day 12 after induction of AA.
`The percentinhibition of inflammation measured on the day of maximal inflammation was computed asfollows:
`
`mean maximal score of test group
`mean maximal score of control group
`
`x 100%
`
`[0033] All four compounds were found to be effective, producing more than 60% inhibition of inflammation whereas
`oleic acid had noeffect. The results are summarized in Table 1.
`
`Twofurther experiments showed that 500 jl of oleyl alcohol (100 I corresponds to about 90 mg oleyl alcohol)
`[0034]
`suppressed the inflammation by 96% and 91%.
`
`Table 1. Effects of various agents onthe inflammation of adjuvant arthritis
`
`Compound Tested
`% Inhibition (100
`wl
`
`Glycerol mono-oleate
`98%
`
`Oleyl alcohol
`78%
`
`Linolenyl alcohol
`75%
`
`66%
`
`
`
`
`Cetyl alcohol
`
`10
`
`15
`
`20
`
`25
`
`
`Example 2. Protection against AA by different doses of oleyl alcohol
`
`30
`
`35
`
`40
`
`45
`
`5a
`
`55
`
`To study the dose responseeffect of oleyl alcohol in AA, oleyl alcohol was administered subcutaneously in
`[0035]
`doses of 10, 50,100 or 500 mg to Lewis rats once 14 days before induction of AA, as described in Example 1 above.
`[0036]
`Fig. 1 shows the dose response effect of oleyl alcohol.
`It can be seen that increasing doses of oleyl alcohol
`suppressed the arthritis. On the day of peak disease, day 26, the inflammation was suppressed by 14% (10 pl), 61%
`(50 wl), 78% (100 yl) and 90% (500 jl).
`
`
`Example 3. Anti-inflammatory effect of oleyl alcohol and other immunomcdulators and protection against EAE
`in DA rats
`
`Experimental autoimmune encephalomyelitis (EAE) is an experimental autoimmune disease inducible in some
`[0037]
`strains of rats by immunization with myelin basic protein (MBP)or proteclipid protein (PLP) in complete Freund’s adjuvant
`(CFA) or with an emulsion of the rat’s spinal cord in either CFA or incomplete Freund’s adjuvant (IFA}. EAE in DA rats
`is considered as a model of chronic EAE. Within two to three weeks the animals develop cellular infiltration of the myelin
`sheaths of the central nervous system resulting in demyelination and paralysis. Most of the animals die, but others have
`milder symptoms, and someanimals develop a chronic form of the disease that resembles chronic relapsing and remitting
`multiple sclerosis (MS) in humans. Therefore, these animals with EAE serve as a model for the human MS autcimmune
`disease. EAE develops in the animal about 12 days after immunization and is characterized by paralysis of various
`degrees due to inflammation of the central nervous system. In somestrains, like the Lewis rat, the paralysis can last up
`to 6-7 days and the rats usually recover unless they die during the peak of their acute paralysis. In other strains of rats
`like the DA rat, the paralysis can be chronic and remitting.
`[0038]
`Forthe induction and clinical assessment of EAE, spinal cord obtained from DArats is frozen, thawed and
`minced thoroughly with a spatula before immunization. Rats are immunized by one subcutaneousinjection (just under
`the skin) into the dorsal base ofthe tail with 200 wl emulsion prepared from 1:1 IFA (Difco, Detroit, MI, USA) and antigen
`(volume/weight, i.e. 100 wl IFA/100 mg of whole spinal cord} or from 1:1 CFA (IFA was complemented with 4 mg/ml of
`Mycobacterium tuberculosis strain 37RA) and antigen (volume/weight, i.e. 100 jl CFA/100 mg of whole spinal cord).
`The emulsion was prepared bytitration with a gas-tight glass syringe and a needle, 1.2 mm diameter. Rats are regularly
`weighed and examinedfor clinical signs of EAE. A four-graded scale was used to assessclinical severity: 0, no paralysis;
`1, tail weakness (hanging); 2, hind limb paralysis; 3, hind and fore limb paralysis; 4, severe total paralysis (Lorentzen et
`al., 1995).
`[0039] Groups of 5 or 7 DAstrain female rats, 8-9 weekold, are immunized in the hind footpads with 0.1 ml per footpad
`of IFA containing 100 mg of whole, homogenized DA spinal cord, for a total of 200 mg per rat. On the dayof immunization,
`the rats are treated by SC injection with oleyl alcohol or other agent according to the invention (100 yl) or with paraffin
`
`5
`
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`

`EP 1 385 500 B1
`
`oil (control). The rats are scored for EAE on a severity scale of 0 - 4 as described above.
`
`Example 4. Anti-inflammatory effect of oleyl alcohol and protection against EAE in Lewis rats
`
`EAE induced in Lewis rats is considered as a model of acute inflammation in the brain (as opposed to the
`[0040]
`chronic disease in DArats}.
`[0041]
`For EAEinduction, three lyophilized guinea pig spinal cord homogenate (GPSCH) emulsions were prepared
`as follows: {i) 25 mg of lyophilized GPSCH (GP2} was suspended in 2.5 ml of sterile PBS (Sigma} and incubated for
`one hour at 37° C; (ii) 54.1 mg of Mycobacterium tuberculosis H37Ra (MT, Difco} was suspendedin 13.5 ml CFA (Sigma)
`containing 1mg/ml MT to obtain 5 mg/ml MT; (iii) 2.5 ml CFA (5 mg/ml MT) was added into vial with 2.5 ml of PBS
`containing 25 mg GPSCH to yield 5 mg/ml GPSCH and 2.5 mg/ml MT. The mixture was transferred into a glass syringe
`connected to a second glass syringe through a Luer lock bridge. The material was mixed well by transferring from one
`syringe to another for about 10 minutes until the materiall was well emulsified. The emulsion of GPSCHat a dose of1
`mg/rat and MT at a dose of 0.5 mg/rat in CFA induced EAEin rats (based on previous titration).
`[0042]
`For the treatment, two groups of eight 9-10 weeks old Lewis rats (Harlan, Israel), were treated with the test
`samples {oleyl alcohol or IFA) 14 days before EAE induction. The group treated with IFA served as the control group.
`The test samples wereinjected at a dose of 0.5 ml/kg once, subcutaneously. A third group of 8 rats was not treated and
`served as non-treated control.
`
`EAE wasinduced in rats of all three groups 14 days after injection of the test samples by injection with 0.1 ml
`[0043]
`of the GPSCH emulsion in CFA into each of the hind leg foot pads (0.2 ml per rat).
`[0044] The EAEclinical signs were observed and scored from the 9th day post-EAE induction until the termination of
`the experiment according to the following five-graded scale to assess clinical severity: 0, normal behavior; 1, weight
`loss; 2, tail weakness; 3, hind legs hypotonia and weakness; 4, hind legs paralysis; 4, severe total paralysis; 5, impaired
`respiration and/or convulsions and/orfull paralysis or death. All rats having scores of 1 and above were considered sick.
`[0045] The calculation of EAE results was carried out as follows:
`
`10
`
`15
`
`20
`
`25
`
`(i) Calculation of the incidence of disease
`
`30
`
`[0046] The numberof sick animals in each group were summed. The incidence of disease and the % activity were
`calculated as follows:
`
`Incidence of disease = No. ofsick rats in group
`No. of rats in group
`
`x 100%
`
`% activity * = 1 _ (disease incidence in treated group )
`disease incidence in control group
`
`x 100%
`
`* = (according to incidence)
`
`(ii) Calculation of the mean maximal score (MMS)
`
`[0047] The maximal score of each rat in the group were summed. The mean maximal score (MMS) and the % activity
`of the group were calculated as follows:
`
`Mean Maximal
`“an
`
`s
`
`>» Maximalscore of each rat
`score = ————___—___-—-
`No.of rats in the group
`
`35
`
`40
`
`45
`
`5a
`
`55
`
`6
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`

`EP 1 385 500 B1
`
`% activity *= (1
`
`MMSoftreated group
`
`MMS