AU2008200812B2 - Nasal and oral compositions of polyunsaturated ketones for the treatment of psoriasis - Google Patents

Description

AUSTRALIA Patents Act 1990 COMPLETE SPECIFICATION Standard Patent Applicant(s): AVEXXIN AS Invention Title: NASAL AND ORAL COMPOSITIONS OF POLYUNSATURATED KETONES FOR THE TREATMENT OF PSORIASIS The following statement is a full description of this invention, including the best method for performing it known to me/us: NASAL AND ORAL COMPOSITIONS OF POLYUNSATURATED KETONES FOR THE TREATMENT OF PSORIASIS This invention relates to the use of certain 5 polyunsaturated long-chain ketones for the treatment of psoriasis and in particular to ketones carrying electron withdrawing substituents alpha to the carbonyl functionality in such treatment. Psoriasis is a common, chronic, inflammatory skin 10 disorder. Psoriatic tissue is characterised by chronic inflammation in both epidermis and dermis, the disease being further characterised by hyperplasia of epidermal keratinocytes, fibroblast activation, alteration of eicosanoid metabolism, and leukocyte infiltration. 15 Effective treatments for psoriasis such as cyclosporin A, steroids, methotrexate and photochemotherapy all have immunosuppressive activity and are thus not ideal treatments due to their side effects. Scientists have therefore pursued other 20 potential treatments for this disease. It has been observed that psoriatic tissue exhibits elevated levels of arachidonic acid and eicosanoids. This suggests that phospholipase

A

2

(PLA

2 ) may be involved in the pathogenesis of psoriasis. 25 The phospholipases are a group of enzymes that release unsaturated fatty acids from the sn2 position of membrane phospholipids. Once released, the fatty acids are converted by various enzymes into biologically very important signalling molecules. Release of arachidonate 30 initiates the arachidonate cascade leading to the synthesis of eicosanoids such as prostaglandins. Eicosanoids are important in a variety of physiological processes and play a central role in inflammation. In Inflammation, Vol. 18, No.1 1994, Andersen et al 35 identify the presence of certain phospholipases in psoriatic human skin. It is therefore believed that inhibition of 2 phospholipase enzymes should have potential in curing some of the inflammatory symptoms, including epidermal hyperproliferation due to increased leukotriene production, related to eicosanoid production and cell 5 activation in both epidermis and dermis in psoriasis. In J. Chem. Soc. Perkin Trans. 1, 2000, 2271-2276 several structurally different compounds are reported as inhibitors of cPLA 2 in vitro. The compounds tested were based around (all-Z)-eicosa-5,8,11,14,17-pentaenoic acid 10 (EPA) and (all-Z)-docosa-4,7,10,13,16,19-hexaenoic acid (DHA). The paper suggests that preliminary studies show that in vitro the compounds are active as enzyme inhibitors. The compounds in J. Chem. Soc. Perkin Trans. 1, 15 2000, 2271-2276 have however not been tested in vivo and there is thus no way of predicting their in vivo effects. In addition, when devising a treatment for a disease it is necessary to ensure selectivity. There are a very large number of phospholipase enzymes known 20 and more enzymes of this type are being discovered as medical science develops. Since phospholipases control a wide variety of different intracellular functions it is necessary to develop inhibitors of these enzymes that are selective for the particular phospholipase whose 25 activity is to be altered. Compounds which inhibit a large number of phospholipase enzymes are of little commercial interest since the benefits of a desired enzymic inhibition will be opposed by the presence of many unwanted and potentially dangerous side effects 30 caused by unwanted enzyme inhibitions. There remains a need therefore, to provide highly selective inhibitors of phospholipase enzymes. The present inventors have surprisingly found that compounds of somewhat similar structure or the same 35 structure as those identified in the Perkin Transactions paper are selective for IVa PLA enzymes and are therefore ideal candidates for the treatment of - 3 psoriasis in the absence of side effects. Given that there are a total of 23 enzymes in the phospholipase ground and each enzyme fulfils a different physiological and pathological function this is surprising. Moreover, s the compounds of the invention have surprisingly been found to be particularly potent in reducing eicosanoid production, by for example, the inhibition of group IVa

PLA

2 . Thus, viewed from one aspect the invention provides a lo nasal or oral pharmaceutical composition comprising a compound of formula (I) R-CO-X (I) is (wherein R is a C 16

-

24 unsaturated hydrocarbon group interrupted a, P, y, or 6 to the carbonyl group by a heteroatom or group of heteroatoms selected from S, 0, N, SO, SO 2 said hydrocarbon group comprising at least 5 non conjugated double bonds; and 20 X is an electron withdrawing group) and a pharmaceutically acceptable excipient. The composition may be prepared through the use of a compound of formula (I) 25 R-CO-X (I) (wherein R is a C 1

-

2 4 unsaturated hydrocarbon group optionally interrupted a, P, y, or 8 to the carbonyl group by a heteroatom or group of heteroatoms selected from S, 30 0, N, SO, SO 2 , said hydrocarbon group comprising at least 5 non-conjugated double bonds; and X is an electron withdrawing group) for the manufacture of a medicament for the treatment of psoriasis. 35 Viewed from another aspect the invention provides a method of treating psoriasis comprising administering to an animal, preferably a mammal, e.g. human, an effective N:\MelbourneCases\Patent\3000-53999\PS3772 AU I\Specis\P53772.AU. I Specification 2008-2-5.1doc - 3a amount of the nasal or oral pharmaceutical composition comprising a compound of formula (I) as hereinbefore described. The composition may be prepared through the use of a 5 compound of formula (I) as hereinbefore described for use in the manufacture of a medicament for inhibiting the enzyme of IVa PLA 2 . Viewed from yet another aspect, the invention relates to a particular form of a pharmaceutical composition 10 comprising a compound of formula (I) as hereinbefore described. The group R preferably comprises 5 to 7 double bonds, preferably 5 or 6 double bonds, e.g. 5 double bonds which should be non-conjugated. It is also 15 N \Melbourne\CasesPatn\3000-53999\P53772 AU I\Specis\P5377:tAU I Specification 2008-2-15 doc 4 preferred if the double bonds do not conjugate with the carbonyl functionality. The double bonds present in the group R may be in the cis or trans configuration however, it is preferred if the 5 majority of the double bonds present (i.e. at least 50%) are in the cis configuration. In further advantageous embodiments all the double bonds in the group R are in the cis configuration or all double bonds are in the cis configuration except the double bond nearest the carbonyl 10 group which may be in the trans configuration. The group R may have between 16 and 24 carbon atoms, preferably 19 to 21 carbon atoms. The group R may carry a heteroatom or group of heteroatoms positioned a, P, y, or 8 to the carbonyl, 15 preferably P or y to the carbonyl. Preferably the heteroatom is 0 or S or a sulphur derivative such as SO. Specifically preferred RCOX groups are those of formula coxx so Cox S CO sCox 20 The R group may carry up to three substituents 2140635_1 (GHMatters) 5 selected from halo or C 1 6 -alkyl. If present the substituents are preferably non-polar, and small, e.g. a methyl group. It is preferred however, it the R group remains unsubstituted. 5 The group X is an electron withdrawing group. Suitable groups in this regard include O-C 1 6 alkyl, CN,

CO

2

-C

1 - alkyl, phenyl, CHal 3 , CHal 2 H, CHalH 2 wherein Hal represents a halogen, e.g. fluorine, chlorine, bromine or iodine, preferably fluorine. 10 In a preferred embodiment the electron withdrawing group is CHal 3 , especially

CF

3 . Highly preferred compounds for use in the invention are EPACOCF 3 , EPASCOCF 3 and AKH 217 as depicted below. Compounds of formula (I) may be manufactured using 15 known chemical synthetic routes. It is convenient to begin synthesis from the commercially available compounds arachidonic acid, EPA or DHA. Conversion of the acid functionality of these compounds into, for example a -COCF 3 group can be achieved readily, e.g. by 20 converting the carboxylic acid into its corresponding acid chloride and reacting the same with trifluoroacetic anhydride in the presence of pyridine. Introduction of a heteroatom into the carbon chain is also achieved readily. Conveniently, for example, 25 the starting acid is reduced to an alcohol and, if required, converted to the corresponding thiol. The nucleophilic thiol may then be reacted with a. group such as BrCH 2

COCF

3 thereby introducing the carbonyl and electron withdrawing species. Complete synthetic 30 protocols may be found in J. Chem. Soc., Perkin Trans 1, 2000, 2271-2276 or J. Immunol., 1998, 161, 3421. The compounds of formula (I) may be formulated into medicaments using conventional techniques well known to the skilled pharmaceutical chemist. Thus, the compounds 35 may be formulated with well known excipients or pharmaceutical carriers. The medicaments of the invention may also comprise - 6 other conventional additives such as antioxidants, preservatives, colouring, flavouring etc. The medicaments of the invention may be formulated as tablets, pills, powder, capsules, emulsions, creams or 5 ointments, for nasal or oral administration. The mode of administration is oral or nasal, although transmucosal, parenteral, topical, intradermal etc. are other modes of administration. The amount of the medicament required to effect a 1o successful treatment will, of course, depend on the patient and on the severity of the psoriasis. The dose will be readily determined by the skilled chemist. The compounds of the invention may be used to treat psoriasis in combination with other known pharmaceuticals 15 for said purpose and this forms a further aspect of the invention. All references, including any patents or patent applications, cited in this specification are hereby incorporated by reference. No admission is made that any 20 reference constitutes prior art. The discussion. of the references states what their authors assert, and the applicants reserve the right to challenge the accuracy and pertinency of the cited documents. It will be clearly understood that, although a number of prior art 25 publications are referred to herein, this reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art, in Australia or in any other country. In the claims which follow and in the description of 30 the invention, except where the context requires otherwise due to express language or necessary implication, the word "comprise" or variations such as "comprises" or "comprising" is used in an inclusive sense, i.e. to specify the presence of the stated features but not to 35 preclude the presence or addition of further features in N We lboureCases\Patent\53000-53999\P53772 A U I\Specis\P53 7 72 A U I Specification 2008-2- 5 doc - 6a various embodiments of the invention. The invention is described further below with reference to the following non-limiting examples and figures. s Figure 1 shows the relative inhibition of IVa PLA 2 enzyme activity for a number of compounds of the invention in comparison to commercial compounds. Recombinant IVa

PLA

2 enzyme was preincubated with inhibitor (5 4M) for 10 minutes and then assayed in the mixed-micelle enzyme 10 activity assay. The control was not pretreated with inhibitor. Results are given as % of control and are mean of duplicate determinations from 1 out of 4 representative experiments. Figure 2 shows concentration dependant inhibition of 15 IVa PLA 2 in the mixed-micelle enzyme activity assay. Increasing inhibition of IVa PLA 2 by EPACOCF 3 , EPASCOCF 3 and AACOCF 3 are shown in Figure B, and increasing inhibition of IVa PLA 2 by EPACH(OH)COCF 3 , DRACOCF 3 and MAFP are shown in Figure A. Results are given as % of control 20 and are mean of duplicate determinations from 7 one out of 2 to 4 representative experiments. Figure 3A shows that calcium ionophore A2 3187 stimulates extracellular release of [3H]-labelled lipid in a concentration-dependent manner. HaCaT cells were 5 treated with A 2 3 18 ? for 1 hour, arachidonic acid and eicosanoids were extracted from cell media using Bond Elut C18 columns and contents of [3H]-labelled lipids in media were determined by scintillation counting. Each column in the figure represents the average of 10 triplicate determinations from one out of 3 representative experiments. Figure 3B shows that calcium ionophore

A

23 18 , dosedependently also stimulates production of prostaglandin

E

2

(PGE

2 ), a product of cyclooxygenase 15 modification of arachidonic acid (AA). PGE 2 accumulation in medium was assayed by PGE 2 enzyme immunoassay (EIA) Figure 4 shows that the fatty acid derivative

EPACOCF

3 dosedependently is more powerful than the commercially available cyclooxygenase 2-selective 20 inhibitor NS-398 in inhibiting A 2 3187 -induced PGE 2 release in LPS-stimulated HaCat cells (IC 5 . values 180 nM and 240 nM, respectively). Figure 5 shows that TNFa or IL-13 stimulated NF-kB activation is dose-dependently inhibited by AKH217 (91% 25 and 81% respectively). MATERIALS AND METHODS Materials Calcium ionophore A 23187 , Sigmacoat, 3-(4,5 30 dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and bovine serum albumin were obtained from Sigma (St. Louis, MO, USA). Phosphatidylcholine, 1-stearoyl 2-arachidonyl and [3HJ arachidonic are from Amersham (Buckinghamshire, UK). Aluminium sheets silica gel 60, 35 ethyl acetate, iso-octane and acetic acid were purchased from Merck (Darmstadt, Germany). TNFa was a generous gift from Professor Terje Espevik, Norwegian University 8 of Science and Technology, NTNU and IL-13 was purchased from Roche Molecular Biochemicals.

AACOCF

3 is from BIOMOL (Plymouth Meeting, PA, USA), and 5 MAFP is from Cayman (Ann Arbor, MI, USA). All fatty acid compounds were stored under N 2 at -80 0 C. Cell culture The spontaneously immortalized human skin keratmocyte 10 cell line HaCaT was kindly provided by Prof. N. E. Fusenig (Heidelberg, Germany). Cells were grown in Dulbecco's Modified Eagle's Medium (DMEM) with 1 g glucose/i (Gibco BRL, Life Technologies Ltd, Paisley, Scotland), supplemented with 5% fetal calf serum (FCS) 15 (HyClone Laboratories, Inc., Utah, USA), 0.3 mg/ml L glutamine (Sigma Chemical Company, St.Louis, Mo, USA), 0.1 mg/ml gentamicine (Sigma) and 1 pg/ml fungizone (Gibco). Confluent cells were stimulated with A 2318 , Il 10 (10mg/ml) or TNFa (10mg/ml) in 0.5% (v/v) FCS for 1 20 hour before harvesting. Passages 40-80 of the cells were used. Generation of HaCat transfectants expressing luciferase under strict control of transcription factor NF-kB is described elsewhere (Anthonsen et al, J. Biol. Chem. 2001, 276, 30527). The reporter plasmid pBIIX 25 contains two copies of the HIV NF-kB sequence cloned upstream of the mouse fos promoter driving expression of the Photinus pyralis luciferase gene. EIA detection was performed according to manufacturer description, Cayman Chemical, MI, USA. A 30 dilution of 1:10 was used for PGE 2 measurement. Microplate Manager Software (Bio-Rad Laboratory) calculated sample data. Luciferase Assay 35 Cells were seeded in 24-round multiwell plates (2.8 x 105 cells/well). Treated cells were washed two times with phosphate-buffered saline and lysed, and luciferase 9 activities were determined using the Luciferase Reporter Assay system (Promega) and Turner Luminometer model TD 20/20 (Turner Designs) as described by the manufacturer. 5 Synthesis of (all-Z)-eicosa-5,8,11,14,17-pentaenoic acid (EPA) and (all-Z)-docosa-4,7,10,13,16,19-hexaenoic acid (DHA) derivatives The derivatives used in the enzyme assays are shown 10 below. EPACOCF 3 was prepared as described in J. Immunol. , 1998, 161, 3421. AACOCF3 and MAFP were bought from suppliers as mentioned above. The remaining derivatives were prepared as described in J. Chem. Soc. Perkin Trans. 1, 2000, 2271-2276. 15 R R= COCF 3 - EPACCCF 3 ___ ___ R= COAC H 3 - EPACOCH 3 R 20 R= COCF 3 - DHACOCF 3 --- R R= COCF 3 - AACOCF3 R= POF-CH 3 - MAFP 25__OX X= CF3 = AKH217 3COX X= CF 3 = EPASCOCF 3 3C0 3 EPACH (OH)

CF

3 35 Mixed-micelle assay of cPLA 2 activity Sources of IV PLA 2 enzyme activity were insect cells over 10 expressing recombinant human IV PLA 2 (10 pg IV PLA 2 protein/10 cells; Bac PAK Baculovirus expression system; CLONTECH Laboratories, Palo Alto, CA, USA). Cytosolic fractions of insect cells were prepared as described in 5 Schalkwijk et al (1992) Eur. J. Biochem. 210, 169-176. The protein contents of the cytosolic fractions were measured with Bio-Rad protein assay (Bio-Rad Laboratories GmbH, Munich, Germany) using bovine serum albumin as standard. The inhibitory derivatives were 10 added 10 minutes prior to substrate addition. The preincubation of inhibitors was performed at room temperature. IV PLA 2 enzyme activity was analyzed using [14C]-L-3 Phosphatidylcholine, 1-stearoyl-2-arachidonyl as substrate according to Wijkander et. al. (Eur. J. 15 Biochem. 202, 873-880, 1991). After 30 min the reaction was stopped and centrifuged, the CHCl 3 phase was evaporated with N 2 gas to dryness and then resuspended in CHCl 3 :MeOH (9:1, v/v). Thin layer chromatography (TLC) separated free arachidonic acid from phospholipids on 20 aluminum sheets silica gel 60 developed with ethyl acetate: iso-octane:acetic acid: water (55: 75: 8:100, v/v/v/v) (Gronnich et al, J. Clin. Invest., 93, 1224 1233, 1994). Phosphor-Imager quantified free arachidonic acid and phospholipids, and IV PLA 2 activity was 25 expressed as decreased arachidonic acid release by enzyme incubated with inhibitor compared to no inhibitor. Arachidonic acid and eicosanoid detection 30 Confluent cells were labelled with 1 piCi/ml [ 3 H] arachidonic acid in media supplemented with 0.5 % (v/v) FCS 24 hours before cell induction and inhibition. About 90 % of the radioactive arachidonic acid were incorporated in the cell membranes. Extracellular

[

3 H] 35 arachidonic acid was removed by washing the cells 3 times in media. The HaCaT cells were then preincubated with inhibitor for 1 hour and stimulated with calcium 11 ionophore for 1 hour. The cell media were collected and cleared by centrifugation. Arachidonic acid and eicosanoids were extracted from media using Bond Elut C18 octadecyl columns (500 mg) (Varian SPP, Harbor City, 5 CA) as described by Powell, Anal. Biochem. 164, 117-131, 1987; with modifications previously described Brekke, Cytokine, 4, 269-280, 1992. The samples were collected in glass tubes precoated with Sigmacoat. The ethyl acetate solution of samples were completely dried with 10 N 2 , redissolved in 0,5 ml fresh ethyl acetate and aliquots of 50 pl (triplicates) samples were subjected to liquid -scintillation counting (Beckman LS 1701) in 5 ml Ready Protein liquid (Beckman). 15 The amount of PGE 2 in cell culture media from calcium ionophore stimulated HaCaT cells was measured using an enzyme immunoassay (EIA; Cayman). The assay is based on the competition between free PGE 2 and a PGE 2 acetylcholinesterase for a limited amount of PGE 2 20 monoclonal antibody. The media were diluted 1:10 before analyzing of the PGE 2 contents. Microplate Manager Software (Bio-Rad Laboratory) calculated the sample data. 25 MTT assay Confluent cells were pretreated with inhibitors in serum free medium for 1 hour, and then treated with stimulating agent for 1 hour. Conversion of substrate [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium 30 bromide] was measured as optical density at 580 nm after 4 hours according to Mosmann [Mosmann T, J.Immunol. Methods 65, 55-63, 1983]. For each concentration of inhibitor nine parallels were measured. 35 RESULTS IV PLA 2 enzyme activity In order to investigate the action of the fatty acid 12 derivatives as inhibitors of IV PLA 2 , we measured the IV

PLA

2 activity in the mixed micelle assay with recombinant IV PLA 2 as enzyme source, as described in materials and methods. The synthetic fatty acid derivatives we made 5 are listed above, together with the commercial available inhibitors, which we used for comparison.

EPACOCF

3 , EPASCOCF 3 and AKH-217 seem to have the same potency as IV PLA 2 inhibitors as AACOCF3 (i.e. 75-80 % inhibition) (Figure 1). MAFP and DHACOCF 3 were poorer IV 10 PLA 2 inhibitors (50% and 30% inhibition respectively) The compound EPACH(OH)CF 3 was also tested which results in severe attenuation of inhibitory effect (10%. inhibition).

EPACOCH

3 was made as a control compound with methyl instead of the trifluoromethyl

(CF

3 ) group. 15 EPACOCH 3 showed no inhibition (figure 1). The IC 50 values of EPACOCF 3 , EPASCOCF 3 and AACOCF3 were measured to be 2.9 + 1.9, 3.5 + 0 pM and 5.8 + 1.9 pM respectively (Figure 2B) . While the IC 50 values of

DHACOCF

3 , MAFP and EPACH(OH)CF 3 were determined to be 20 21.3 + 1.5 pM, 24± 1.4 pM and 43 + 7.1 pM respectively (Figure 2A). Kinetic studies with the inhibitors in the mixed micelle assay were performed in order to see if the time course was linear. A peak was achieved in two minutes 25 (results not shown), indicating that the inhibitors are very fast acting. In summary EPACOCF 3 , EPASCOCF 3 and AKH217 seem to have similar or perhaps slightly higher potency as the commercially available compound AACOCF 3 in inhibiting IVa 30

PLA

2 Arachidonic acid and eicosanoid detection. In order to evaluate the effect of EPA and DHA 35 derivatives in a more biological system, we utilized the HaCaT cells as a model system [Sjursen et al, Cytokine, 12, 8, 1189-1194, 2000] . The calcium ionophore

A

23187 has 13 been shown to induce arachidonic acid release in many cell types, probably by increasing the intracellular Ca2+-concentration and thereby inducing the association of cPLA 2 with cellular membranes [Kramer and Sharp, FEBS 5 Lett, 410, 49-53, 1997] . In HaCaT cell, the ionophore induced a dose response release of [3H] -labelled arachidonic acid (Figure 3A). Concentrations higher than 10 pM of A 231 7 were toxic as determined by MTT assay. 10 The next step in evaluating our synthetic fatty acid inhibitors was to examine their ability to reduce the extracellular release of PGE 2 in response to A 231 7 in HaCaT cells. Before the cell experiments were performed, we evaluated the toxicity of the inhibitors. MTT assay 15 showed that concentrations of 25 pM and higher of the fatty acid compounds are toxic to HaCaT cells (results not shown). HaCat cells upregulate expression of cyclooxygenase 2 message when treated with LPS (200 ng/ml, 5% human 20 serum) for 30 min. (unpublished results). Upon ionophore stimulation for one hour, PGE 2 accumulates in medium (Figure 3B) . The fatty acid derivative

EPACOCF

3 was compared to NS-398, a cox-2 selective inhibitor, in its potency to reduce

PGE

2 production by preventing release 25 of its precursor AA. HaCat cells were pretreated with LPS (200 ng/ml, 5% human serum) for 30 min., treated with either inhibitors at different concentrations (0.2 25 pM) for another 30 min and stimulated with A 23187 , 30 min. Finally,

PGE

2 EIA was performed on cell media. The 30 dose-response curve was constructed with respect to percent

PGE

2 production without inhibitor. As can be observed in Figure 4, EPACOCF 3 is much more potent than NS-398 in inhibiting

PGE

2 production

(IC

50 values of 180 nM and 240 nM, respectively), and indicates that 35 substrate deprivation may be more powerful than activity inhibition for the cyclooxygenase. In order to determine if inhibition of IVa PLA 2 has 14 any biologic consequence, HaCaT cells were stimulated with the proinflammatory cytokines IL-13 or TNFa. As a measure of inflammation, activation of the transcription factor NF-kB was analysed. We have shown earlier that TNFa or 5 IL-13 activates NF-kB in HaCaT cells (Thommesen et al, J. Immunol., 1998, 161, 3421). NF-kB activation was analysed as luciferase expression. Treatment of the stably transfected HaCat-pBIIX cells with TNFL or IL-13 for 1 h enhanced NF-kB-dependent expression (not shown). In the 10 presence of inhibitors AKH217, IL-13 stimulated luciferase expression was dose-dependently inhibited by 81%. TNFa stimulated NF-kB activation was inhibited dose-dependently AKH217 by 91% (Figure 5) thus confirming that our synthetic fatty acid inhibitors may be useful in 15 inhibiting inflammatory responses. In the claims which follow and in the preceding description of the invention, except where the context requires otherwise due to express language or necessary implication, the word "comprise" or variations such as 20 "comprises" or "comprising" is used in an inclusive sense, i.e. to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention. It is to be understood that, if any prior art 25 publication is referred to herein, such reference does not constitute an admission that the publication forms a part of the common general knowledge in the art, in Australia or any other country. 2140635_1 (GHMatters)

Claims (14)

1. A nasal or oral pharmaceutical composition comprising a compound of formula (I) 5 R-CO-X (I) (wherein R is a C 1624 unsaturated hydrocarbon group interrupted a, P, y, or 8 to the carbonyl group by a heteroatom or group of heteroatoms selected from S, 0, N, 10 SO, S02 said hydrocarbon group comprising at least 5 non conjugated double bonds; and X is an electron withdrawing group) and a pharmaceutically acceptable excipient. 15

2. A composition as claimed in claim 1 wherein said hydrocarbon group has 5 to 7 double bonds.

3. A composition as claimed in claim 2 wherein said hydrocarbon group comprises 5 double bonds. 20

4. A composition as claimed in claims 1 to 3 wherein no double bond is conjugated with the carbonyl group.

5. A composition as claimed in any one of claims 1 to 4 25 wherein all double bonds are in the cis configuration.

6. A composition as claimed in any one of claims 1 to 4 wherein all double bonds are in the cis configuration except the double bond nearest the carbonyl. 30

7. A composition as claimed in any one of claims 1 to 6 wherein the R group comprises 19 to 21 carbon atoms.

8. A composition as claimed in any one of claims 1 to 7 35 wherein the R group comprises a heteroatom or group of heteroatoms P or y to the carbonyl. 2140635_1 (GHMatters) 16

9. A composition as claimed in any one of claims 1 to 8 wherein said heteroatom or group of heteroatoms is 0, S or So. 5

10. A composition as claimed in any one of claims 1 to 9 wherein the RCOX group is: "'Cox so cox S cOx ---- sCox 10

11. A composition as claimed in any one of claims 1 to 10 wherein X is a O-Cl-6 alkyl, CN, C0 2 -C 1 .- alkyl, phenyl, CHal 3 ,CHal 2 H, CHalH 2 wherein Hal represents a halogen. is

12. A composition as claimed in claim 11 wherein X is CHal 3 .

13. A composition as claimed in claim 1 wherein RCOX is S COCF 3 or S 3 1 COCF 3 20

14. A nasal or oral pharmaceutical composition comprising a compound of formula (I) substantially as herein described with reference to the Figures. 2140635_1 (GHMners)