WO2013081452A1 - Phosphatidylinositol - Google Patents

Abstract

The invention relates to a new pharmaceutical compound, diacyl phosphatdylinositol in which both the sn-1 and the sn-2 place are taken by stearic acid (18:0) (diacyl [18:0; 18:0] phosphatidylinositol), more preferably, wherein said diacyl phosphatidylinositol is compound 1 as depicted in Fig. 10 or a racemate of compounds 1 and 2 as depicted in Fig. 10. Said pharmaceutical compound and pharmaceutical compositions comprising this compound are specifically useful for the treatment of a disease or a condition wherein suppression of T-cell activation is desirable, such as asthma, diabetes Type 1, rheumatoid arthritis, inflammatory bowel disease or psoriasis. Also part of the invention are food items containing the compound(s) of the invention and use thereof in a diet to treat or prevent the disease or condition mentioned above

Description

Title: Phosphatidylinositol Field of the invention

The invention relates to the field of pharmaceutics, more specifically to pharmaceutical preparations that affect the immune system by interference with antigen-presenting molecules and T-cell activation. More specifically, it relates to pharmaceutical preparations for prevention or treatment of a disease or a condition wherein suppression of T-cell activation is desirable, for example an auto-immune disease, allergic disorder or chronic inflammatory disease, such as asthma, diabetes Type 1, rheumatoid arthritis, inflammatory bowel disease and psoriasis.

Background

The immune system defends the body from attack by invaders recognized as foreign. At the heart of the system is the ability to recognize and respond to substance called antigens, whether they are infectious (foreign) agents or part of the body (self-antigens). T cells are critical immune cells that help to destroy infected cells and coordinate the overall immune response. The T cell bears the T-cell receptor (TCR) that interacts with antigen-presenting molecules, such as MHC (major histocompatibility complex).

It has been shown (e.g. in WO 2007/142510) that phosphatidyl inositols, Pi's) have beneficial effects on the immune system, where closely related molecular species, like phosphatidyl ethanolamine (PE) and phosphatidyl serine (PS), which together with PI make up the phospholipid mixture lecithin, are not effective. However, PI as it occurs in nature is also a mixture of inositol phospholipids, being a group of acidic (anionic) phospholipids consisting of a phosphatidic acid backbone, linked to an inositol moiety via the phosphate group. In most cases, the inositol moiety of the inositol phospholipids is in the ID-l-myo-inositol configuration and the glycerol moiety is in the sn-glycero-3-phospho configuration. The fatty acid chains at the sn-1 and sn-2 positions of the glycerol backbone are generally ester-linked and the fatty acid chains can be saturated or non-saturated fatty acyls. An analysis of PI from different sources shows that the content of the individual fatty acids in PI varies largely (see Table 1). Especially the difference between plant derived PI (in this case from Arabidopsis thaliana) and mammalian derived PI is striking. However, even within the same species there are differences in PI fatty acids relating to the tissue source of the PI.

Table 1. Fatty acid composition of phosphatidylinositol (wt % of the total) in animal and plant tissues.

Tissue Fatty acids

16:0 18:0 18:1 18:2 18:3 20:3 20:4 22:3 22:5 22:6

Bovine heart

40 14 1 1 1 31 1 1 2

[1]

Bovine liver

5 32 12 6 1 7 23 4 3 5

[21

Rat liver [3] 5 49 2 2 4 35 1

Arabidopsis

48 3 2 24 24

thaliana [4]

[1] = Thompson, W. and acDonald, G., Eur. J. Biochem., 65, 107-111 (1976). [2] = Thompson, W. and MacDonald, G., J. Biol. Chem., 250, 6779-6785 (1975). [3] = Wood, R. and Harlow, R.D. Arch. Biochem. Biophys., 135, 272-281 (1969). [4] = Browse, J., Warwick, N., Somerville, C.R. and Slack, C.R. Biochem. J., 235, 25-31 (1986).

In animal sources of PI, the fatty acid composition is characterized by a high content of stearic (C 18:0) and arachidonic (C20:4) acids, wherein all the stearic acid is linked to position sn-1 and the arachidonic acid to position sn-2, and as much as up to 80% of the total lipid may consist of the single molecular species sn-l-stearoyl-sn2- arachidonoyl-glycerophosphorylinositol. In plant phosphatidylinositol, plamitic (C16:0) acid is the main saturated fatty acid, while linoleic (C 18: l) and linolenic (C I 8:2) acids are the main unsaturated components. Again, many of the saturated fatty acids are in position sn-1 and the unsaturated in position sn-2. However, the sn- 1-fatty acyl residue may also be replaced by O-alkyl or O-alkenyl to yield alkyl-acyl PI or alkenyl-acyl PI, respectively. Also comprised in the group of Pi's are lyso- inositolphospholipids which lack the 2-fatty acyl, and thus have a free hydroxyl group at the glycerol-2 position. Table 2. Distribution of fatty acids (mol % of the total) in positions sn-1 and sn-2 in phosphatidylinositol (PI) and the phosphatidylinositol mono- and diphosphates of ox brain. I PI monophosphate PI diphosphate

Fatty acids

sn-1 sn-2 sn-1 sn-2 sn-1 sn-2

16:0 15 9 7

18:0 74 69 69

18:1 10 10 20 13 21 10

18:2 1 2 trace 1 1 1

20:3(n-9) 5 10 10

20:3(n-6) 5 11 12

20:4(n-6) 67 49 52

22:3 7 10 7

22:6(n-3) trace trace trace

Holub, B.J., Kuksis, A. and Thompson, W. J. Lipid Res., 11, 558-564 (1970).

Thus far only naturally occurring PI has been tested and/or administered to check for its pharmaceutical properties. Since this naturally occurring PI is a mixture of different PI species, it would be beneficial to have a medicament which contains only one of the species. Yet, it is not known which of the species of PI that are present in the natural occurring isolates is pharmacologically active.

SUMMARY OF THE INVENTION

The present inventors now have discovered that diacyl phosphatidylinositol in which both the sn-1 and the sn-2 place are taken by stearic acid (18:0), in this invention also denominated as diacyl [18:0; 18:0] phosphatidylinositol or PI (18:0), is useful in medicine. Specifically, the invention relates to ad iacyl phosphatdylinositol molecule in which both the sn-1 and the sn-2 place are taken by stearic acid (18:0) (diacyl [18:0; 18:0] phosphatidylinositol), more preferably, wherein said diacyl phosphatidylinositol is compound 1 as depicted in Fig. 10 or a racemate of compounds 1 and 2 as depicted in Fig. 10. The invention further relates to said diacyl [18:0; 18:0] phosphatidylinositol for use in the treatment of a disease or a condition wherein suppression of T-cell activation is desirable.

Further, the invention pertains to said diacyl [18:0; 18:0] phosphatidylinositol for use in the treatment of asthma, diabetes Type 1 , rheumatoid arthritis, inflammatory bowel disease or psoriasis.

In another embodiment, the invention relates to a pharmaceutical composition comprising diacyl [18:0; 18:0] phosphatidylinositol as defined above where said pharmaceutical composition comrpises no other phosphatidylinositol species.

Preferably, such a pharmaceutical composition further comprises excipients for topical application, preferably for topical application on the skin. Alternatively, such a pharmaceutical composition further comprises excipients for mucosal application, preferably for mucosal application in the airways or in the intestine.

Further, said pharmaceutical composition preferably further comprises one or more other active ingredients, these not being other phosphatidylinositol species.

In a further preferred embodiment, said diacyl [18:0; 18:0] phosphatidylinositol is present in said composition in a suspended form in a physiologically acceptable aqueous carrier, preferably in physiological saline.

Also preferred is a pharmaceutical composition wherein said diacyl [18:0 ; 18:0] phosphatidylinositol makes up less than 50 percent, preferably less than 30 percent, more preferably less than 25 percent of the dry weight of the composition.

Further preferred is a pharmaceutical composition as defined above, herein said composition is adapted for mucosal administration, preferably for rectal

administration. Alternatively, said mucosal composition is adapted for pulmonary administration, for example in the form of a nebuliser or aerosol.

A further part of the invention is a method for preparing a pharmaceutical composition comprising as active ingredient diacyl [18:0; 18:0] phosphatidylinositol as defined above and no other phosphatidylinositol species, comprising preparing a non-liposomal suspension of said diacyl [18:0; 18:0] phosphatidylinositol in an aqueous physiologically acceptable carrier, preferably in saline.

Also part of the invention is the use of diacyl [18:0; 18:0] phosphatidylinositol as defined above for the manufacture of a medicament for the treatment or prevention of asthmatic conditions in a mammal. Alternatively, part of the invention is the use of diacyl [18:0; 18:0] phosphatidylinositol as defined above for the manufacture of a medicament for the prevention or treatment of an inflammatory bowel disease in a mammal, preferably ulcerative colitis or Crohn's disease.

Further part of the invention is a food item or food supplement enriched with diacyl [18:0; 18:0] phosphatidylinositol as defined above.

In this respect, the invention also comprises the use of diacyl [18:0; 18:0]

phosphatidylinositol as defined above for the manufacture of a food item or food supplement capable of preventing an inflammatory disease, in particular allergic asthma or ulcerative colitis.

In an alternative embodiment, the invention relates to a method of preventing the occurrence of an inflammatory disease in a healthy subject, in particular allergic asthma or ulcerative colitis, comprising providing said subject a diet with diacyl [18:0; 18.0] phosphatidylinositol as defined above or a pharmaceutically acceptable salt thereof.

Alternatively, the invention relates to a method of preventing the occurrence of an inflammatory disease in a healthy subject, in particular allergic asthma or ulcerative colitis, comprising administrating to said subject a pharmaceutical composition as defined above.

Also comprised in the invention is a method of treatment of a subject suffering from an inflammatory disease, in particular allergic asthma or ulcerative colitis, said method comprising administrating to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition as defined above.

The invention further comprises the use of a food item with diacyl [18:0; 18:0] phosphatidylinositol as defined above for the prevention and/or treatment of an inflammatory disease, in particular allergic asthma or ulcerative colitis, more preferably said use is in the form of a diet.

Next, the invention comprises the use of a pharmaceutical composition as defined above for the prevention and/or treatment of an inflammatory disease, in particular allergic asthma or ulcerative colitis.

Lastly, the invention comprises a pharmaceutical composition as defined above for topical administration, more specifically for the prevention or treatment of psoriasis. LEGENDS TO THE FIGURES

Fig. 1. synthetic PI (18:0) inhibits IL-2 release by PMA/Calcium ionophore activated T cells

Fig. 2. Synthetic PI (18:0) inhibits T cell activation without inducing toxicity

Fig. 3. Activity of synthetic PI (18:0) and bovine liver PI (Avanti) at signal transduction level. Phospho-p38 and phospho-JNK are inhibited.

Fig. 4. Synthetic PI (18:0) does not inhibit ERK phosphorylation

Fig. 5. Dose-dependent inhibition of inflammatory T cell response by PI (18:0).

Fig. 6. Effects of PI analogs on T cell response of human PBMCs.

Fig. 7. Effects of PI (18:0) and dexamethasone in an in vitro psoriasis model.

Fig. 8. Comparison of the effects of bovine PI and PI (18:0) in an in vitro psoriasis model.

Fig. 9. Dose response behaviour of PI (18:0) in the in vitro psoriasis model.

Fig. 10. Chemical structure of two diastereoisomers of PI (18:0).

DETAILED DESCRIPTION

The inventors have identified a new species of phosphatidylinositol, diacyl [18:0; 18:0] phosphatidylinositol, which is characterised in that it contains two stearic acid moieties at the sn-1 and sn-2 position of the glycerol moiety of the

phosphatidylinositol molecule. Although stearic acid moieties are abundant in the phosphatidylinositol species found in nature (see Table 1), they are - at least as far is known - exclusively found at the snl position (see Table 2).

As will be shown in the experimental part, this compound has an activity which is comparable with the activity demonstrated by a mixture of phosphatidylinositols as can be provided e.g. by a bovine liver extract. However, such an extract has the disadvantage that it probably will contain phosphatidylinositol species that do not have any pharmacological activity or even species that may counteract each other. Further, isolation of phosphatidylinositols from biological tissue is complex and purity is debatable. One of the advantages of the present invention is that by chemical synthesis, as is exemplified in the experimental part, a pure preparation of this one phosphatidylinositol species can be obtained.

The invention therefore relates to a composition for the prevention or treatment of a condition associated with unwanted T cell activation comprising as an active ingredient diacyl [18:0; 18:0] phosphatidylinositol, herein also abbreviated as "PI 18:0". The composition is for example a pharmaceutical composition or a food product with immunomodulatory properties. In eukaryotic cells, inositol

phospholipids such as phosphatidylinositol are quantitatively minor but vital components of membrane lipids with critical structural, metabolic and signalling roles. Thus, in contrast to known immunosuppressive agents, inositol phospholipids are natural constituents of the body. While diacyl [18:0; 18:0] phosphatidylinositol, however, does not occur in nature (at least it has not yet being described as such), it does not differ greatly from the Pis that are found in biological tissues, and thus no major problems with biocompatibility are to be expected. Accordingly, also the risk of unwanted side effects of a composition of the invention is lower compared to the 'foreign' immunomodulators, which are currently used in medicine, such as corticosteroids, non-steroidal anti-inflammatory drugs (NSAIDs) and (monoclonal) antibodies against cytokines, in particular against TNFa or interleukins, such as IL-13. Other aspects of the invention relate to the use of diacyl [18:0; 18:0]

phosphatidylinositol for the manufacture of a medicament for the treatment of any disorder associated with unwanted or excessive T cell activation, such as immune disease, allergic disorder or chronic inflammatory disease. It also relates to methods for suppressing the immune system of a subject, comprising administering a suitable dose of diacyl [18:0 ; 18:0] phosphatidylinositol to said subject. The invention is of particular use for the treatment of asthma, diabetes Type I, rheumatoid arthritis (RA), psoriasis and inflammatory bowel disease (IBD). Furthermore, the invention provides a food item or food supplement having immunomodulatory properties.

As shown herein, diacyl [18:0; 18:0] phosphatidylinositol is capable of blocking IFN- γ release by peripheral blood mononuclear cells (PBMC). Furthermore, it was found that IPL can directly inhibit T cell activation induced by phorbol ester and calcium ionophore to mimic downstream T cell receptor signalling as well as by stimulation of the T cell receptor-complex (CD3) and co-stimulatory molecule CD28 (Fig. 3B). The above effects were found to be dose-dependent. The presence of a phosphodiester bridge and a lipid moiety appear crucial for these effects, since myo- inositol had no significant suppressive effect (Figure 2). Moreover, diacyl glycerol had no significant suppressive effect.

Accordingly, a composition comprising diacyl [18:0; 18:0] phosphatidylinositol is suitably used to suppress or inhibit the activation of T cells and therewith suppress the unwanted effects of T cell activation, in particular cytokine (interferon) production. A pharmaceutical or food composition according to the invention is advantageously used for the treatment or prevention of a condition that is associated with or caused by unwanted (e.g. excessive) activation of cells bearing a T cell receptor, like T lymphocytes or NKT cells. For example, a condition associated with unwanted T cell activation by an antigen-presenting molecule, either a peptide or lipid antigen- presenting molecule, can be ameliorated by a treatment with a composition comprising diacyl [18:0; 18:0] phosphatidylinositol. In a specific embodiment, said condition is an MHC-associated condition. MHC class II molecules play a central role in the pathophysiology of several pulmonary diseases including asthma, sarcoidosis, hypersensitivity pneumonitis, and lung transplant rejection. Other MHC-associated diseases include Type I diabetes Mellitus, Crohn's disease, ulcerative colitis,

Rheumatoid arthritis, Multiple Sclerosis, psoriasis and solid organ transplant rejections (such as liver and kidney). However, the immunosupressive effects of diacyl [18:0; 18:0] phosphatidylinositol are not restricted to processes involving antigen-presenting molecules. Rather, they extend to the direct modulation of downstream T cell signalling, including the induction of proliferation and cytokine production. In one embodiment, the invention provides a composition comprising diacyl [18:0; 18:0] phosphatidylinositol for the suppression of T cell activation, wherein said composition modulates an intracellular T cell event that requires de novo protein synthesis

The effects of phosphatidylinositol are specific for the inositol headgroup. The same dose of phosphatidyl serine (PS), also an acidic phospholipid, was not only found to be ineffective in suppressing T cell activation but even enhanced PMA/Ca-induced IL-2 production (see WO 2007/142510). In said document it is also shown that PS appears toxic to activated T cells in a dose-dependent fashion. This is in contrast to earlier studies on the immunomodulatory effects of acidic phospholipids. For example, Ponzin et al. (Immunopharmacology. 1989 Nov-Dec; 18(3): 167-76.) disclose immunosuppressive effects of intravenously administered PS vesicles in mice. Along that same line, Caselli et al. (Immunopharmacology. 1992 May-Jun;23(3):205-213) reported the inhibition of DNA synthesis in activated peripheral blood cells by PS. As PI was only partially effective (and even caused a stimulation of DNA synthesis at low concentrations), the effect was contributed to the phosphorylserine headgroup. Furthermore, only PS species containing unsaturated fatty acyl chains were active. Although the inositol headgroup seems to provide for the pharmacological effect, it appears from the experimental section of this application that a PI analogue with short fatty acyl chains at the sn-1 and sn-2 positions (both pentyl: PI 5:0) is hardly effective. On the other hand, a PI (18:0) analogue, in which the inositol moiety has been replaced with a dimethyl moiety (indicated as PX 18:0 in the present application), shows T-cell suppressant activity, but at high dosages.

Further, it has been established that the pharmacological effect of the diacyl [ 18:0; 18:0] phosphatidylinositol is stereo selective. One of the diastereoisomers (indicated as compound 1 in Fig. 10 and potentially written as [ 1R,2S,3R,4R,5S,6S] diacyl [ 18:0; 18:0] phosphatidylinositol) is at least ten times as potent as the diastereoisomer indicated as compound 2 in Fig. 10 ([\S,2R,3S,4S,5R,6R] diacyl [ 18:0; 18:0] phosphatidylinositol). In the present application the term diacyl [18:0; 18:0] phosphatidylinositol means the racemate or the diasteroeoisomer indicated as compound 1 of Fig. 10.

Conditions that would benefit from a composition comprising diacyl [18:0; 18:0] phosphatidylinositol include any condition in which attenuation of antigen-presenting molecules and/or excessive or unwanted T cell activation is desirable. In one embodiment, a composition of the invention is used for the treatment or prophylaxis of a condition in which attenuation of (auto reactive) T cell activation is desirable. Preferably, the disease or symptoms associated therewith allow topical treatment with a composition of the invention, more preferably by mucosal administration. However, topical skin administration or systemic administration of a diacyl [18:0; 18:0] phosphatidylinositol-containing composition can also be envisaged.

Autoimmune diseases arise from an overactive immune response of the body against substances and tissues normally present in the body. Examples of auto-immune diseases are: systemic lupus erythematodes (SLE), rheumatoid arthritis (RA), postpartum thyroid dysfunction, auto-immune thromocytopenia, psoriasis, psoriatic arthritis, alopecia aerate, scleroderma, dermatitis heφetiformis, polymyositis, dermatomyositis, pemphigus vulgaris, spondyloarthropathies such as ankylosing spondylitis, vitiligo, Sjogren's syndrome, multiple sclerosis (MS), coeliac disease, Crohn's disease, myasthenia gravis, ulcerative colitis, autoimmune neuropathies such as Guillain-Barre, primary biliary cirrhosis, autoimmune hepatitis, vasculitis, autoimmune uveitis, Type 1 or immune-mediated diabetes mellitus (DM1), autoimmune hemolytic anemia, pernicious anemia, Graves' disease, autoimmune thrombocytopenia, Goodpasture's syndrome, narcolepsy, Hashimoto's thyroiditis, Hashimoto's encephalitis, autoimmune oophoritis and orchitis, autoimmune disease of the adrenal gland, anti-phospholipid syndrome, Miller-Fisher syndrome, and vasculitides such as Wegener's granulomatosis and Behcet's disease.

Allergic disorders occur when the immune system overreacts to exposure to antigens (either foreign or auto-antigens) in the environment. The substances (antigens) that provoke such attacks are called allergens. The immune response can cause symptoms such as swelling, watery eyes, and sneezing, and even a life-threatening reaction called anaphylaxis. Taking medications such as antihistamines can relieve most symptoms. Some examples of allergic disorders are asthma and eczema. Asthma is a respiratory disorder that can cause breathing problems, and frequently involves an allergic response by the lungs. If the lungs are oversensitive to certain allergens (like pollen, molds, animal dander, or dust mites), it can trigger breathing tubes in the lungs to become narrowed, leading to reduced airflow and making it hard for a person to breathe. Eczema is a scaly, itchy rash also known as atopic dermatitis. Environmental allergies (to dust mites, for example), seasonal allergies (such as hay fever), drug allergies (reactions to specific medications or drugs), food allergies (such as to nuts), and allergies to toxins (bee stings, for example) are the common conditions people usually refer to as allergies.

In one aspect the invention provides a pharmaceutical composition comprising as an active ingredient diacyl [18:0; 18:0] phosphatidylinositol and no other

phosphatidylinositol species, said composition being adapted or formulated for pulmonary administration. Also provided is the use of diacyl [18:0; 18:0] phosphatidylinositol, for the manufacture of a medicament for the treatment or prevention of asthmatic conditions in a mammal, preferably in a human subject. Phospholipids, including IPL, have been used before in pulmonary compositions to facilitate the delivery of active agents. Typically the lipid is incorporated in liposomes or other type of lipid body allowing for the delivery and dispersion of proteinaceous active agents. WO2005/055994 for example discloses a composition comprising a surfactant mixture comprising phospholipids and a lung-surfactant polypeptide of a specific amino acid sequence that maximizes its interaction with the alveoli. In contrast, the anti-asthma composition of the present invention comprises diacyl [18:0; 18:0] phosphatidylinositol rather than a lung-surfactant polypeptide as active agent. Accordingly, in one embodiment the composition for the pulmonary administration of diacyl [18:0; 18:0] phosphatidylinositol does not contain a lung- surfactant polypeptide or other kind of proteinaceous therapeutic molecule.

Eosinophilia refers to conditions in which abnormally high amounts of eosinophils are found in either the blood or in body tissues. Eosinophilia occurs in a wide range of conditions, including allergic diseases such as asthma, hay fever and eosinophilic esophagitis. The invention therefore also relates to methods and compositions for preventing or treating eosinophilia. Worldwide the main cause of eosinophilia is parasitic infection. It can also occur in relation to common skin diseases, medicine reactions, and parasitic infections. Increased numbers of eosinophils are associated with allergic disease or parasitic infections. This is helpful in combating parasitic infections but in cases of allergic diseases they accumulate in tissues and cause damage. For example, in asthma, eosinophilia causes damage to the airways of the lung.

The exact mechanism by which diacyl [18:0; 18:0] phosphatidylinositol exerts its suppressive effects in autoimmune diseases is at present unclear. Most likely, it is blocking downstream TCR signalling per se, irrespective of how the TCR is triggered. .

A pharmaceutical composition comprising diacyl [18:0 ; 18:0] phosphatidylinositol or a pharmaceutically acceptable salt (e.g. sodium or ammonium) thereof may be in the form of a capsule, tablet, lozenge, enema, dragee, pill, droplet, suppository, powder, spray, vaccine, ointment, paste, cream, inhalant, patch, aerosol, and the like.

Pharmaceutically acceptable salts can be, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like. A thorough discussion of pharmaceutically acceptable excipients is available in

Remington's Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991).

As pharmaceutically acceptable carrier, any solvent, diluent or other liquid vehicle, dispersion or suspension aid, surface active agent, isotonic agent, thickening or emulsifying agent, preservative, encapsulating agent, solid binder or lubricant can be used which is most suited for a particular dosage form and which is compatible with the diacyl [18:0; 18:0] phosphatidylinositol.

Pharmaceutically acceptable carriers in therapeutic compositions may contain liquids such as water, saline, glycerol and ethanol. Saline is preferred. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles. Typically, the therapeutic compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared.

For therapeutic treatment, diacyl [18:0; 18:0] phosphatidylinositol is applied to the subject in need thereof. The diacyl [18:0; 18:0] phosphatidylinositol may be administered to a subject by any suitable route, preferably in the form of a pharmaceutical composition adapted to such a route and in a dosage that is effective for the intended treatment. A mucosal route of aclministration, e.g. intrarectal or intratracheal, is preferred as this reduces the risk of unwanted side-effects when compared to systemic administration. Therapeutically effective dosages of the diacyl [18:0; 18:0] phosphatidylinositol required for treating the disorder, for instance for prevention and/or treatment of a disorder selected from the group consisting of asthma, rheumatoid arthritis, diabetes, psoriasis and inflammatory bowel disease in the body of a human or animal subject, can easily be determined by the skilled person, for instance by using animal models. Of course, diacyl [18:0; 18:0] phosphatidylinositol can also be used in combination with one or more immunosuppressant or antiinflammatory (drug) therapies known in the art, including corticosteroids and nonsteroidal anti-inflammatory drugs (NSAIDs).

The term "therapeutically effective amount" as used herein refers to an amount of diacyl [18:0; 18:0] phosphatidylinositol to reduce or prevent unwanted or excessive T cell activation, or to exhibit a detectable therapeutic or prophylactic effect. The effect can be detected by, for example, measurement of cytokine production or by any other suitable method of assessing the progress or severity of inflammatory responses which methods are known per se to the skilled person. The precise effective amount for a subject will depend upon the subject's size and health, the nature and extent of the condition, and the therapeutics or combination of therapeutics selected for administration. Thus, it is not useful to specify an exact effective amount in advance. However, the effective amount for a given situation can be determined by routine experimentation and is within the judgment of the clinician or experimenter.

Specifically, the compositions of the present invention can be used to reduce or prevent psoriasis, asthma or ulcerative colitis (UC) and/or accompanying biological or physical manifestations. Methods that permit the clinician to establish initial dosages are known in the art. The dosages to be administered must be safe and efficacious. For purposes of the present invention, an effective daily dose will be from about 0.01 μg/kg to 1 g/kg and preferably from about 0.5 μg kg to about 400 mg/kg of diacyl [18:0; 18:0] phosphatidylinositol in the individual to which it is administered.

Yet in another alternative embodiment, the diacyl [18:0 ; 18:0] phosphatidylinositol of the invention may be administered from a controlled or sustained release matrix inserted in the body of the subject.

A composition wherein said diacyl [18:0 ; 18:0] phosphatidylinositol makes up less than 50 percent, preferably less than 30 percent, more preferably less than 25 percent of the dry weight of the composition was found to be effective in reducing or preventing inflammatory symptoms.

Given the broad spectrum of immunomodulatory effects of inositol phospholipids as disclosed herein and in WO 2005/142510, the invention also relates to a food item, food supplement or food additive comprising diacyl [18:0; 18:0] phosphatidylinositol. The food item can be a solid or a liquid food product. The food additive is defined as a composition that can be used for the manufacture of a (functional) food item. A food supplement is defined as a composition that can be consumed in addition to the normal food intake and which comprises elements or components that are not or in only minor amounts, present in the normal diet and of which sufficient or increased consumption is desired. The composition of a food item does not necessarily differ much from that of a food supplement or food additive.

A diacyl [18:0; 18:0] phosphatidylinositol-enriched food item or food supplement as described above may suitably comprise 0.01 to 99.9 wt.% of diacyl [18:0; 18:0] phosphatidylinositol. In a preferred embodiment such a food item or food supplement comprises from 0.01 to 50 wt.%, preferably from 0.01 to 10 wt.%, more preferably from 0.01 to 5 wt.% of diacyl [18:0 ; 18:0] phosphatidylinositol or acceptable salt thereof.

In order to make a food item or food supplement comprising an elevated amount of diacyl [18:0; 18:0] phosphatidylinositol suitable for human or animal consumption, the nutritional value, texture, taste or smell may be improved by adding various compounds to said item or supplement. The skilled person is well aware of the different sources of protein, carbohydrate and fat that may be used in food items or food supplements according to the invention and of the possible sweeteners, vitamins, minerals, electrolytes, coloring agents, odorants, flavoring agents, spices, fillers, emulsifiers, stabilizers, preservatives, antioxidants, food fibers, and other components for food items that may be added to improve its nutritional value, taste or texture. The choice for such components is a matter of formulation, design and preference. The amount of such components and substances that can be added is known to the skilled person, wherein the choice may e.g. be guided by recommended daily allowance dosages (RDA dosages) for children and adults and animals.

Portions for intake of the food item or food supplement may vary in size and are not limited to the values corresponding to the recommended dosages. The term "food supplement" is herein not intended to be limited to a specific weight or dosage. A composition of a food item or food supplement as described above may in principle take any form suited for consumption by man or animal. In one embodiment the composition is in the form of a dry powder that can be suspended, dispersed, emulsified or dissolved in an aqueous liquid such as water, coffee, tea, milk, yogurt, stock or fruit juice and alcoholic drinks. To this end, the powder may be provided in unit-dosage form.

In an alternative preferred embodiment an diacyl [18:0; 18:0] phosphatidylinositol- comprising composition in the form of a dry powder is tableted. To that end, a composition for a food supplement according to the invention may very suitably be provided with fillers, such as microcrystalline cellulose (MCC) and mannitol, binders such as hydroxypropylcellulose (HPC), and lubricants such as stearic acid or other excipients.

A composition of a food item or food supplement as described above may also be provided in the form of a liquid preparation wherein the solids are suspended, dispersed or emulsified in an aqueous liquid. Such a composition may be admixed directly through a food item or may e.g. be extruded and processed to grains or other shapes.

In an alternative embodiment a food item or food supplement may take the shape of a solid, semi-solid or liquid food item, such as a bread, a bar, a cookie or a sandwich, or as a spread, sauce, butter, margarine, dairy product, and the like. Preferably, diacyl [18:0; 18:0] phosphatidylinositol is applied in a dairy product, such as for instance a butter or margarine, custard, yogurt, cheese, spread, drink, or pudding or other dessert. Preferably the food item is a drink, as one can easily estimate the intake of lipid this way. The diacyl [18:0; 18:0] phosphatidylinositol can also be used in butters or fats used for frying and baking, because it is relatively resistant to degradation at high temperatures. This characteristic also enables use of the diacyl [18:0; 18:0] phosphatidylinositol in food items or food supplements that undergo a pasteurization or sterilization treatment. Diacyl [18:0; 18.0] phosphatidylinositol-containing diet or slimming products also constitute preferred embodiments of food items or food supplements according to the invention.

If a food item according to the invention is used as an animal feed, the food item may e.g. be prepared in the form of a powder, a grain, a waffle, a porridge, a block, a pulp, a paste, a flake, a cook, a suspension or a syrup.

For administering to humans the food item of the invention may very suitably be prepared in the form of a food supplement.

The present invention further relates to a method for the preparation of a food item or food supplement according to the invention, comprising enriching a food item or food supplement with diacyl [18:0; 18:0] phosphatidylinositol or an acceptable salt thereof. In one embodiment the invention provides a method for the preparation of a food item or food supplement enriched with diacyl [18:0 ; 18:0] phosphatidylinositol, comprising processing diacyl [18:0 ; 18:0] phosphatidylinositol in a food item or food supplement, preferably to an amount of 0.01 to 99.9 wt.%, more preferably to an amount of from 0.01 to 50 wt.%, even more preferably to an amount of from 0.01 to 10 wt.%, and most preferably to an amount of from 0.01 to 5 wt.%.

In a method for preparing a food item according to the invention the food item may first be prepared separately and then be joined with diacyl [18:0 ; 18:0]

phosphatidylinositol to provide a food item according to the invention wherein said diacyl [18:0; 18:0] phosphatidylinositol is incorporated in the food item. The food item may be separately prepared by conventional methods such as by mixing, baking, frying, cooking, steaming or poaching and may, if necessary, be cooled prior to joining with diacyl [18:0; 18:0] phosphatidylinositol. According to another suitable embodiment, diacyl [18:0; 18:0] phosphatidylinositol is incorporated as a component in the food item during the preparation thereof.

A food item or food supplement according to the present invention may very suitably be defined as a nutraceutical composition. Nutraceuticals can be defined as natural products that are used to supplement the diet by increasing the total dietary intake of important nutrients. This definition includes nutritional supplements such as vitamins, minerals, herbal extracts, antioxidants, amino acids, and protein supplements.

Nutraceutical products fit into the newly created product category of "Dietary

Supplements" as established by the F.D.A. in the Dietary Supplement Act of 1994. This act specifically defined dietary supplements to include: vitamins, minerals, herbs or other botanicals, antioxidants, amino acids, or other dietary substances used to supplement the diet by increasing the total daily intake.

A "nutraceutical composition" is defined herein as a food composition fortified with ingredients capable of producing health benefits. Such a composition in the context of the present invention may also be indicated as foods for special dietary use; medical foods; and dietary supplements. The food item and/or food supplement of the present invention is a nutraceutical composition since it is fortified with diacyl [18:0; 18:0] phosphatidylinositol according to the invention and since it is capable of treating or preventing diseases or conditions associated with excessive or unwanted T cell activation. For example, the food item or supplement may help to prevent or reduce symptoms associated with an inflammatory condition such as allergies (e.g. hay fever) and the like.

As with the pharmaceutical composition, the amount of diacyl [18:0; 18:0] phosphatidylinositol in the food or food additive will depend on several factors. The food product will generally comprise a concentration of diacyl [18:0; 18:0] phosphatidylinositol that is sufficient to provide a consumer with an effective amount of lipid upon consumption of a regular (e.g. daily) portion of the food product. For example, the food product may be a beverage which is typically consumed as a 200- ml portion wherein diacyl [18:0; 18:0] phosphatidylinositol is present in an amount of 0.0005 to 2 mg per litre, preferably etc. In another embodiment, the invention provides a spread comprising diacyl [18:0; 18:0] phosphatidylinositol. It will be recognized by those skilled in the art that the optimal quantity and spacing of individual dosages for achieving the therapeutic effects of the pharmaceutical composition, food item or food supplement described herein may easily be determined by the skilled person. They will be determined by the nature and extent of the condition being treated, as well as by the form, route and site of administration, and the particular individual undergoing treatment, and that such optima can be determined by conventional techniques. Typically, the treatment will involve the administration of diacyl [18:0; 18:0] phosphatidylinositol in amount of approximately

0.001 to 1 gram, preferably 0.005 to 0.5 gram, more preferably 0.01 to 0.1 gram per kilogram of body weight per 24 hours. If applied as topical composition to the skin of a patient, the administration will amount to approximately 0.001 to 1 gram per cm² of the skin, preferably 0.005 to 0.5 gram, more preferably 0.01 to 0.1 gram per cm². It will also be appreciated by those skilled in the art that the optimal dosage regimen,

1. e. the number of doses, can be ascertained using conventional course of treatment determination tests well known in the art. Generally speaking, a therapeutic dosing regimen will involve the administration of the selected dosage formulation comprising diacyl [18:0; 18:0] phosphatidylinositol, e.g. an aerosol spray for asthma patients, at least once daily, preferably one to four times daily, until the symptoms have subsided, or given in times of remission, in order to prevent a disease flare-up (exacerbation). For purposes of the present invention, an effective dose will be from about 0.01-5% of the dry food weight in the individual to which it is administered, meaning that for an adult human being the daily dose will be between about 0.04 and 35 grams of diacyl [18:0; 18:0] phosphatidylinositol. Preferably, a pharmaceutical composition is formulated such that it is suitable for the topical and/or mucosal administration to a subject in need thereof, e.g. a human or animal subject suffering from an immune disease. Mucosal routes of drug delivery include the mucosal linings of the nasal, rectal, vaginal, ocular and oral cavity. Sites for topical mucosal administration include the airway passage (e.g. nasal or intratracheal delivery) and the gastrointestinal tract. In one embodiment, the invention provides a spray comprising diacyl [18:0; 18:0] phosphatidylinositol. In particular, it provides the use of an aerosol spray comprising diacyl [18:0; 18:0]

phosphatidylinositol for the treatment or prevention of symptoms associated with asthma. In another embodiment, it provides a rectal enema or suppository comprising diacyl [18:0; 18:0] phosphatidylinositol. These are particularly useful in the treatment or control of inflammatory bowel diseases. The two main categories of inflammatory bowel disease are ulcerative colitis and Crohn's disease. Ulcerative colitis is characterized by recurring episodes of inflammation of the mucosal layer of the large bowel not related to an intestinal infection. The inflammation involves the rectum and may extend proximally in a continuous fashion. Crohn's disease is characterized by recurring episodes of suppurative inflammation of any part of the bowel, from the mouth to the anus. This inflammation is transmural, and can result in strictures, microperforations, and fistulae. The inflammation is non-contiguous and thus can produce skip lesions throughout the bowel. A composition of the invention comprising diacyl [18:0; 18:0] phosphatidylinositol for use in the treatment of inflammatory bowel disease may of course also contain one or more therapeutic substances and excipients commonly used. For example, it may contain other anti- inflammatory drugs such as an aminosalicylate (e.g.5-ASA) or (cortico-) steroid. In yet another embodiment, the present invention provides an ointment or creme comprising diacyl [18:0; 18:0] phosphatidylinositol. These are particularly useful in the treatment or control of inflammatory diseases of the skin such as psoriasis or pemphigus vulgaris. Also for these indications combinations with other drugs, such as corticosteroids are contemplated.

A known issue relating to the administration of lipids to a subject, be it in foods or in pharmaceutical compositions, is that they can be metabolized, e.g. by general or specific lipases. This is particularly relevant for application of phospholipids in the digestive tract. Also within the present invention, metabolism (e.g. degradation or conversion) of diacyl [18:0; 18:0] phosphatidylinositol administered to a subject may occur prior to the moment the lipid reaches its site of action, such as APCs and/or T cells. This issue may be addressed by administering diacyl [18:0; 18:0]

phosphatidylinositol or a pharmaceutically acceptable salt thereof, alone or in combination, as a so-called precursor compound which compound comprises certain substituents as a result of which the diacyl [18:0; 18:0] phosphatidylinositol can no longer, or only at reduced rates, be metabolized. These precursors are preferably resistant to hydrolysis in the upper parts of the digestive tract (e.g. mouth, stomach), and are for instance split relatively easy in the lower part of the digestive tract (e.g. coecum, colon), if diacyl [18:0; 18:0] phosphatidylinositol should have its working especially there. Preferably, when the intake of the precursor is via the oral route, the intact or metabolized precursors are taken up into the blood stream and transported to the target site(s) where they may be activated in order to exert their beneficial effect. Thus, it is possible that activation occurs when the compound has been absorbed from the digestive tract, e.g. in the blood or the liver. As a result, the amount of the compound is raised at those locations where the diacyl [18:0; 18:0]

phosphatidylinositol has its action. For instance, a diacyl [18:0; 18:0]

phosphatidylinositol precursor may be used that can be split or activated in vivo by a suitable enzyme so that the diacyl [18:0; 18:0] phosphatidylinositol is liberated that may suppress T cell activation in the subject.

Diacyl [18:0; 18:0] phosphatidylinositol or a pharmaceutically acceptable salt thereof, may be provided to a subject in need thereof for prophylactic or therapeutic reasons. Diacyl [18:0; 18:0] phosphatidylinositol or a pharmaceutically acceptable salt thereof, may be provided to a subject in need thereof in the form of a food item or food supplement, or in the form of a pharmaceutical preparation, all such administration forms being capable of preventing the development and/or to alleviate the symptoms of unwanted T cell activation. In particular, the development and/or severity of asthma, diabetes and inflammatory bowel disease are considered.

Diacyl [18:0; 18:0] phosphatidylinositol or a pharmaceutically acceptable salt thereof, may be used in a food item or food supplement. A food supplement is defined as a composition that can be consumed in addition to the normal food intake and which comprises elements or components that are not or in only minor amounts, present in the normal diet and of which sufficient or increased consumption is desired. The composition of a food item does not necessarily differ much from that of a food supplement.

A food item or food supplement with diacyl [18:0; 18:0] phosphatidylinositol is advantageously used for the prevention and/or treatment of an inflammatory disease, in particular allergic asthma or ulcerative colitis. The food item or supplement also finds its use in a diet for treating and/or preventing an inflammatory disease, in particular allergic asthma or ulcerative colitis. Thus, the invention provides an immunosuppressive food item or food supplement comprising diacyl [18:0; 18:0] phosphatidylinositol. Also provided herein is a method of preventing the occurrence of an inflammatory disease, in particular allergic asthma, psoriasis or ulcerative colitis, in a healthy subject comprising providing said subject a diet with enhanced levels of diacyl [18:0; 18:0] phosphatidylinositol or a pharmaceutically acceptable salt thereof. Alternatively, or in addition, said subject may be administered (e.g. via the topical and/or mucosal route) a pharmaceutical composition according to the invention. The invention furthermore provides the use of a pharmaceutical composition according to the invention for the prevention and/or treatment of an inflammatory disease, in particular allergic asthma, psoriasis or inflammatory bowel diseases.

A further aspect relates to a method of treatment of a subject suffering from an inflammatory disease, in particular allergic asthma, psoriasis or inflammatory bowel diseases, such as ulcerative colitis, said method comprising administrating to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition according to the invention. EXAMPLES

Example 1 - synthesis of diacyl [18:0; 18:0] phosphatidylinositolSynthesis

The chiral lipid building block (5) was prepared starting from commercially available (S)-(+)-2,2-dimethyl-l,3-d ioxolane-4-methanol (1 ), by a number of straight-forward synthetic steps (scheme 2).

Scheme 2

THF

Pd/C

EtOH, AcOH

First, the free hydroxyl group is reacted with benzylbromide (2) to afford benzyl ether 3. Next, the d ioxolane moiety is cleaved under acidic conditions, yielding diol 4, after which the liberated hydroxyl groups are esterified with stearoic acid to afford 5. Finally, the benzyl ether is cleaved by hydrogenolysis, leading to the desired lipid building block 5.

The required chiral inositol building block (13) was prepared starting from commercially available myo-inositol (7), as depicted in scheme 3.

Scheme 3

TBAF, THF

13

Desymmetrization of myo-inositol was achieved by reaction of 7 with D-( + )- camphor dimethyl acetal (8) under acidic conditions, affording enantiomerically pure ketal 9, after crystallization. Next, one of the remaining hydroxyl groups was selectively protected using fert-butyldiphenylsilylchloride (TBDPSCI), after which the chiral camphor auxiliary was removed by treatment with mercapto-ethanol and a Lewis acid. The resulting pentaol 11 was treated with excess of methoxymethyichioride (MOMCI) and subsequently with TBAF to remove the silyl protecting group, leading to the desired inositol building block (13).

Coupling of the building blocks 7 and 13 and further elaboration to PI 18 : 0 was achieved using the multistep procedure depicted in scheme 4.

13.

tetrazole

Wmelfiflamine

18 P1 18:0 ammonium salt

First, reaction of lipid building block 6 with /v,/ -diisopropylmethylphosphonamidic chloride (14) afforded intermediate 15 which was isolated but immediately reacted with inositol building block 13, to afford intermediate phosphite 16. Again, this intermediate was not isolated but reacted fu rther with

mefachloroperbenzoic acid (mCPBA) to afford phosphate 17. Demethylation of the phosphate was achieved with trimethylamine, leading to tetramethylammonium salt 18. Finally, deprotection of the MOM groups by means of ethanethiol and a Lewis acid, followed by treatment with ammonium hydroxide and silica gel column chromatography, afforded the desired PI 18 : 0 as the corresponding ammonium salt.

Example 2 - Inhibition of interferon gamma release by PBMC

For analysis of PI 18:0 inhibitory capacity, cells of a T-cell line DN32.D3 (hereafter referred to as DN32) were used. DN32 cells were stimulated with 5 ng/mL phorbol ester 4-phorbol- 12-myristate-13-acetate (PMA, Sigma-Aldrich) and 100 ng/mL CAI (A23187, Sigma-Aldrich) in the presence of a concentration range from 0 to 100 microg/mL bovine PI (Avanti) or synthetic PI 18:0. At 24 h IL-2 concentrations were measured in the supernatant by means of ELISA. The results in Fig. 1 show that low concentrations of PI 18:0 are capable of inhibiting IL-2 release. In the low concentration range, the inhibitory effect is as strong as that of bovine liver PI.

To test the toxicity of the compound, DN32 cells were stimulated with 5 ng mL phorbol ester 4-phorbol-12-myristate-13-acetate (PMA, Sigma-Aldrich) and 100 ng mL CAI (A23187, Sigma-Aldrich) in the presence of a concentration range from 0 to 100 microg/mL bovine PI (Avanti) or synthetic PI 18:0. After 18h of culture viability of the cells was determined with trypan blue exclusion. The results are shown in Fig. 2, where it can be concluded that PI 18:0 has the same low toxicity as bovine liver PI: nearly 100% of the cells survived, and there was no clear dosis-effect response between doses of 0.5 and 100 μg/mL.

In order to test the actual signal transduction mechanism, DN32 cells were stimulated with 5 ng/mL phorbol ester 4-phorbol-l 2 -myristate- 13 -acetate (PMA, Sigma-Aldrich) and 100 ng/mL CAI (A23187, Sigma-Aldrich) in the presence of a concentration range from 0 to 200 microg/mL bovine PI (Avanti) or synthetic PI 18:0. After lh of culture phosphorylation of p38 and J K was determined by CBA analysis.

Quantitative differences were obtained by determining phosphoproteins in cell lysates with the BD-phosphoprotein-cytometric bead array (BD Biosciences) that was performed according to the manufacturers' instructions (Fig. 3). It can be observed that both phospho-p38 and phospho-JNK are inhibited by both PI 18:0 and bovine liver PI. Especially in the case of phospho-JNK it appears that lower doses of PI 18:0 are more effective, and an inverse dose-relationship was observed. For bovine PI and for inhibition of phosphor-p38 the dose-effect relation was less pronounced, both for PI 18:0 and bovine liver PI. For testing the effect on ERK phosphorylation, DN32 cells were stimulated with 5 ng/mL phorbol ester 4-phorbol-l 2-myristate- 13 -acetate (PMA, Sigma-Aldrich) and 100 ng/mL CAI (A23187, Sigma-Aldrich) in the presence of a concentration range from 0 to 200 microg/mL bovine PI (Avanti) or synthetic PI 18:0. After lh of culture phosphorylation of ERKl and ERK2 were determined by Western blot: Proteins from whole cell lysates were separated by SDS-PAGE and transferred to immobilon-P transfer membrane. Western blots were stained with antibodies to Phospho-p44/42 MAPK (ERK1/2), actin (Cell Signalling, Boston, MA, USA) and HRP-conjugated secondary antibody. Detection was performed with Luminescence Supersignal West Femto (Pierce, Rockford, IL, USA). Intensity of the staining was assessed using Gene-Tools (Syngene, Frederick, MD, USA). Data were expressed (see fig. 4) as percentage phosphorylated protein relative to actin on the same blot. Bovine PI showed a dose-dependent inhibition of ERK phosphorylation both for p42 and p44 MAPK, PI 18:0 had no effects, neither with lower doses nor with higher doses. The PI from soy protein, which also was tested in this assay showed a slight inhibitory effect at low doses, similar to the effect of the same doses of bovine PI, but at the highest dose tested (200 μg/mL) the dose-dependency disappeared and in the case of p44 MAPK even a stimulation was observed.

Thus, with respect to PI 18:0 the following conclusions can be drawn from the above experiments:

• PI (18:0) inhibits T-cell activation.

• PI (18:0) suppression reaches a plateau level leaving residual IL-2 release · The inhibition remains effective at low concentrations.

• PI (18:0) inhibits P38 and JNK phosphorylation.

The anti-inflammatory capacities of PI 18:0 were tested in comparison with dexamethasone. For this, human peripheral blood mononuclear cells (PBMC) were isolated from buffy coats of healthy donors. PBMC 3xl0⁵ cells/ml were cultured in flat bottom culture plates with anti-CD3 anti-CD28 coated beads (stimulator beads, Dynal) at a ratio of 1 bead per 2 cells in the presence of 50 μ¾½Ι, or 100 μ /πιί of PI 18:0 or 2 x 10^e"8 M dexamethasone. At 48h after culture human interferon-gamma concentrations were measured in the supernatant by means of ELISA (see Fig. 5). PI 18:0 showed a clear dose-dependent effect on the IFNy release of the PBMCs where the 100 μg/mL dose of PI 18:0 showed a stronger inhibitory effect than the tested concentration of dexamethasone. Example 3 - Inhibition of psoriasis development by diacyl [18:0; 18:0] phosphatidylinositol

Human peripheral blood mononuclear cells (PBMC) were isolated from buffy coats of healthy donors. PBMC were cultured at a concentration of 3x10⁵ cells/ml in the bottom chamber of transwell plates (12 well) and stimulated with anti-CD3 anti - CD28 coated beads (stimulator beads, Dynal) at a ratio of 1 bead per 2 cells. The compound to be tested was added to the bottom chamber. In the top chamber a skin biopsy from a healthy volunteer was placed. At 72h after culture the human biopsy was immersed in Tissue-Tek® O.C.T™ Compound (Sakura, Cat. No. 4583, www.sakuraeu.com) and snapfrozen in liquid nitrogen. Cryostat sections of 6 micrometer were stained with a mouse anti human ICAM primary antibody (R&D systems) and a biotinylated secondary horse-anti-rabbit secondary antibody. Staining was visualized with the Vectastain ABC Elite Kit (Vector Laboratories) and 3,3'- diaminobenzidine tetrahydrochloride (Sigma-Aldrich, St. Louis, MO, USA). Sections were counterstained with Hematoxylin. Scoring of the staining intensity and localization was performed by two independent individuals in blinded fashion. The score ranged from a minimum of 0 to a maximum of 4.

Fig. 7 shows the experimental set-up (left panel) and the effects of PI (18:0) 100 g/mL in comparison with the effects of the known psoriasis inhibitor dexamethason on the stained cryostat sections. The skin stimulation as performed in the above described experimental set-up normally induces expression of ICAM- 1 in the skin. With both treatments of PI (18:0) and dexamethason, no ICAM molecules are visible.

Fig. 8 shows effect of PI (18:0) in the psoriasis model in comparison to the effects of bovine PI. The skin of three different human donors was treated as described above, and either no, 100 μg mL bovine PI (Avanti) or 50 pg/ml PI (18:0) was tested. It shows that in all three experiments PI (18:0) outperforms the effect of bovine PI, even with a lower dose.

From the transwell cultures with skin biopsies as described above, supernatants were collected and human interferon-γ concentrations were measured (ELISA). As is shown in Fig. 9 a dose-dependent relation with the concentration of PI (18:0) could be established, providing more proof that PI (18:0) inhibits the interferon-γ induced expression of ICAM1 in the skin.

Example 4 - Analogues of diacyl [18:0; 18:0] phosphatidylinositol

The anti-inflammatory capacities of PI 18:0 were tested in comparison with several analogues. For this, human peripheral blood mononuclear cells (PBMC) were isolated from buffy coats of healthy donors. PBMC 3xl0⁵ cells/ml were cultured in flat bottom culture plates with anti-CD3 anti-CD28 coated beads (stimulator beads, Dynal) at a ratio of 1 bead per 2 cells in the presence of 10, 25, 50, 100 or 150 g/mL of PI 18:0 or the indicated dose of PI analogue (see Fig. 6). At 48h after culture human interferon-gamma concentrations were measured in the supernatant by means of ELISA.

The analogues that were tested were PI (5:0), which is a phosphatidylinositol with two fatty acids of only 5 C -atoms, PX (18:0) is a molecule that does not contain the inositol headgroup and epi-PI (18:0), which is ([ IS,2R,3S,4S,5R,6R] diacyl [18:0; 18:0] phosphatidylinositol (compound 2 in Fig. 10).

Furthermore, also myoinositol from two different sources and distearoyl glycerol have been tested. Moreover, as chloroform methanol was used in the storage procedure of some of the compounds a solvent control, obtained by the exact same method, (Mock Chlor/MeOH) was included.

As can be seen in Fig. 6, only bovine PI and PI (18:0) show efficacy. Some activity is observed with epi-PI (18:0) and PX (18:0), whereas the other compounds are not or only marginally active.

Claims

1. Diacyl phosphatdylinositol in which both the sn- 1 and the sn-2 place are taken by stearic acid (18:0) (diacyl [18:0; 18:0] phosphatidylinositol), more preferably, wherein said diacyl phosphatidylinositol is compound 1 as depicted in Fig. 10 or a racemate of compounds 1 and 2 as depicted in Fig. 10.

2. Diacyl [18:0; 18:0] phosphatidylinositol as defined in claim 1 for use in the treatment of a disease or a condition wherein suppression of T-cell activation is desirable.

3. Diacyl [18:0; 18:0] phosphatidylinositol as defined in claim 1, for use in the treatment of asthma, diabetes Type 1, rheumatoid arthritis, inflammatory bowel disease or psoriasis.

4. Pharmaceutical composition comprising diacyl [18:0; 18:0] phosphatidylinositol as defined in claim 1 and no other phosphatidylinositol species.

5. Pharmaceutical composition according to claim 4, further comprising excipients for topical application, preferably for topical application on the skin.

6. Pharmaceutical composition according to claim 4, further comprising excipients for mucosal application, preferably for mucosal application in the airways or in the intestine.

7. Pharmaceutical composition according to any of claims 4-6, further comprising one or more other active ingredients, these not being other phosphatidylinositol species.

8. Pharmaceutical composition according to any of claims 4-7, wherein said diacyl [18:0; 18:0] phosphatidylinositol is present in said composition in a suspended form in a physiologically acceptable aqueous carrier, preferably in physiological saline.

9. Pharmaceutical composition according to any of claims 4-8, wherein said diacyl [18:0 ; 18:0] phosphatidylinositol makes up less than 50 percent, preferably less than 30 percent, more preferably less than 25 percent of the dry weight of the composition.

10. Pharmaceutical composition according to any one of claim 4-9, wherein said composition is adapted for mucosal administration.

11. Composition according to claim 11 , wherein said composition is adapted for rectal administration.

12. Composition according to claim 11, wherein said composition is adapted for pulmonary administration, for example in the form of a nebuliser or aerosol.

13. A method for preparing a pharmaceutical composition comprising as active ingredient diacyl [18:0; 18:0] phosphatidylinositol as defined in claim 1 and no other phosphatidylinositol species, comprising preparing a non-liposomal suspension of said diacyl [18:0; 18:0] phosphatidylinositol in an aqueous physiologically acceptable carrier, preferably in saline.

14. Use of diacyl [18:0; 18:0] phosphatidylinositol as defined in claim 1 for the manufacture of a medicament for the treatment or prevention of asthmatic conditions in a mammal.

15. Use of diacyl [18:0; 18:0] phosphatidylinositol as defined in claim 1 for the manufacture of a medicament for the prevention or treatment of an inflammatory bowel disease in a mammal, preferably ulcerative colitis or Crohn' s disease.

16. A food item or food supplement enriched with diacyl [18:0; 18:0]

phosphatidylinositol as defined in claim 1.

17. Use of diacyl [18:0; 18:0] phosphatidylinositol as defined in claim 1 for the manufacture of a food item or food supplement capable of preventing an

inflammatory disease, in particular allergic asthma or ulcerative colitis.

18. A method of preventing the occurrence of an inflammatory disease in a healthy subject, in particular allergic asthma or ulcerative colitis, comprising providing said subject a diet with diacyl [18:0; 18:0] phosphatidylinositol as defined in claim 1 or a pharmaceutically acceptable salt thereof.

19. A method of preventing the occurrence of an inflammatory disease in a healthy subject, in particular allergic asthma or ulcerative colitis, comprising administrating to said subject a pharmaceutical composition according to any one of claims 4 to 12.

20. A method of treatment of a subject suffering from an inflammatory disease, in particular allergic asthma or ulcerative colitis, said method comprising administrating to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition according to any one of claims 4 to 12.

21. Use of a food item with diacyl [18:0; 18:0] phosphatidylinositol as defined in claim 1 for the prevention and/or treatment of an inflammatory disease, in particular allergic asthma or ulcerative colitis.

22. Use of a food item with diacyl [18:0; 18:0] phosphatidylinositol as defined in claim 1 in a diet for treating and/or preventing an inflammatory disease, in particular allergic asthma or ulcerative colitis.

23. Use of a pharmaceutical composition according to any one of claims 4 to 12 for the prevention and/or treatment of an inflammatory disease, in particular allergic asthma or ulcerative colitis.

24. A pharmaceutical composition according to any of claims 4-9 for topical administration.

25. A pharmaceutical composition according to claim 24 for the prevention or treatment of psoriasis.